Issue published October 1, 1985 Previous issue | Next issue
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Research Articles
×Abstract
In many species, including humans, pulmonary alveoli are formed after birth by septal subdivision of the large gas-exchange saccules present at birth. In rats septation occurs mainly between the 4th and 14th postnatal days (Burri, P. H. 1974. Anat. Rec. 180:77-98), but little is known about the regulation of this process. We found that dexamethasone (0.1 micrograms daily) given to rats from age 4 to 13 d markedly impaired saccule septation to at least age 60 d and also diminished the extent of the increase of alveolar surface area (Sa). Underfeeding from birth to age 14 d did not diminish saccule septation but did result in diminished Sa. We conclude dexamethasone-treated rats have a critical period during which the gas-exchange saccules present at birth must be subdivided. Since Sa increased in dexamethasone-treated rats without a change in alveolar size, and, the enlargement of Sa was diminished in underfed rat pups without a deficit of saccule septation, we postulate new alveoli were formed by means other than septation of the large gas-exchange saccules present at birth. Furthermore, these various means of forming alveoli, and hence of increasing Sa, were differently regulated: dexamethasone decreased the enlargement of Sa brought about by both septation of the gas-exchange saccules present at birth and by other, as yet unidentified, means of forming alveoli; underfeeding did not diminish Sa increases produced by saccule septation but did decrease the extent of Sa enlargement due to the other means of forming alveoli.
Authors
D Massaro, N Teich, S Maxwell, G D Massaro, P Whitney
×Abstract
Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.
Authors
Y Matzner, M Bar-Ner, J Yahalom, R Ishai-Michaeli, Z Fuks, I Vlodavsky
×Abstract
The relationship between the expression of HLA-DR antigens and the HLA-DR alpha gene methylation was examined in systemic lupus erythematosus (SLE). Using permanent B cell lines, we found reduced DR expression in SLE. The low DR expression was correlated with high anti-DNA antibody titers in patients' sera. The amounts of DR alpha message were lower in SLE cells than in normal controls, suggesting that the low expression of DR antigens is associated with gene functions. The extent of DNA methylation was examined at five CCGG sites in the HLA-DR alpha locus. DNA from both SLE and normal cells showed variable methylation patterns. Since the DR alpha gene is a single-copy gene, such a variability is the result of assaying a mixture of transformed clones containing methylated DR alpha gene, with other clones containing unmethylated DR alpha gene. A distinctive feature of normal cells was a consistent methylation pattern: 12 normal cell lines showed exactly the same pattern. In contrast, 28 SLE cell lines showed a cell-line-specific methylation, and hypermethylation at the DR alpha locus. The hypermethylation is often associated with transcriptionally inactive genes. Thus, our results suggest that (a) B cells with hypermethylated DR genes might express no or few DR antigens; (b) the ratio of cells with differently methylated DR genes is consistent in normal individuals, while, in SLE patients, cells with hypermethylated DR genes predominate, resulting in apparently reduced DR antigen expression; and (c) the aberrant DR expression could be associated directly with immunoregulatory dysfunctions in SLE disease.
Authors
H Sano, L J Compton, N Shiomi, A D Steinberg, R A Jackson, T Sasaki
×Abstract
We have characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with less than 0.1 microM MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 microM) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 microM) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 microM drug. Two cell lines with resistance to 10 microM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 microM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.
Authors
G A Curt, J Jolivet, D N Carney, B D Bailey, J C Drake, N J Clendeninn, B A Chabner
×Abstract
A novel platelet-agglutinating protein (PAP) was purified approximately 2,000-fold from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) by ammonium sulfate fractionation, DEAE-Sephacel and concanavalin A-Sepharose chromatographies. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with and without reduction, this preparation revealed a major protein band with a molecular weight of 37,000, and a minor band with a molecular weight of 32,000-34,000. After elution from the gel, only the 37,000-mol wt protein corresponding to the major band induced the platelet agglutination. When four normal plasmas and the recovery plasma from the same TTP patient were subjected to the similar purification steps, the 37,000-mol wt major band was absent. The 125I-PAP bound to the platelets in a concentration-dependent manner. The platelet agglutination induced by PAP was not inhibited by hirudin, heparin in the presence of antithrombin III, phenylmethylsulfonyl fluoride, apyrase, aspirin, or prostaglandin I2. However, it was inhibited by IgG from normal adults and from the same TTP patient after recovery. The anti-37,000-mol wt PAP antiserum prepared in the rabbit formed a single precipitin line against the highly purified PAP. Using this antiserum in the Western immunoblotting, the 37,000-mol wt protein band was found in the three TTP plasmas, of which the platelet-agglutinating activity was inhibited by the anti-37,000-mol wt PAP IgG. The 37,000-mol wt immunoprecipitin band was absent in the plasmas obtained from another two TTP patients, two normal subjects, two patients with idiopathic thrombocytopenic purpura, and two patients with disseminated intravascular coagulation. These results suggest that the 37,000-mol wt PAP is present only in certain cases of TTP, and is likely to be responsible for the formation of platelet thrombi in the microcirculation.
Authors
F A Siddiqui, E C Lian
×Abstract
A method has been developed for the analysis of hepatitis B surface antigen (HBsAg) antigenic structure at the molecular level that creates "fingerprints" or "signatures" of various hepatitis B viral (HBV) strains. This technique employs high affinity IgM and IgG monoclonal antibodies (anti-HBs) directed against distinct and separate determinants on HBsAg. In performing this antigenic structural analysis, separate binding curves for different monoclonal anti-HBs are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum by radioimmunoassay. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples are aligned by an iterative least-squares procedure to generate the numerical signature characteristic of the viral strain. The numerical signatures are then displayed on a computer-graphic plot. The signature profiles of HBsAg subtypes are a true reflection of their antigenic structure, and in vertical and horizontal transmission studies the molecular characteristics of the viral epitopes are conserved. By signature analysis we found substantial antigenic heterogeneity among the ayw3 strain both in the U.S. and France, as well as in populations of the Far East and Africa. Populations in Ethiopia, Gambia, and the Philippines were infected with two antigenically distinct HBV strains. In some newly identified HBV strains, it was found that epitopes identified by some monoclonal antibodies were absent or substantially reduced, which suggested that a genetic mutation may have occurred. Thus this study suggests that there is far more antigenic heterogeneity in HBV than previously recognized. These variants are antigenically distinct from each other at the epitope level, and were heretofore unrecognized by polyvalent anti-HBsAg antibodies.
Authors
E Ben-Porath, J R Wands, R A Marciniak, M A Wong, L Hornstein, R Ryder, M Canlas, A Lingao, K J Isselbacher
×Abstract
The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.
Authors
K H Krause, W Schlegel, C B Wollheim, T Andersson, F A Waldvogel, P D Lew
×Abstract
Arachidonate lipoxygenation to monohydroxylated eicosatetraenoic acids (HETE) was studied in rat nephrotoxic serum nephritis (NSN). A single infusion of nephrotoxic serum enhanced conversion of [3H]arachidonic acid ([3H]C20:4) to [3H]12-HETE in glomeruli isolated from nephritic rats compared with controls. The percent conversion of [3H]arachidonic acid was 1.95 +/- 0.2% in control glomeruli and 14.2 +/- 2% in nephritic glomeruli 2 d after induction of disease. No significant changes in the conversion of [3H]C20:4 to [3H]5-, 8-, and 9-HETE were noted. Extraction of glomerular HETE by alkaline hydrolysis, to evaluate possible reacylation of HETE after their production, confirmed the presence of 12-HETE and did not provide evidence of 5-HETE synthesis. Increased glomerular 12-HETE synthesis in nephritic rats was also demonstrated by high pressure liquid chromatography-UV detection and by 12-HETE radioimmunoassay. The enhanced glomerular 12-HETE synthesis commenced as early as 3-5 h after administration of nephrotoxic serum and peaked at day 2 with 10-fold enhancement of 12-HETE production. Increments of glomerular 12-HETE persisted on day 7 and returned toward control levels by day 14. Platelet depletion, induced by antiplatelet antisera, did not decrease glomerular 12-HETE synthesis in NSN, thereby eliminating platelets as the cellular origin of 12-HETE. Glomerular epithelial and mesangial cells are the most likely sources of enhanced 12-lipoxygenase activity. The enhanced arachidonate 12-lipoxygenation in glomerular immune injury could have important proinflammatory effects in the evolution of glomerulonephritis since 12-HETE has important effects on leukocyte function.
Authors
E A Lianos, M A Rahman, M J Dunn
×Abstract
Rat proximal convoluted tubules were perfused in vivo to examine the active and passive components of chloride absorption. Chloride flux was a linear function of the transepithelial electrochemical driving force, yielding a permeability coefficient of 20.6 X 10(-5) cm/s. In the absence of an electrochemical driving force, chloride absorption persisted at the rate of 131 peq/mm X min, thus demonstrating active absorption of chloride. Addition of luminal cyanide to tubules absorbing chloride inhibited net chloride absorption. In tubules perfused with a low luminal chloride concentration in which there was net chloride secretion, addition of luminal cyanide increased the magnitude of net chloride secretion. These studies demonstrate that transepithelial chloride transport involves two components: a passive paracellular flux and an active transcellular flux. Cyanide affects net chloride flux by inhibiting active transcellular chloride absorption.
Authors
R J Alpern, K J Howlin, P A Preisig
×Abstract
Glomerular fibrin deposition is important in the pathogenesis of renal failure and crescent formation in glomerulonephritis. The mechanisms of glomerular fibrin deposition are unknown. The current studies explored the role of macrophages in this process. Methods were developed for measuring glomerular fibrin deposition and glomerular procoagulant activity in a passive model of the autologous phase of antiglomerular basement membrane antibody-induced glomerulonephritis in rabbits. Significant fibrin deposition was observed to be associated with glomerular macrophage accumulation. Leukocyte ablation with mustine hydrochloride prevented both glomerular macrophage accumulation and fibrin deposition without affecting the coagulation system or the deposition of disease-inducing antibodies and complement. Repletion with mononuclear inflammatory cells produced significant fibrin deposition. To examine the role of tissue injury per se in glomerular fibrin deposition, a macrophage-independent model of glomerular injury (heterologous phase glomerulonephritis) was also studied. Although a similar degree of glomerular injury occurred, there was no significant fibrin deposition. This suggests that macrophages, rather than injury alone, are responsible for fibrin deposition. Lysates of isolated glomeruli containing macrophages demonstrated greatly enhanced procoagulant activity compared with lysates of glomeruli without macrophages. Thus macrophages appear to be directly responsible for glomerular fibrin deposition in antiglomerular basement membrane antibody-induced glomerulonephritis, and this appears to be due to their ability to express procoagulant activity rather than their propensity to cause glomerular injury.
Authors
S R Holdsworth, P G Tipping
×Abstract
The purpose of our study was to assess the effect of cold, dry air (CDA) on the nasal mucosa of selected individuals in relation to the release of inflammatory mediators associated with mast cells. 12 subjects with a history of nasal symptoms of rhinorrhea and congestion upon cold or dry environmental exposure were challenged by nasal breathing of CDA and warm, moist air (WMA). Each subject was tested on two occasions with the order of the challenges reversed. Symptom scores were recorded, and the levels of histamine, prostaglandin (PG) D2, kinins, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity in nasal lavage fluids were measured. CDA caused a significant increase in mediator levels and in symptom scores as compared to baseline or to WMA. No significant increase in symptom scores or mediators was noted after WMA challenge, with the exception of a marginal increase in kinins. The response to CDA was similar, regardless of challenge order. Changes in mediators correlated with one another, and symptom scores correlated significantly with the levels of histamine, kinins, and PGD2. Five subjects without a history of nasal symptoms on cold air exposure had no change in mediators or symptom scores after CDA or WMA challenge. We conclude that CDA causes the release of inflammatory mediators possibly associated with mast cells and speculate that such a mechanism may be involved in the bronchospasm induced by CDA in asthmatics.
Authors
A G Togias, R M Naclerio, D Proud, J E Fish, N F Adkinson Jr, A Kagey-Sobotka, P S Norman, L M Lichtenstein
×Abstract
Viral infections in humans are frequently associated with granulocytopenia and/or granulocytosis. Such changes in myelopoiesis could result from infection of the granulocyte-macrophage colony-forming cell (CFC-GM) or changes in the production of colony-stimulating activity (CSA). Endothelial cells are a known source of CSA and may be transiently or persistently infected during a number of viral infections, including infection with herpes simplex virus type I (HSV-I) and measles virus. Therefore, we examined the effect of endothelial cell infection with these two viruses on the production of CSA. Uninfected passaged endothelial cells produce CSA when stimulated by the continual presence of a factor present in medium conditioned by peripheral blood monocytes (MCM). Within 4 h of infection with HSV-I, endothelial cells no longer produced CSA in response to MCM. In contrast, measles virus infection induced CSA production by passaged endothelial cells even in the absence of MCM. Measles virus-induced CSA production was maximal at 24 h and required the presence of live virus within the endothelial cells. The effects of HSV-I and measles virus on CSA production were not dependent on alterations in the production of alpha- or gamma-interferon by the infected endothelial cells. Infection with HSV-I did not stimulate endothelial cells to release any detectable interferon. In contrast, the supernatants of the measles-infected cells contained only beta-interferon, a known inhibitor of CFC-GM development. These studies suggest that CSA production by endothelial cells is directly altered by infection with HSV-I and measles virus. An alteration in CSA production might contribute to changes in myelopoiesis that frequently accompany viral infection in humans.
Authors
S L Gerson, H M Friedman, D B Cines
×Abstract
The polymorphic Ia epitope recognized by monoclonal antibody 109d6 is detectable on the leukemic cells of a significantly increased number of individuals with acute myelogenous leukemia, compared with its frequency in normal healthy control individuals. In control individuals, the presence of the 109d6 epitope is closely correlated with but not identical to the DRw53 allo-specificity. However, the frequency of particular conventional Ia allodeterminants, including DRw53, is not significantly elevated in the leukemia group. Considerable evidence supports the conclusion that the high frequency of the 109d6 epitope reflects an inherited basis for susceptibility to the development of acute myelogenous leukemia and not a differentiation event occurring in the leukemic lineage. The 109d6 determinant is expressed by leukemic myeloblasts as well as by homologous normal B cells and monocytes obtained from the same individuals during remission of the leukemia. Furthermore, in healthy family members the 109d6 epitope is encoded by Ia haplotypes that are shared with the patient. Of special interest, certain of these haplotypes have combinations of the 109d6 epitope and Ia specificities not commonly seen in normal individuals; here, also, healthy family members share these haplotypes.
Authors
S Seremetis, J Cuttner, R Winchester
×Abstract
Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimulate in vivo processes) revealed (a) a rapid decline in proinsulin concommitant with formation of conversion intermediates, (b) formation of des-Arg31, Arg32-proinsulin and des-Lys64,Arg65-proinsulin in the ratio 3.3:1 at steady state, and (c) complete conversion of the precursor to insulin during extended incubation. Studies on normal human pancreas identified a similar ratio of des-Arg31,Arg32-proinsulin to des-Lys64,Arg65-proinsulin (approximately 3:1), whereas two insulinomas contained sizable amounts of des-Arg31,Arg32-proinsulin, but barely detectable amounts of des-Lys64,Arg65-proinsulin. None of the tissues contained measurable quantities of Arg32/Glu33- or Arg65/Gly66-split proinsulin. Analysis of plasma from three diabetic subjects managed by the intravenous infusion of human proinsulin revealed less than 1% processing of the circulating precursor to conversion intermediates and no processing of the precursor to human insulin. Nevertheless, analysis of plasma from the same subjects managed by the subcutaneous infusion of proinsulin revealed 4-11% processing of the precursor to intermediates that had the properties of des-Arg31,Arg32-proinsulin and Arg65/Gly66-split proinsulin. We conclude that (a) processing of proinsulin to insulin in vivo as in vitro likely occurs by preferential cleavage at the Arg32-Glu33 peptide bond in proinsulin, (b) proinsulin is inefficiently processed in the vascular compartment, and (c) subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.
Authors
B D Given, R M Cohen, S E Shoelson, B H Frank, A H Rubenstein, H S Tager
×Abstract
Factor VIII antigen (VIII:CAg) exhibits molecular weight heterogeneity in normal plasma. We have compared the relative quantities of VIII:CAg forms present in normal individuals (n = 22) with VIII:CAg forms in renal dysfunction patients (n = 19) and in patients with disseminated intravascular coagulation (DIC; n = 7). In normal plasma, the predominant VIII: CAg form, detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was of molecular weight 2.4 X 10(5), with minor forms ranging from 8 X 10(4) to 2.6 X 10(5) D. A high proportion of VIII:CAg in renal dysfunction patients, in contrast, was of 1 X 10(5) mol wt. The patients' high 1 X 10(5) mol wt VIII: CAg level correlated with increased concentrations of serum creatinine, F1+2 (a polypeptide released upon prothrombin activation), and with von Willebrand factor. Despite the high proportion of the 1 X 10(5) mol wt VIII:CAg form, which suggests VIII:CAg proteolysis, the ratio of Factor VIII coagulant activity to total VIII:CAg concentration was normal in renal dysfunction patients. These results could be simulated in vitro by thrombin treatment of normal plasma, which yielded similar VIII:CAg gel patterns and Factor VIII coagulant activity to antigen ratios. DIC patients with high F1+2 levels but no evidence of renal dysfunction had an VIII:CAg gel pattern distinct from renal dysfunction patients. DIC patients had elevated concentrations of both the 1 X 10(5) and 8 X 10(4) mol wt VIII:CAg forms. We conclude that an increase in a particular VIII:CAg form correlates with the severity of renal dysfunction. The antigen abnormality may be the result of VIII:CAg proteolysis by a thrombinlike enzyme and/or prolonged retention of proteolyzed VIII:CAg fragments.
Authors
M J Weinstein, L E Chute, G W Schmitt, R H Hamburger, K A Bauer, J H Troll, P Janson, D Deykin
×Abstract
This study was designed to examine: (a) the effects of adenosine and its analogues on renin release in the absence of tubules, glomeruli, and macula densa, and (b) whether adenosine may be involved in a macula densa-mediated renin release mechanism. Rabbit afferent arterioles (Af) alone and afferent arterioles with macula densa attached (Af + MD) were microdissected and incubated for two consecutive 30-min periods. Hourly renin release rate from a single arteriole (or an arteriole with macula densa) was calculated and expressed as ng AI X h-1 X Af-1 (or Af + MD-1)/h (where AI is angiotensin I). Basal renin release rate from Af was 0.69 +/- 0.09 ng AI X h-1 X Af-1/h (means +/- SEM, n = 16) and remained stable for 60 min. Basal renin release rate from Af + MD was 0.20 +/- 0.04 ng AI X h-1 X Af + MD-1/h (n = 6), which was significantly lower (P less than 0.0025) than that from Af. When adenosine (0.1 microM) was added to Af, renin release decreased from 0.72 +/- 0.16 to 0.24 +/- 0.04 ng AI X h-1 X Af-1/h (P less than 0.025; n = 9). However, when adenosine was added to Af + MD, no significant change in renin release was observed. N6-cyclohexyl adenosine (an A1 adenosine receptor agonist) at 0.1 microM decreased renin release from Af from 0.69 +/- 0.14 to 0.39 +/- 0.12 ng AI X h-1 X Af-1/h (n = 5, P less than 0.05). However, 5'-N-ethylcarboxamide adenosine (an A2 adenosine receptor agonist) either at 0.1 microM or at 10 microM had no effect. Theophylline, at a concentration (10 microM) that does not block phosphodiesterase but does block adenosine receptors, increased renin release from Af + MD from 0.21 +/- 0.03 to 0.46 +/- 0.08 ng AI X h-1 X Af + MD-1/h (P less than 0.05; n = 8). The results are consistent with the hypotheses that adenosine decreases renin release via the activation of A1 adenosine receptors, and that adenosine may be an inhibitory signal from the macula densa to juxtaglomerular cells.
Authors
S Itoh, O A Carretero, R D Murray
×Abstract
C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.
Authors
M T Aguado, J D Lambris, G C Tsokos, R Burger, D Bitter-Suermann, J D Tamerius, F J Dixon, A N Theofilopoulos
×Abstract
We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.
Authors
T Ganz, M E Selsted, D Szklarek, S S Harwig, K Daher, D F Bainton, R I Lehrer
×Abstract
The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.
Authors
M E Selsted, S S Harwig, T Ganz, J W Schilling, R I Lehrer
×Abstract
Postischemic acute renal failure (ARF) induced by cardiac surgery is commonly prolonged and may be irreversible. To examine whether persistence of postischemic, tubular cell injury accounts for delayed recovery from ARF, we studied 10 patients developing protracted (36 +/- 4 d) ARF after cardiac surgery. The differential clearance and excretion dynamics of probe solutes of graded size were determined. Inulin clearance was depressed (5.0 +/- 1.7 ml/min), while the fractional urinary clearance of dextrans (radii 17-30 A) were elevated above unity. Employing a model of conservation of mass, we calculated that 44% of filtered inulin was lost via transtubular backleak. The clearance and fractional backleak of technetium-labeled DTPA ([99mTc]DTPA, radius = 4 A) were identical to those of inulin (radius 15 A). The time at which inulin or DTPA excretion reached a maximum after an intravenous bolus injection was markedly delayed when compared with control subjects with ARF of brief duration, 102 vs. 11 min. Applying a three-compartment model of inulin/DTPA kinetics (which takes backleak into account) revealed the residence time of intravenously administered inulin/DTPA in the compartment occupied by tubular fluid and urine to be markedly prolonged, 20 vs. 6 min in controls, suggesting reduced velocity of tubular fluid flow. We conclude that protracted human ARF is characterized by transtubular backleak of glomerular ultrafiltrate, such that inulin clearance underestimates true glomerular filtration rate by approximately 50%, and by sluggish tubular fluid flow, which strongly suggests the existence of severe and generalized intraluminal tubular obstruction. Because all patients also exhibited extreme hyperreninemia (16 +/- 2 ng/ml per h) that was inversely related to inulin clearance (r value = -0.83) and urine flow (r value = -0.70), we propose that persistent, angiotensin II-mediated renal vasoconstriction may have delayed healing of the injured tubular epithelium.
Authors
S M Moran, B D Myers
×Abstract
Factor B and C2 are structurally and functionally similar complement proteins encoded by genes that are closely linked within the class III region of the major histocompatibility complex (MHC). In this study, restriction endonuclease digestion of cosmid DNAs isolated from an H-2d murine genomic library indicated that the chromosomal organization of these two genes was similar in mouse to that in man. To further characterize their expression, cosmid DNAs encoding human and murine factor B and C2 were introduced into mouse L cells by DNA-mediated gene transfer. Factor B expression was demonstrated in cells transfected with either the human or the murine gene, but not in cells transfected with a control plasmid. Synthesis and secretion of factor B by L cells transfected with the human and murine cosmids was similar to that of human and murine cells in primary culture. An interspecies variation between human and murine factor B was identified and reproduced with extraordinary fidelity by the mouse fibroblast. In contrast, C2 RNA and protein were expressed by L cells alone and by L cells transfected with a control plasmid, as well as by L cells transfected with cosmids encoding human and murine complement genes. Expression of the transferred human C2 gene was demonstrated by the presence of a new distinct C2 RNA transcript and secretion of biologically active human C2. These results demonstrate the similarity of organization of the murine and human class III MHC regions. Expression of the two closely linked gene products, C2 and factor B, after DNA-mediated gene transfer provides a system for further analysis of regulation in both normal and deficient states.
Authors
D H Perlmutter, H R Colten, D Grossberger, J Strominger, J G Seidman, D D Chaplin
×Abstract
One reason that some people are prone to calcium oxalate nephrolithiasis is that they produce urine that is subnormal in its ability to inhibit the growth of calcium oxalate crystals. We have identified in human urine a glycoprotein (GCI) that inhibits calcium oxalate crystal growth strongly, and at low concentrations (10(-7) M); in this study, we have isolated GCI molecules from the urine of normal people and patients with calcium oxalate stones. GCI from stone formers is abnormal in three ways: it contains no detectable gamma-carboxyglutamic acid (Gla), whereas normal GCI contains 2-3 residues of Gla per mole; about half of the GCI in urine of patients inhibits crystal growth 4-20 times less than normal GCI as judged by its performance in a kinetic growth assay, in vitro; at the air-water interface, patient GCI has a film collapse pressure approximately half of normal. GCI molecules from the urine of patients with calcium oxalate nephrolithiasis are intrinsically abnormal, and these abnormalities could play a role in the genesis of stones.
Authors
Y Nakagawa, V Abram, J H Parks, H S Lau, J K Kawooya, F L Coe
×Abstract
In hemochromatosis and other disorders associated with iron overload, a significant fraction of the total iron in plasma circulates in the form of low molecular weight complexes not bound to transferrin. Efficient and unregulated clearance of this form of iron by the liver may account for the hepatic iron loading and toxicity that characterize these diseases. We tested this possibility by examining the hepatic removal process for representative iron complexes in the single-pass perfused rat liver. Hepatic uptake of both ferrous and ferric 55Fe from ultrafiltered human serum was found to be highly efficient and effectively irreversible (single-pass extraction of 1 microM iron, 58-75%). Similar high efficiencies were seen for iron complexed to specific physiologic and nonphysiologic coordinators, including histidine, citrate, fructose, oxalate and glutamate, and tricine. Because of lower plasma flow rates, single-pass extraction of these iron complexes in vivo should be even greater. Autoradiography confirmed that most iron had been removed by parenchymal cells. Hepatic removal from Krebs-tricine buffer was saturable with similar kinetic parameters for ferrous and ferric iron (apparent Km, 14-22 microM; V max, 24-38 nmol min-1 g liver-1). These findings suggest that high levels of non-transferrin-bound iron in plasma may be an important cause of hepatic iron loading in iron overload states.
Authors
P Brissot, T L Wright, W L Ma, R A Weisiger
×Abstract
To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage-like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors.
Authors
R G Spragg, D B Hinshaw, P A Hyslop, I U Schraufstätter, C G Cochrane
×Abstract
The purified human monocytes of a responding donor preincubated with heat-inactivated serum T1264 or T1295 derived from pregnant women for 30 min at 37 degrees C expressed allogeneic suppressive effects on the proliferative response of the lymphocytes from the same donor in the allogeneic mixed lymphocyte reaction (MLR). The pregnancy serum in our experiments was found not to contain any antibodies to DR and DQ antigens on monocytes of the responding donor. Accordingly, the suppressive effects mediated by monocytes were not based on the blocking of DR and DQ antigens on monocytes of the responding donor by DR and DQ antibodies in the serum. These highly reproducible allogeneic suppressive effects by monocytes of the responding donor were demonstrated in the MLR specific for DRw9-positive stimulating cells, whereas no inhibition was seen in the cultures with other stimulating cells of different DR phenotypes. Additionally, these suppressive effects appeared on the monocytes of a DR2-positive responding donor, but not on the monocytes of a DR2-negative responding donor. These suppressive effects were abolished when the absorbed pregnancy serum by monocytes of the DR2-positive responding donor was used. In this suppression phenomenon that we discovered, monocytes of the responding donor appear to act as regulatory cells on the proliferative response of the allogeneic MLR. This regulatory function of monocytes could be expressed through the specific molecules distinct from DR and DQ determinants on monocytes in cooperation with antibodies (IgG class) in the pregnancy serum.
Authors
M Nieda, T Juji, S Imao
×Abstract
Serotonin stimulates aldosterone secretion both in vitro and in vivo, and serotonin antagonism decreases plasma aldosterone levels in patients with idiopathic aldosteronism. This study was designed to assess the effects of the serotonin precursor, 5-hydroxytryptophan (5HTP), upon aldosterone secretion in man, and to determine whether stimulatory effects of 5HTP are mediated through the central nervous system. Oral 5HTP, administered as a single 200-mg dose, increased plasma aldosterone levels from 4.7 +/- 0.6 to 13.3 +/- 2.8 ng/dl in dexamethasone-pretreated, normal volunteers. Peripheral inhibition of decarboxylation of 5HTP, achieved by pretreatment with carboxydopa, 25 mg three times daily for 3 d, significantly increased the stimulatory effects of 5HTP on aldosterone levels (P less than 0.001). No change in aldosterone levels occurred in subjects who received placebo after pretreatment with dexamethasone and carboxydopa. Increased aldosterone was not accompanied by increases in plasma levels of renin activity, potassium, or ACTH. Plasma levels of 5HTP were markedly increased by carboxydopa pretreatment, but peak plasma levels of serotonin were not significantly altered. Four patients with idiopathic aldosteronism all had an increase in plasma aldosterone levels after 5HTP administration, whereas the response in four patients with aldosterone-producing adenoma was variable. Incubation of isolated human and rat adrenal glomerulosa cells with serotonin resulted in increased aldosterone secretion by both sets of cells, whereas 5HTP was ineffective in stimulating aldosterone secretion in vitro. We conclude that central serotonergic pathways are involved in the stimulation of aldosterone induced by administration of 5HTP. This mechanism may be an important etiologic factor in the hypersecretion of aldosterone that occurs in patients with idiopathic aldosteronism.
Authors
Y Shenker, M D Gross, R J Grekin
×Abstract
Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.
Authors
W E Fowler, L J Fretto, K K Hamilton, H P Erickson, P A McKee
×Abstract
The plasma protein apolipoprotein (apo) E is an important determinant of lipid transport and metabolism in mammals. In the present study, immunocytochemistry has been used to identify apo E in specific cells of the central and peripheral nervous systems of the rat. Light microscopic examination revealed that all astrocytes, including specialized astrocytic cells (Bergmann glia of the cerebellum, tanycytes of the third ventricle, pituicytes of the neurohypophysis, and Müller cells of the retina), possessed significant concentrations of apo E. In all of the major subdivisions of the central nervous system, the perinuclear region of astrocytic cells, as well as their cell processes that end on basement membranes at either the pial surface or along blood vessels, were found to be rich in apo E. Extracellular apo E was present along many of these same surfaces. The impression that apo E is secreted by astrocytic cells was confirmed by electron microscopic immunocytochemical studies, which demonstrated the presence of apo E in the Golgi apparatus. Apo E was not present in neurons, oligodendroglia, microglia, ependymal cells, and choroidal cells. In the peripheral nervous system, apo E was present within the glia surrounding sensory and motor neurons; satellite cells of the dorsal root ganglia and superior cervical sympathetic ganglion as well as the enteric glia of the intestinal ganglia were reactive. Apo E was also present within the non-myelinating Schwann cells but not within the myelinating Schwann cells of peripheral nerves. These results suggest that apo E has an important, previously unsuspected role in the physiology of nervous tissue.
Authors
J K Boyles, R E Pitas, E Wilson, R W Mahley, J M Taylor
×Abstract
Neutrophils are often seen first at sites of granulomatous inflammation but their contribution to monocyte recruitment and granuloma formation is unknown. We tested the hypothesis that neutrophils release chemotaxins which attract monocytes. We found that rapid accumulations of fluid and influxes of neutrophils followed by monocytes occurred in bacillus Calmette--Guérin (BCG)-sensitized rabbits given BCG intrapleurally but did not occur in nitrogen mustard-treated (neutropenic) BCG-sensitized rabbits given BCG intrapleurally--unless the rabbits were also given intrapleural injections of neutrophils. We also found monocyte chemotaxins in pleural spaces of control and neutrophil-reconstituted neutropenic but not in neutropenic rabbits given BCG intrapleurally. Moreover, pleural fluid monocyte chemotaxins had molecular weights (12,000-15,000 and 1,000) that were similar to molecular weights of monocyte chemotaxins present in supernatants from mixtures of neutrophils and BCG in vitro. In addition, intrapleural injection of neutrophils and BCG or supernatants from in vitro mixtures of neutrophils and BCG (but not neutrophils or BCG alone) increased the numbers of monocytes and 3H cell pellet activity in pleural fluids from untreated neutropenic rabbits or neutropenic rabbits previously injected intravenously with 3[H]methyl thymidine-labeled monocytes. Furthermore, fewer BCG were recovered from pleural fluids of BCG-sensitized control compared to neutropenic rabbits given BCG, and at autopsy 10 d after instillation of BCG, control but not neutropenic rabbits had well-defined granulomas without adhesions on their pleural surfaces. Our results suggest that BCG stimulates neutrophils to release chemotaxins that recruit monocytes, and that these responses might contribute to granuloma formation in tuberculous pleurisy.
Authors
V B Antony, S A Sahn, A C Antony, J E Repine
×Abstract
We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by beta-thromboglobulin and platelet factor 4 release. The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/IIIa) complex totally inhibits the SPA. The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification of the patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia.
Authors
H R Grainick, S B Williams, L P McKeown, M E Rick, P Maisonneuve, C Jenneau, Y Sultan
×Abstract
We studied the effect of an arginine vasopressin (AVP) analogue, (1-[beta-mercapto-beta, beta-cyclopentamethylenepropionic acid],2-O-ethyltyrosine, 4-valine)AVP(d[CH2]5Tyr[Et]VAVP), on the stimulation of adenylate cyclase by various hormones in the isolated nephron segments and 3H-AVP binding to renal papillary membranes from the rat. The net water flux across the renal cortical collecting tubules of the rabbit was also examined. We found that d(CH2)5Tyr(Et)VAVP significantly inhibited adenylate cyclase activation by AVP in cortical, medullary, and papillary collecting tubules and in the medullary thick ascending limb. In contrast, the AVP analogue did not alter the stimulation of adenylate cyclase by parathyroid hormone in the cortical thick ascending limb, by glucagon in the medullary thick ascending limb, and by calcitonin in cortical collecting tubules. In addition, d(CH2)5Tyr(Et)VAVP blocked [3H]AVP binding to renal papillary membranes. The enhanced net water transport induced by AVP in isolated, perfused rabbit cortical collecting tubules also was completely blocked by this AVP analogue. These results indicate that d(CH2)5Tyr(Et)VAVP specifically antagonizes the cellular action of AVP on the medullary thick ascending limb and on the cortical, medullary, and papillary collecting tubules. Evidence is also presented for competitive antagonism as the cellular mechanism of action.
Authors
J K Kim, M A Dillingham, S N Summer, S Ishikawa, R J Anderson, R W Schrier
×Abstract
An evaluation of surgically obtained skin (age range, 8-92 yr) revealed that there is an age-dependent decrease in the epidermal concentrations of provitamin D3 (7-dehydrocholesterol). To ascertain that aging indeed decreased the capacity of human skin to produce vitamin D3, some of the skin samples were exposed to ultraviolet radiation and the content of previtamin D3 was determined in the epidermis and dermis. The epidermis in the young and older subjects was the major site for the formation of previtamin D3, accounting for greater than 80% of the total previtamin D3 that was produced in the skin. A comparison of the amount of previtamin D3 produced in the skin from the 8- and 18-yr-old subjects with the amount produced in the skin from the 77- and 82-yr-old subjects revealed that aging can decrease by greater than twofold the capacity of the skin to produce previtamin D3. Recognition of this difference may be extremely important for the elderly, who infrequently expose a small area of skin to sunlight and who depend on this exposure for their vitamin D nutritional needs.
Authors
J MacLaughlin, M F Holick
×Abstract
We have previously described a subpopulation of patients with septic shock who had a reversible depression of radionuclide-determined left ventricular ejection fraction (EF). To investigate the mechanism of this myocardial depression, an in vitro model of mammalian myocardial cell performance was established employing primary spontaneously beating rat myocardial cells. The contraction of a single cardiac cell was quantitated by recording the changes in area occupied by the cell during contraction and relaxation. In 20 septic shock patients during the acute phase, the mean left ventricular EF was decreased (mean = 0.33, normal mean = 0.50), and serum obtained during this acute phase induced a mean (+/- standard error of the mean) 33 +/- 4% decrease in extent and 25 +/- 4% decrease in velocity of myocardial cell shortening during contraction (P less than 0.001). In contrast, serum obtained from 11 of these same patients before shock (n = 2) or after recovery (n = 9) of the left ventricular EF (mean = 0.50) showed a return toward normal in extent and velocity of shortening (P less than 0.001). Sera from 17 critically ill nonseptic patients, from 10 patients with structural heart disease as a cause for a depressed EF, and from 12 healthy laboratory personnel, induced no significant changes in in vitro myocardial cell performance. In 20 patients during the acute phase of septic shock, the decreased EF in vivo demonstrated a significant correlation (r = +0.52, P less than 0.01) with a decrease in the extent of myocardial cell shortening in vitro. The quantitative and temporal correlation between the decreased left ventricular EF and this serum myocardial depressant substance argues for a pathophysiologic role for this depressant substance in producing the reversible cardiomyopathy seen during septic shock in humans.
Authors
J E Parrillo, C Burch, J H Shelhamer, M M Parker, C Natanson, W Schuette
×Abstract
We describe an English family with an atypical gamma delta beta-thalassemia syndrome. Heterozygosity results in a beta-thalassemia phenotype with normal hemoglobin A2. However, unlike previously described cases, no history of neonatal hemolytic anemia requiring blood transfusion was obtained. Gene mapping showed a deletion that extended from the third exon of the G gamma-globin gene upstream for approximately 100 kilobases (kb). The A gamma-globin, psi beta-, delta-, and beta-globin genes in cis remained intact. The malfunction of the beta-globin gene on a chromosome in which the deletion is located 25 kb away suggests that chromatin structure and conformation are important for globin gene expression.
Authors
P Curtin, M Pirastu, Y W Kan, J A Gobert-Jones, A D Stephens, H Lehmann
×Abstract
The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.
Authors
H Kather, W Bieger, G Michel, K Aktories, K H Jakobs
×Abstract
The mechanisms of aberrant conduction at the onset of induced orthodromic tachycardia in the Wolff-Parkinson-White syndrome were analyzed in 20 consecutive patients in whom this tachycardia was initiated by the atrial (A2) and/or right ventricular (V2) extrastimulus techniques. Of 13 patients in whom orthodromic tachycardia was induced by the A2 method, functional right bundle branch block occurred at tachycardia onset in four (31%) and left bundle aberrancy in two (15%), one of whom also manifested right bundle aberrancy. The occurrence of bundle branch block at the onset of tachycardia was linked to aberrant conduction of the initiating A2 impulse which, in turn, was associated with attainment of relatively short His1His2 intervals within the tachycardia initiation zone. Aberrant conduction of A2 was also more common in patients without manifest preexcitation. In contrast, of 14 patients in whom orthodromic tachycardia was induced by the V2 method, left bundle aberrancy occurred at the onset of tachycardia in 11 (79%), one of whom manifested right bundle branch block as well. Left bundle aberrancy was more likely to occur when the interval from the initiating V2 (or macro-reentrant V3) impulse to the first anterograde His deflection was less than 300 ms. This suggests that left bundle aberrancy at the onset of orthodromic tachycardia induced by the V2 method results from concealed retrograde penetration of the His-Purkinje system, with the left bundle being last to recover. Our findings provide the conceptual basis for a physiologic approach to the deliberate induction of specific types of aberrant conduction at onset of orthodromic tachycardia in patients with Wolff-Parkinson-White syndrome.
Authors
M H Lehmann, S Denker, R Mahmud, P Tchou, J Dongas, M Akhtar
×Abstract
In previous studies we have demonstrated that 3,5,3'-triiodothyronine (T3) in vitro produces a prompt increase in the uptake of the sugar analogue 2-deoxyglucose (2-DG) by freshly isolated rat thymocytes. This effect is prompt, being evident at 20 min after addition of T3, is independent of new protein synthesis, and can be elicited by physiologic concentrations of the hormone. In the present studies, we have sought to determine whether physiologic doses of T3 are capable of inducing an increase in 2-DG uptake in the thymocytes of the living animal. Therefore, 26-28-d-old female rats were injected with increasing doses of i.v. T3, followed 60 min later by 3H-labeled 2-DG. 30 min later, animals were killed, thymocytes were isolated, and their 3H content determined. Uptake of [3H]2-DG was increased by T3 in a dose-dependent manner. The lowest effective dose was 10 ng/100 g of body weight (30% above control) and the maximally effective dose 1 microgram/100 g of body weight (116% above control). The effect of T3 was independent of new protein synthesis in that it was not blocked by a dose of cycloheximide that inhibited the incorporation of [3H]leucine into thymocyte protein by 92-95%. Comparable studies with various thyronine analogues revealed the following rank order of potency: L-T3 greater than L-3,5,3'5'-tetraiodothyronine (L-T4) greater than D-T3 greater than or equal to D-T4 greater than L-3,3'5'-triiodothyronine greater than 3'-isopropyl-3,5-L-diiodothyronine (T2) = 3,5-L-T2. DL-thyronine was without effect. These studies indicate that T3 in physiologic doses acts in vivo to increase the uptake of sugar by rat thymocytes by a mechanism that is extranuclear in origin, in that it is independent of new protein synthesis. The findings support the conclusion that the previously demonstrated effects of T3 on thymocyte sugar uptake in vitro, which seem clearly to be mediated at the level of the plasma membrane, have physiologic relevance.
Authors
J Segal, S H Ingbar
×Abstract
In families with X-linked chronic granulomatous disease (CGD), heterozygous females have two stable populations of polymorphonuclear leukocytes (PMN) in their blood; one normal, the other, deficient in oxygen metabolism. The two types of PMN can be distinguished by the ability or lack of ability to reduce nitroblue tetrazolium dye. The variation in the percent normal PMN among 11 CGD heterozygotes was shown to follow a binomial distribution based on eight independent trials and a chance of success of 50%. This is consistent with the occurrence of X-chromosome inactivation (lyonization) when eight embryonic founder cells for the hematopoietic system are present. Serial determinations of the percent normal PMN in individual heterozygotes showed very limited variability (standard deviations ranged from 2.0% to 5.2%) most of which could be ascribed to experimental error. An estimate of the remaining variation (residual variance) was introduced into a well-known formula to calculate the appropriate number of pluripotent stem cells necessary to support hematopoiesis and a figure exceeding 400 was obtained. Thus, the data indicate that in humans there is a highly polyclonal system of hematopoiesis.
Authors
E S Buescher, D W Alling, J I Gallin
×Abstract
Keratinocytes produce a molecule, epidermal-derived thymocyte activating factor (ETAF), which is biologically and physiochemically similar to the polypeptide hormone interleukin 1 (IL-1). Because the stratum corneum (SC) is composed of terminally differentiated keratinocytes, we questioned whether ETAF/IL-1 could be isolated from this tissue. The extraction of normal human SC with a physiologic saline solution yielded a large amount of ETAF/IL-1 activity, as measured by the in vitro thymocyte co-stimulator assay. SC-derived ETAF/IL-1 (scETAF/IL-1) eluted from a sizing column with an approximate molecular weight of 15,000, and demonstrated three isoelectric point forms after separation on a chromatofocusing column. By these physiochemical characteristics, scETAF/IL-1 was found to be similar, if not identical to human keratinocyte- and macrophage-derived ETAF/IL-1. Further, a number of biologic effects known to occur in vivo after the administration of ETAF/IL-1, such as fever, neutrophilia, and an increase in plasma levels of acute-phase proteins, were all induced by the injection of scETAF/IL-1 into endotoxin-nonresponsive mice. scETAF/IL-1 was also found to stimulate collagenase production by human fibroblasts in vitro. In summary, our studies have established that normal human SC contains a large quantity of scETAF/IL-1. Whether scETAF/IL-1 integrates into the earliest afferents phases of local inflammatory responses, or merely represents a means of disposal of excessively produced hormone is currently unresolved.
Authors
L C Gahring, A Buckley, R A Daynes
×Abstract
Previous studies in experimental animals indicate an important inhibitory interaction between cardiopulmonary and arterial baroreflexes. In the dog, for example, cardiopulmonary vagal afferents modulate carotid baroreflex control of vascular resistance. On the other hand, previous studies in human subjects have not produced convincing evidence of a specific interaction between these baroreceptor reflexes. The purpose of this study was to determine whether unloading of cardiopulmonary baroreceptors in humans with nonhypotensive lower body negative pressure selectively augments the reflex vasoconstrictor responses to simulated carotid hypotension produced by neck pressure. In nine healthy subjects, we measured forearm vascular responses with plethysmography during lower body negative pressure alone (cardiopulmonary baroreflex), during neck pressure alone (carotid baroreflex), and during concomitant lower body negative pressure and neck pressure (baroreflex interaction). Lower body negative pressure produced a greater than twofold augmentation of the forearm vasoconstrictor response to neck pressure. This increase in resistance was significantly greater (P less than 0.05) than the algebraic sum of the increase in resistance from lower body negative pressure alone plus that from neck pressure alone. In contrast, lower body negative pressure did not potentiate the forearm vasoconstrictor responses either to intra-arterial norepinephrine or to the cold pressor test. Thus, the potentiation of the vasoconstrictor response to neck pressure by lower body negative pressure cannot be explained by augmented reactivity to the neurotransmitter or to a nonspecific augmentation of responses to all reflex vasoconstrictor stimuli. In conclusion, nonhypotensive lower body negative pressure selectively augments carotid baroreflex control of forearm vascular resistance. These experiments demonstrate a specific inhibitory cardiopulmonary-carotid baroreflex interaction in humans.
Authors
R G Victor, A L Mark
×Abstract
This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.
Authors
D A Bushinsky, G S Riera, M J Favus, F L Coe
×Abstract
Increased adherence of sickle erythrocytes to vascular endothelium has been suggested by Hebbel and his colleagues to play a role in vasocclusive events of sickle cell disease. To define the role of cell membrane changes and plasma factors in cell adherence, a micropipette technique previously developed by us to obtain a direct, quantitative measure of cell adherence was used to evaluate the adhesivity of different morphologic classes of sickle cells to endothelial cells in various suspending media. Irregularly shaped, deformable sickle cells were four- to fivefold more adherent than discoid sickle cells, whereas rigid irreversibly sickled cells were least adherent. Sickle erythrocytes adhered to endothelial cells when suspended in autologous citrated or heparinized plasma but were totally nonadherent when suspended in autologous EDTA plasma. Removal of the divalent cation chelator and addition of calcium to EDTA plasma restored its ability to promote adhesion, implying an absolute requirement for divalent cations in sickle cell adherence. Sickle cells also did not adhere to endothelial cells in protein-free media containing divalent cations, suggesting an additional requirement for plasma proteins. Removal of collagen-binding proteins from citrated sickle plasma resulted in a three- to fivefold reduction in its ability to promote cell adhesion, suggesting an important role for these plasma proteins in sickle cell adherence. The results of this study imply that sickle cell adherence to vascular endothelium is a complex process in which temporal changes in the numbers of cells identified to be most adhesive and the plasma concentration of protein(s) involved in the adhesive process determine the extent of in vivo sickle cell adherence.
Authors
N Mohandas, E Evans
×Abstract
Medium conditioned by activated T lymphocytes stimulates the in vitro proliferation of pluripotent hematopoietic stem cells (spleen colony-forming units [CFU-S]) but the factors involved have not been identified. Because the lymphokine interleukin 3 (IL-3) enhances in vitro colony formation by committed hematopoietic progenitor cells, we examined the effect of IL-3 on the in vitro proliferation of CFU-S using an 11-d spleen colony assay. When mouse marrow cells were placed in liquid culture, CFU-S content declined progressively and by 96 h only 13% of the CFU-S remained. By contrast, after 96 h in the presence of 20 U/ml of IL-3, the number of CFU-S were the same as that in the initial inoculum. Although the number of CFU-S eventually declined, they could still be recovered after 264 h of culture. In the absence of IL-3, the number of CFU-S synthesizing DNA was negligible; in its presence, greater than 20% of the CFU-S were in cycle. IL-3 stimulated CFU-S proliferation at a concentration of 0.2 U/ml. The dose-response curve was similar to that observed for other biologic effects of the lymphokine, and as little as 1 h of exposure to IL-3 enhanced the survival of CFU-S in vitro. Treatment of marrow cells with anti-Thy 1.2 antibody and complement before exposure to IL-3 did not inhibit spleen colony formation, but treatment of the cells with anti-Thy 1.2 antibody and complement after exposure to IL-3 reduced CFU-S recovery after 96 h of culture by 45%. The cell composition of day 11 spleen colonies formed by IL-3-treated marrow cells was similar to that of colonies formed by untreated marrow cells. Finally, day 11 CFU-S persisting in the marrow of mice treated with 5-fluorouracil required IL-3 for proliferation in vitro. Taken together, these data indicate that IL-3 promotes the proliferation of CFU-S in vitro, increases the number of CFU-S synthesizing DNA, but does not alter their commitment program, and the target cell population includes CFU-S with self-renewal and marrow-repopulating ability.
Authors
J L Spivak, R R Smith, J N Ihle
×Abstract
To determine whether intracerebrally localized iodothyronines produce thyroid hormone-related functional effects, heart rate responses were compared in conscious hypothyroid rats given triiodothyronine (T3) by either the intrathecal or the intravenous route. A significant increase in heart rate occurred within 18 h after 1.5 nmol T3/100 g body wt was delivered intrathecally through a cannula previously placed in the lateral cerebral ventricle. Injection of the same T3 dose intravenously through an indwelling jugular catheter or injection of vehicle only by either route produced no significant increase in heart rate during the 48-h postinjection period of observation. These differences were observed even though integrated serum T3 concentrations were significantly lower after intrathecal than after intravenous T3 injection. The results indicate that thyroid hormone effects on heart rate are exerted within the brain as well as within the heart.
Authors
M Goldman, M B Dratman, F L Crutchfield, A S Jennings, J A Maruniak, R Gibbons
×Abstract
We studied the incorporation and metabolism of eicosapentanoic (EPA) and docosahexaenoic acid in six human volunteers who supplemented their normal Western diet for 5 mo daily with 10-40 ml of cod liver oil, rich in omega-3 polyunsaturated fatty acids. EPA and docosahexaenoic acid were incorporated into the total phospholipids of plasma, platelets, and erythrocytes in a dose- and time-dependent manner. During omega-3 fatty acid ingestion serum triacylglycerols were lowered and platelet aggregation upon low doses of collagen was reduced. Concomitantly, formation and excretion of prostanoids showed a characteristic change. As measured in serum from whole clotted blood, thromboxane A3 was formed in small amounts, whereas thromboxane A2 formation was reduced to 50% of control values. Excretion of the main urinary thromboxane A metabolites was unaltered in subjects with low basal excretion rates, but decreased markedly in two subjects with high control values. As determined from the main urinary metabolite, prostaglandin I3 was formed from EPA at rates up to 50% of unaltered prostaglandin I2 formation. The biochemical and functional changes observed lasted for the entire supplementation period of 5 mo and were reversible within 12 wk after cessation of cod liver oil intake. Favorable changes induced by long-chain omega-3 fatty acids include a dose-related and sustained shift of the prostaglandin I/thromboxane A balance to a more antiaggregatory and vasodilatory state.
Authors
C von Schacky, S Fischer, P C Weber
×Abstract
The effects of intermittent infusions of dobutamine were studied in young normal male subjects during a period of bedrest deconditioning to determine whether this synthetic catechol affects physical conditioning processes in humans. 24 volunteers were placed at bedrest and randomized to daily 2-h treatments of saline infusions (control), dobutamine infusions, or maintenance exercise (control). Exercise, hemodynamic, and metabolic studies were performed at base line and at the termination of the 3-wk treatment period. Maximal exercise (duration, oxygen consumption, and workload) fell for the saline group and remained unchanged for the dobutamine and exercise groups. Hemodynamics during exercise were maintained the same as pretreatment base line for the dobutamine and exercise groups, whereas stroke volume and cardiac output dropped and heart rate rose for the saline group. The metabolic profile showed an increased blood lactate response at rest and during submaximal exercise after 3 wk of bedrest for the saline group, and essentially no change for the exercise and the dobutamine groups. Extraction of oxygen across the exercising lower limb rose for the dobutamine group, as did the activity of the skeletal muscle oxidative enzymes, citrate synthetase, and succinate dehydrogenase. In contrast to the exercise control group, the saline and dobutamine groups developed orthostatic hypotension, tachycardia, and accentuation of the renin-aldosterone response over the 3-wk treatment period; for the saline group, this is best explained by the observed fall in blood volume and for the dobutamine group, by the blunting of vascular vasoconstrictive responses. During a period of bedrest deconditioning in humans, infusions of dobutamine maintain many of the physiologic expressions of physical conditioning.
Authors
M J Sullivan, P F Binkley, D V Unverferth, J H Ren, H Boudoulas, T M Bashore, A J Merola, C V Leier
×Abstract
In the present study, the erythropoietic activity of fetal serum was characterized. Using fetal bovine serum (FBS) as a source of the erythropoietic activity and serum-free cultures of fetal mouse livers (FMLC assay) as a detection system, we found that FBS stimulated colony formation from late erythroid progenitor cells (CFU-E) in a dose-dependent fashion. The slope of the dose-response curve, however, was significantly different from that for erythropoietin (Ep), the best-characterized erythropoietic activity so far. The erythropoietic activity of FBS was found in the 120-160- and 40-70-kD range at neutral pH. In the presence of 1 M acetic acid, however, the erythropoietic activity had an apparent molecular mass between 3 and 13 kD. From ion exchange experiments with DEAE-cellulose, the isoionic point of the activity was estimated to about pH 5. Furthermore, the erythropoietic activity of FBS was found to be co-eluted on Sephadex G-150 with the binding proteins of insulinlike growth factors (IGF). The IGF I concentration determined by radioimmunoassay was 70 ng IGF I/ml. The Ep activity of FBS was less than 5 mU/ml when determined with the posthypoxic polycythemic mouse assay for Ep. These results suggest that the erythropoietic activity of FBS is related to IGF and not to Ep. The erythropoietic activity of FBS was abolished by an antiserum against IGF I. Furthermore, IGF I was a factor of approximately 40 more potent than IGF II in stimulating erythroid colony formation. All of these findings suggest that the erythropoietic activity of FBS is IGF I.
Authors
A Kurtz, W Härtl, W Jelkmann, J Zapf, C Bauer
×Abstract
We have recently isolated and characterized a human monoclonal autoantibody, MOR-h1 (multiple organ-reactive human 1), that reacts with antigens in multiple organs and have shown that this antibody binds to human growth hormone and a 35,000-mol wt protein. In the present study we generated three monoclonal anti-idiotypic antibodies (4E6, 3E5, and 3F6) against MOR-h1. These anti-idiotypic antibodies specifically reacted with MOR-h1 and not with 26 other multiple organ-reactive monoclonal IgM autoantibodies nor with pooled human IgM (myeloma proteins). The binding of the anti-idiotypic antibodies to MOR-h1 was inhibited by both human growth hormone and the 35,000-mol wt protein, which strongly suggests that these antibodies react with epitopes at or near the paratope on MOR-h1. The results of competitive binding experiments revealed that the epitope recognized by 4E6 is distinct from that recognized by 3E5 and 3F6. Using these anti-idiotypic antibodies, lymphocytes and sera from normal individuals were tested for the presence of the 4E6 and 3E5/3F6 idiotopes. By indirect immunofluorescence, the 4E6 idiotope was detected on an average of 1.1% of normal circulating B lymphocytes, and by enzyme-linked immunosorbent assays, the 4E6 and to a lesser extent the 3E5/3F6 idiotopes were found on IgG molecules in sera of normal individuals. In spite of the expression of idiotopes known to be present on MOR-h1, no MOR-h1-like antibody activity was detected in normal sera. Examination of sera from patients with several autoimmune diseases failed to show an increased expression of the 4E6 idiotope as compared with normal controls. These data suggest that anti-idiotypic antibody 4E6 recognizes a public idiotope, the expression of which is not restricted to autoimmune disease.
Authors
K Essani, J Satoh, B S Prabhakar, P R McClintock, A L Notkins
×Abstract
Phenotypic expression of 5-phosphoribosyl 1-pyrophosphate (PRPP) synthetase superactivity was examined in lymphoblast lines derived from six unrelated male patients. Fibroblasts from these individuals have increased rates of PRPP and purine nucleotide synthesis and express four classes of kinetic derangement underlying enzyme superactivity: increased maximal reaction velocity (catalytic defect); inhibitor resistance (regulatory defect); increased substrate affinity (substrate binding defect); and combined catalytic and regulatory defects. Lymphoblast lines from three patients with catalytic defects and from three normal individuals were indistinguishable with respect to enzyme activities, PRPP concentrations and generation, and rates of purine synthesis. Enzyme in lymphoblasts from a patient with combined defects also showed normal maximal reaction velocity but expressed purine nucleotide inhibitor resistance. A second regulatory defect and a substrate binding defect were also demonstrable in lymphoblasts and were identical to the enzyme defects in fibroblasts from the respective patients. Regulatory and substrate binding defects in lymphoblasts were accompanied by increased rates of PRPP and purine nucleotide synthesis. Among explanations for selective expression of enzyme superactivity, reduced concentrations of catalytically superactive enzymes seemed unlikely: immunoreactive PRPP synthetase was comparable in normal-derived and patient-derived cells. Activation of normal enzyme in transformed lymphocytes was also unlikely because absolute specific activities of lymphoblast PRPP synthetases corresponded to those of normal fibroblast and erythrocyte enzymes. Abnormal electrophoretic mobilities and thermal stabilities, identified in certain catalytically superactive fibroblast PRPP synthetases, were not found in the corresponding lymphoblast enzymes. Thus, lymphoblast PRPP synthetases from patients with catalytic superactivity appeared to differ structurally and functionally from their fibroblast counterparts.
Authors
M J Losman, D Rimon, M Kim, M A Becker
×Abstract
D-Pantethine is a conjugate of the vitamin pantothenic acid and the low-molecular-weight aminothiol cysteamine. Pantethine is an experimental hypolipemic agent and has been suggested as a source of cysteamine in the treatment of nephropathic cystinosis. We treated four cystinotic children with 70-1,000 mg/kg per d oral D-pantethine and studied its metabolism. Pantethine was rapidly hydrolyzed to pantothenic acid and cysteamine; we could not detect pantethine in plasma after oral administration. The responsible enzyme, "pantetheinase," was highly active in homogenates of small intestinal mucosa and plasma. The Michaelis constant of the rat intestinal enzyme was 4.6 microM and its pH profile showed a broad plateau between 4 and 9. Pantothenate pharmacokinetics after orally administered pantethine followed an open two-compartment model with slow vitamin elimination (t1/2 = 28 h). Peak plasma pantothenate occurred at 2.5 h and levels over 250 microM were seen at 300 times normal. Apparent total body storage of pantothenate was significant (25 mg/kg), and plasma levels were elevated threefold for months after pantethine therapy. Plasma cysteamine concentrations after pantethine were similar to those reported after equivalent doses of cysteamine. However, at best only 80% white blood cell cystine depletion occurred. We conclude that pantethine is probably less effective than cysteamine in the treatment of nephropathic cystinosis and should only be considered in cases of cysteamine intolerance. Serum cholesterol was decreased an average of 14%, which supports the potential clinical significance of pantethine as a hypolipemic agent. Rapid in vivo hydrolysis of pantethine suggests that pantothenate or cysteamine may be the effectors of its hypolipemic action.
Authors
C T Wittwer, W A Gahl, J D Butler, M Zatz, J G Thoene
×Abstract
Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.
Authors
J E Fox
×Abstract
Atrial natriuretic factor (ANF), a recently sequenced cardiac peptide, has been shown to have potent natriuretic, diuretic, and vasodilating effects in several species. We have developed a radioimmunoassay to measure the levels of immunoreactive ANF in human plasma. Plasma levels of ANF in healthy volunteers on a low sodium diet were 9.8 +/- 1.4 pmol/liter and increased to 21.9 +/- 3.0 on a high sodium diet. The levels of atrial natriuretic factor correlated directly with urinary sodium and inversely with plasma renin activity and plasma aldosterone levels. Patients with marked edema due to congestive heart failure had plasma levels of atrial natriuretic factor five times higher than normal (P less than 0.05), whereas patients with cirrhosis and edema had levels that were not different from normal. These results suggest that atrial natriuretic factor plays an important role in the adaptation to increased sodium intake.
Authors
Y Shenker, R S Sider, E A Ostafin, R J Grekin
×Abstract
It is thought that cysts in polycystic kidneys originate from nephron segments and function in a manner similar to the segment or origin. The indirect evidence for this derives from studies of microanatomy and cyst fluid composition. Cysts with low Na+ have been classified as distal, whereas cysts with high Na+ have been classified as proximal. In order to directly determine the transport characteristics of cyst epithelium, cysts from a human polycystic kidney were studied in vitro using Ussing chamber techniques. Composition of cyst fluid was determined in parallel with these studies. Cysts with low Na+ (gradient cysts) demonstrate characteristics consistent with distal nephron origin including elevated potential difference (PD), short-circuit current (Isc), and low conductance. PD and Isc of gradient cysts were amiloride sensitive. Nongradient cysts, however, require additional characterization. At least two types of nongradient cysts were identified, one with characteristics consistent with proximal nephron origin and another apparently without function. These studies are the first direct evidence for active transport of cysts from human polycystic kidney and provide strong evidence to support the concept that cysts function in the same manner as the nephron segment of origin.
Authors
R D Perrone
×Abstract
Injections of media conditioned by concanavalin A-activated spleen cells from acutely diabetic rats accelerated the appearance of diabetes in young Bio-Breeding/Worcester (BB/W) rats. Activity was also found in media conditioned by spleen cells from nondiabetic, W-line Wistar Furth and Buffalo rats. Unconditioned media containing mitogen had no activity. Conditioned media also induced diabetes in resistant W-line BB/W rats but not in Wistar Furth rats. A soluble factor may activate a BB lymphocyte population that promotes diabetes.
Authors
E S Handler, J P Mordes, J Seals, S Koevary, A A Like, K Nakano, A A Rossini
×Abstract
The human erythrocyte (RBC) is a target organ for parathyroid hormone (PTH) and the hormone increases RBC osmotic fragility and induces their hemolysis. The present study was undertaken to examine whether elevated blood levels of PTH affect RBC survival, and therefore whether PTH, being an extracorpuscular factor, is responsible for the shortened RBC survival in chronic renal failure. 51Cr-labeled RBC survival was elevated in six normal dogs, in six animals with chronic renal failure and secondary hyperparathyroidism (NPX), and in six thyroparathyroidectomized dogs (NPX-TPTX) with comparable degree and duration of chronic renal failure. In the normal dogs, 51Cr-labeled RBC survival ranged between 22 and 35 (25.6 +/- 1.9) d. In the NPX dogs, 51Cr-labeled RBC survival was shortened and the values ranged between 16 and 20 (18.4 +/- 0.6) d, a value significantly (P less than 0.01) lower than normal dogs. In NPX-TPTX dogs, 51Cr-labeled RBC survival ranged between 20 and 33 (25.2 +/- 1.8) d, a value not different from that in normal dogs but significantly higher (P less than 0.01) than that in NPX animals. Our data demonstrate that excess blood levels of PTH and not other consequences of the uremic state are responsible for the shortened RBC survival in chronic renal failure.
Authors
M Akmal, N Telfer, A N Ansari, S G Massry
×Abstract
Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu-self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS.
Authors
G M Shearer, S Z Salahuddin, P D Markham, L J Joseph, S M Payne, P Kriebel, D C Bernstein, W E Biddison, M G Sarngadharan, R C Gallo
×Abstract
To determine whether atrial natriuretic factor (ANF) is a circulating hormone in men, a radioimmunoassay suitable for the estimation of ANF in human plasma was developed and the nature of plasma ANF was characterized. Plasma ANF was extracted before radioimmunoassay by affinity chromatography on a column of ANF antibody-coupled agarose. When plasma ANF extract was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with the radioimmunoassay of the eluted gel slices for ANF, almost all of the ANF activities ran in the 3,000-mol-wt area, while three peaks of ANF were observed in human atrial tissue extract, molecular weights of which corresponded to 14,000, 6,000, and 3,000, respectively. Reversed-phase high performance liquid chromatography of atrial tissue extract resolved multiple forms of ANF. In contrast, one major peak was observed in human plasma extract, and its retention time coincided with that of synthetic human alpha-atrial natriuretic polypeptide. When 500 ml of 0.9% saline was infused into six healthy subjects over 45 min, plasma levels of ANF were unequivocally elevated. The mean plasma ANF concentrations rose from the baseline (23.0 +/- 2.5 pg/ml, mean +/- SEM, n = 6) to the peak (41.8 +/- 4.9 pg/ml, mean +/- SEM) at 75 min postinfusion. No significant change in plasma ANF, on the other hand, was found in the control group. These results suggest that ANF is a circulating hormone in men and is secreted in response to isotonic volume expansion.
Authors
T Yamaji, M Ishibashi, F Takaku
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ISSN: 0021-9738 (print), 1558-8238 (online)