Plasma membrane receptors.

• Text
• PDF

Abstract

Authors

R J Lefkowitz, T Michel

Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.

• Text
• PDF

Abstract

We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.

Authors

J E Griffin, J E Zerwekh

Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture.

• Text
• PDF

Abstract

Using a propagating cell culture system of adipocyte precursors from 70-400-g rats, we explored the possibility that regional variations in properties of adipose tissue may reflect site-specific characteristics intrinsic to the cells, rather than extracellular influences. Initially, studies were made of the nature of the fibroblastlike cells from perirenal adipose tissue stroma. Using colony-forming techniques, it was shown that these cells were adipocyte precursors; each confluent colony that was derived from a single cell displayed differentiated adipocytes. This characteristic was evident in cells from rats of all ages and persisted during secondary culture. At all ages of rats studied, perirenal cells replicated more rapidly than epididymal precursors, e.g., for 179-g rats the population-doubling times were 19.3 +/- 0.7 vs. 25.5 +/- 1.2 h (means +/- SEM, P less than 0.03). With aging of the rats, the replication rate of their perirenal cells decreased progressively. Under clonal conditions, the colony size distribution of both perirenal and epididymal precursors revealed heterogeneity in their capacity for replication, perirenal cells showing greater proliferation. These also differentiated more extensively by morphologic and enzymatic criteria. Age and site had effects that persisted through many cell generations; however, high-fat feeding had no perpetuating influence. The dissimilar properties of perirenal as compared with epididymal precursors may reflect differences in regulation of gene expression. The data are also compatible with a later development in embryological life of perirenal tissue. We suggest that the composition of the adipocyte precursor pool is an important determinant of the growth of adipose tissue that occurs in response to a nutrient load. Interregional or interindividual variation in composition may explain regional and individual differences in fat accumulation.

Authors

P Djian, A K Roncari, C H Hollenberg

Central nervous system site of action for the respiratory depressant effect of diacetylmorphine (heroin) in the cat.

• Text
• PDF

Abstract

The purpose of our study was to identify central nervous system sites involved in the respiratory depressant effect of drugs that stimulate opioid receptors. Diacetylmorphine (heroin) was administered into several cerebroventricular regions of chloralose-anesthetized cats, while monitoring pulmonary ventilation with a Fleisch pneumotachograph. Administration of heroin (17, 50, 150, and 450 micrograms) into the forebrain ventricles, which was restricted to these ventricles, resulted in no significant respiratory effects. In contrast, administration of heroin into either the fourth ventricle or the cisterna magna resulted in a significant (P less than 0.05) decrease in respiratory minute volume (VE). In the fourth ventricle this was because of a decrease in frequency (f) and in the cisterna magna, to a decrease in tidal volume (VT). Intravenous administration of heroin in the same dose-range produced a decrease in VE, which was primarily due to a decrease in f. Bilateral application of heroin (70 micrograms/side) to each of three ventral medullary surface sites (Mitchell's, Schlaefke's, and Loeschcke's areas) known to influence respiration elicited a decrease in VE only at Mitchell's area. This decrease was due to decreases in f and VT. The role of this site in the action of intravenously administered heroin was tested by topical application of naloxone to this area in animals with respiratory depression evoked by intravenous heroin. Bilateral application of naloxone (15 micrograms/side) to Mitchell's area restored breathing to normal. These results lead us to suggest that the site of heroin-induced respiratory depression is a specific area (Mitchell's area) on the ventral surface of the medulla.

Authors

A M Taveira da Silva, J D Souza, J A Quest, F D Pagani, J M Moerschbaecher, A Buller, P Hamosh, R A Gillis

Stimulation of histamine release from human basophils by human platelet factor 4.

• Text
• PDF

Abstract

Human basophils were stimulated to release histamine noncytotoxically by purified human platelet factor 4 (PF4) and the synthetic substituent peptide PF4(59-70). Histamine release was augmented significantly by 10(-7) M PF4 and 10(-5) M PF4(59-70), increased in a concentration-dependent manner, and attained a maximum at 3 X 10(-5) M PF4 and 3 X 10(-4) M PF4(59-70) similar to that achieved by goat anti-human myeloma IgE. PF4 (1-60) failed to initiate the release of histamine, which confirmed that the critical determinant of activity is in the carboxy-terminal sequence. Histamine release from basophils by optimally effective concentrations of PF4 and PF4(59-70) reached a plateau by 1-3 min, as contrasted with 10 min or longer for anti-IgE. The elimination of calcium and magnesium from the buffer suppressed the release of histamine by anti-IgE by 79-83%, but had no effect on that elicited by PF4(59-70). The rate of uptake of [125I]PF4 by purified basophils was similar on a molar basis to the rate of release of histamine by the same concentrations of PF4. The noncytotoxic release of histamine from human basophils by PF4 thus is temporally and biochemically distinct from that mediated by IgE and may be similar to that evoked by other polycationic stimuli.

Authors

L L Brindley, J M Sweet, E J Goetzl

Cyanide-induced cytochrome a,a3 oxidation-reduction responses in rat brain in vivo.

• Text
• PDF

Abstract

The sensitivity of the brain to cyanide-induced histotoxic hypoxia and the protective effects of known cyanide antagonists, have been assessed in vivo by reflectance spectrophotometry. Cyanide-related changes in cytochrome a,a3 (cytochrome c oxidase) oxidation-reduction (redox) state, tissue hemoglobin saturation, and local blood volume were continuously monitored in cerebral cortex of rats. Noncumulative, dose-dependent inhibition of the in situ mitochondrial respiratory chain was evaluated directly by measuring increases in reduction levels of the terminal oxidase. These transient cytochrome a,a3 reductions were accompanied by increases in regional cerebral hemoglobin saturation and blood volume. Cytochrome redox responses were not altered either in magnitude or kinetics by hyperoxia; however, the cyanide-cytochrome dose-response curve was greatly shifted to the right by pretreatment with sodium nitrite, and the recovery rate of cytochrome a,a3 from cyanide-induced reduction was enhanced fourfold by pretreatment with sodium thiosulfate.

Authors

C A Piantadosi, A L Sylvia, F F Jöbsis

Formation of diiodotyrosine from thyroxine. Ether-link cleavage, an alternate pathway of thyroxine metabolism.

• Text
• PDF

Abstract

Studies were performed to elucidate the nature of the pathway of hepatic thyroxine (T4) metabolism that is activated by inhibitors of liver catalase. For this purpose, the metabolism of T4 in homogenates of rat liver was monitored with T4 labeled with 125I either at the 5'-position of the outer-ring (125I-beta-T4) or uniformly in both the outer and inner rings (125I-U-T4). In homogenates incubated with 125I-beta-T4 in an atmosphere of O2, the catalase inhibitor aminotriazole greatly enhanced T4 degradation, promoting the formation of large proportions of 125I-labeled iodide (125I-I-) and chromatographically immobile origin material (125I-OM), but only a minute proportion of 125I-labeled 3,5,3'-triiodothyronine (125I-T3) (T3 neogenesis). In an atmosphere of N2, in contrast, homogenates produced much larger proportions of 125I-T3, and aminotriazole had no effect. In incubations with 125I-U-T4, under aerobic conditions, control homogenates degraded T4 slowly; formation of 125I-labeled 3,5-diiodotyrosine (125I-DIT) was seen only occasionally and in minute proportions. However, in homogenates incubated under O2, but not N2, aminotriazole consistently elicited the formation of large proportions of 125I-DIT, indicating that the ether link of T4 was being cleaved by an O2-dependent process. Formation of 125I-DIT in the presence of aminotriazole and O2 was markedly inhibited by the substrates of peroxidase, aminoantipyrine, and guaiacol. GSH greatly attenuated the increase in DIT formation induced by aminotriazole, whereas the sulfhydryl inhibitor N-ethylmaleimide (NEM) activated the DIT-generating pathway, even in the absence of aminotriazole. Activation of the in vitro formation of 125I-DIT from 125I-U-T4 was also produced by the in vivo administration of aminotriazole or bacterial endotoxin, an agent that reduces hepatic catalase activity. Studies with 125I-DIT as substrate revealed it to be rapidly deiodinated by liver homogenates under aerobic conditions. Recovery of 125I-DIT from 125I-U-T4 was increased by the addition of the inhibitor of iodotyrosine dehalogenase, 3,5-dinitrotyrosine. However, as judged from studies conducted in parallel with radioiodine-labeled DIT and 125I-U-T4 as substrates, none of the factors that altered the proportion of 125I-DIT found after incubations with 125I-U-T4 did so by altering the degradation of the 125I-DIT formed. The factors that influenced DIT formation from T4 in rat liver had opposite effects on T3 neogenesis. Thus, aminotriazole, endotoxin, NEM, and an aerobic atmosphere, all of which enhanced DIT formation, were inhibitory to T3 neogenesis. In contrast, anaerobiosis and GSH inhibited ether-link cleavage of T4, but facilitated T3 neogenesis. The foregoing results suggest that a pathway for the ether-link cleavage of T4 to yield DIT is present in rat liver. Activity of this pathway, which appears to be peroxidase mediated, is inversely related to activity of the pathway for the T3 neogenesis. It is further suggested that this reciprocity reflects a reciprocal relationship between hepatic GSH and H2O2, the former increasing T3 formation and inhibiting DIT formation, and the latter producing opposite effects.

Authors

A Balsam, F Sexton, M Borges, S H Ingbar

In vitro insulin resistance of human adipocytes isolated from subjects with noninsulin-dependent diabetes mellitus.

• Text
• PDF

Abstract

To assess possible cellular mechanisms of in vitro resistance in noninsulin-dependent diabetes mellitus (NIDDM), maximum insulin-stimulated glucose transport and utilization and insulin binding were measured in adipocytes isolated from weight-matched normal glycemic subjects and patients with NIDDM. Glucose transport rate was determined by measuring the amount of [U-14C]-D-glucose taken up by incubating adipocytes at trace concentrations of glucose (300 nM), and glucose metabolism by estimating the amount of lactate, CO2, triglyceride, and total glucose carbons retained in the cells following incubating at 5.5 mM glucose. Insulin binding was measured at 50, 100, and 200 pM [mono125I-tyrosinyl A14]insulin. Both maximum insulin-stimulated glucose transport and utilization in adipocytes from diabetic subjects were 40% (P less than 0.01) and 32% (P less than 0.05) lower, respectively, than values obtained for subjects with normal glucose tolerance. In addition, the maximum capacity of glucose transport was correlated with the maximum capacity of glucose utilization (r = 0.81, P less than 0.001). Furthermore, fasting plasma glucose concentrations of diabetic subjects were negatively correlated with both maximum insulin-stimulated glucose transport (r = -0.56, P less than 0.05) and glucose utilization (r = -0.67, P less than 0.05). Since basal glucose transport in adipocytes from diabetic subjects was also 33% lower than in adipocytes from normal subjects, there was no change in the relative ability of insulin to stimulate glucose transport. However, there was a 64% decrease in the sensitivity of the glucose transport system to insulin (P less than 0.05), unrelated to concomitant changes in insulin binding. These results demonstrate that both maximal insulin-stimulated glucose transport and utilization, and the sensitivity of the glucose transport system to insulin, was decreased in adipocytes isolated from subjects with NIDDM. These in vitro defects were associated with impaired glucose metabolism in vivo, consistent with the view that the metabolic alterations observed at the cellular level may contribute to the in vivo insulin resistance of NIDDM.

Authors

A Kashiwagi, M A Verso, J Andrews, B Vasquez, G Reaven, J E Foley

Prostacyclin biosynthesis in cultured vascular endothelium is limited by deactivation of cyclooxygenase.

• Text
• PDF

Abstract

Primary monolayer cultures of human umbilical vein endothelium produce prostacyclin (PGI2) in response to stimulation by thrombin, ionophore A23187, arachidonic acid, and the prostaglandin endoperoxide, PGH2. None of these treatments had a significant effect on the capacity of the endothelium to produce PGI2 in response to subsequent stimulation by PGH2. By contrast, endothelium initially exposed to thrombin, A23187, or arachidonic acid produced approximately 37, 68, and 84% less PGI2, respectively, upon subsequent stimulation by arachidonic acid. These findings suggest that PGI2 biosynthesis in cultured endothelium results in deactivation of cyclooxygenase-hydroperoxidase but not PGI2 synthetase. To test the hypothesis that PGI2 biosynthesis alone causes deactivation of cyclooxygenase, thrombin, A23187, and arachidonic acid were added to monolayers that had been preincubated with ibuprofen (250 microM), a rapidly reversible, competitive inhibitor of this enzyme. After removal of the ibuprofen and the initial stimulus, PGI2 production in response to subsequent stimulation by arachidonic acid was maximal. These findings suggest that the metabolism of arachidonic acid itself causes a direct deactivation of cyclooxygenase. After an initial exposure to arachidonic acid, PGI2 production in response to a second stimulation by arachidonic acid was restored to approximately 34, 69, and 74% of maximal, after recovery periods of 1, 24, and 48 h, respectively. We conclude that the regulation of PGI2 biosynthesis in normal vascular endothelium may be in part a function of the activity and biosynthesis of cyclooxygenase-hydroperoxidase and the deactivation of this enzyme may be a primary factor limiting the capacity of the endothelium to produce PGI2.

Authors

A F Brotherton, J C Hoak

Restriction fragment length polymorphism associated with the pro alpha 2(I) gene of human type I procollagen. Application to a family with an autosomal dominant form of osteogenesis imperfecta.

• Text
• PDF

Abstract

One cloned complementary DNA and one genomic subclone were used to detect restriction fragment length polymorphism associated with the pro alpha 2(I) gene for human type I procollagen. The restriction fragments obtained from examination of 30-122 chromosomes confirmed previous indications that the pro alpha 2(I) gene is found in a single copy in the human haploid genome. One highly polymorphic site was detected with EcoRI in the 5'-half of the gene. The restriction site polymorphism at the site had an allelic frequency of 0.38, and it generated two fragments of 10.5 and 3.5 kilobase in homozygous individuals. The restriction fragment length polymorphism generated at the EcoRI site was used to study affected and non-affected individuals in four generations of a family with an autosomal dominant form of osteogenesis imperfecta. The data demonstrated a linkage of the phenotype to a pro alpha 2(I) allele with a lod score of 2.41 at a recombination fraction (theta) of 0. The data therefore provided presumptive evidence that osteogenesis imperfecta in this family is caused by a mutation in the pro alpha 2(I) gene or some contiguous region of the genome. The relatively high frequency of polymorphism at the EcoRI site makes it useful for studying a broad range of genetic disorders in which mutations in type I procollagen are suspected. In addition, the polymorphic site should provide useful markers for linkage studies with other loci located on human chromosome 7.

Authors

P Tsipouras, J C Myers, F Ramirez, D J Prockop

Cytochalasin B is a potent mitogen for chronic lymphocytic leukemia cells in vitro.

• Text
• PDF

Abstract

It is widely accepted that the neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) respond poorly to common mitogens. The fungal metabolite cytochalasin B (0.5 micrograms/ml) is a weak mitogen for normal lymphocytes. However, when peripheral blood lymphocytes from 19 patients with CLL of B cell origin (B-CLL) were cultured with 0.5 micrograms cytochalasin B/ml, significant new DNA synthesis ( [14C]thymidine incorporation) occurred in 18. Stimulation indices with cytochalasin B varied widely (range = 1.9-28.2, mean +/- SD = 10.6 +/- 7.5; delta cpm range = 1,157-153,818; n = 26) but in 11 cases exceeded those seen with concanavalin A (Con A), phytohemagglutinin, or pokeweed mitogen. In all 11, the mitogenic response to cytochalasin B exceeded that to pokeweed mitogen, which is believed to be a T cell-dependent B cell mitogen. In three cases, the responses to cytochalasin B were 8.6, 3.5, and 2.3 times greater than those to Con A. As with other mitogens, the DNA synthetic response to cytochalasin B was time and dose dependent. Peak thymidine incorporation occurred at 72-88 h and declined thereafter. Significant mitogenic effects were observed with 0.1-5 micrograms cytochalasin B/ml with a peak at 0.5-2 micrograms/ml. Stimulated DNA synthesis was abolished by 1 mM hydroxyurea. Cells from two patients with B-CLL were separated by rosetting with sheep erythrocytes (E). Depletion of E-rosette-positive cells from the CLL cell population abolished the response to Con A but did not affect the response to cytochalasin B. Cytochalasin B is a potent mitogen for B-CLL cells and may be useful in cytogenetic studies of this often indolent neoplasm.

Authors

R A Larson, S Yachnin

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

• Text
• PDF

Abstract

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

Authors

H J Sander, J W Slot, B N Bouma, P A Bolhuis, D S Pepper, J J Sixma

Identification of a new structural variant of human apolipoprotein E, E2(Lys146 leads to Gln), in a type III hyperlipoproteinemic subject with the E3/2 phenotype.

• Text
• PDF

Abstract

A type III hyperlipoproteinemic subject having the apolipoprotein E (apo E) phenotype E3/2 was identified. From isoelectric focusing experiments in conjunction with cysteamine treatment (a method that measures cysteine content in apo E), the E2 isoform of this subject was determined to have only one cysteine residue, in contrast to all previously studied E2 apoproteins, which had two cysteines. This single cysteine was shown to be at residue 112, the same site at which it occurs in apo E3. From amino acid and sequence analyses, it was determined that this apo E2 differed from apo E3 by the occurrence of glutamine rather than lysine at residue 146. When phospholipid X protein recombinants of the subject's isolated E3 and E2 isoforms were tested for their ability to bind to the human fibroblast apo-B,E receptor, it was found that the E3 bound normally (compared with an apo E3 control) but that the E2 had defective binding (approximately 40% of normal). Although they contained E3 as well as E2, the beta-very low density lipoproteins (beta-VLDL) from this subject were very similar in character to the beta-VLDL from an E2/2 type III hyperlipoproteinemic subject; similar subfractions could be obtained from each subject and were shown to have a similar ability to stimulate cholesteryl ester accumulation in mouse peritoneal macrophages. The new apo E2 variant has also been detected in a second type III hyperlipoproteinemic subject.

Authors

S C Rall Jr, K H Weisgraber, T L Innerarity, T P Bersot, R W Mahley, C B Blum

Chlorpropamide action on renal concentrating mechanism in rats with hypothalamic diabetes insipidus.

• Text
• PDF

Abstract

To determine vasopressin (VP)-potentiating effect of chlorpropamide (CPMD), we studied the effect of CPMD in vivo and in vitro in kidneys and in specific tubule segments of rats with hypothalamic diabetes insipidus, homozygotes of the Brattleboro strain (DI rats). Rats on ad lib. water intake were treated with CPMD (20 mg/100 g body wt s.c. daily) for 7 d. While on ad lib. water intake, the urine flow, urine osmolality, urinary excretion of Na +, K +, creatinine, or total solute excretion did not change. However, corticopapillary gradient of solutes was significantly increased in CPMD-treated rats. Higher tissue osmolality was due to significantly increased concentration of Na +, and to a lesser degree urea, in the medulla and papilla of CPMD-treated rats. Consequently, the osmotic gradient between urine and papillary tissue of CPMD-treated rats (delta = 385 +/- 47 mosM) was significantly (P less than 0.001) higher compared with controls (delta = 150 +/- 26 mosM). Minimum urine osmolality after water loading was higher in CPMD-treated DI rats than in controls. Oxidation of [14C]lactate to 14CO2 coupled to NaCl cotransport was measured in thick medullary ascending limb of Henle's loop (MAL) microdissected from control and CPMD-treated rats. The rate of 14CO2 production was higher (delta + 113% +/- 20; P less than 0.01) in CPMD-treated MAL compared with controls, but 14CO2 production in the presence of 10(-3) M furosemide did not differ between MAL from control and from CPMD-treated rats. These observations suggest that CPMD treatment enhances NaCl transport in MAL. Cyclic AMP metabolism was analyzed in microdissected MAL and in medullary collecting tubule (MCT). MCT from control and from CPMD-treated rats did not differ in the basal or VP-stimulated accumulated of cAMP. The increase in cAMP content elicited by 10(-6) M VP in MAL from CPMD-treated rats (delta + 12.0 +/- 1.8 fmol cAMP/mm) was significantly (P less than 0.02) higher compared with MAL from control rats (delta + 5.1 +/- 1.0 fmol cAMP/mm). Preincubation of MAL dissected from Sprague-Dawley rats with 10(-4) M CPMD in vitro increased cAMP accumulation in the presence of VP, but no such enhancement was found in preincubated MCT. Adenylate cyclase activity, basal or stimulated by VP, 5'guanylimidodiphosphate, or by NaF, assayed in isotonic medium did not differ between MAL or MCT from control rats and MAL or MCT from CPMD-treated rats. When assayed in hypertonic medium (800 mosM), the adenylate cyclase activity in the presence of 10(-6) M VP was significantly higher in MAL of CPMD-treated rats. MAL and MCT from control and CPMD-treated rats did not differ in the activities of cAMP phosphodiesterase. The rate of [(14)C]prostaglandin E2 by medullary and papillary microsomes was not different between the control and CPMD-treated rats; likewise, there was no difference in accumulation of immunoreactive prostaglandin E2 in the medium of in vitro incubated medullary or papillary slices prepared from control and CPMD-treated rats. Based on the findings recounted above, we propose a hypothesis that CPMD administration enhances the antidiuretic effect of VP, primarily by increasing medullary and papillary tonicity dye to increased NaCl reabsorption in MAL. There is no evidence that CPMD sensitizes collecting tubules to the action of VP, at least at the camp-generation step. Therefore, increased antidiuretic response to VP in the kidneys of CPMD-treated DI rats is due to enhanced osmotic driving force for water reabsorption (lumen-to-interstitium osmotic gradient) in collecting tubules, rather than due to increased VP-dependent water permeability of tubular epithelium.

Authors

E Kusano, J L Braun-Werness, D J Vick, M J Keller, T P Dousa

Regulation of transferrin receptor expression on human leukemic cells during proliferation and induction of differentiation. Effects of gallium and dimethylsulfoxide.

• Text
• PDF

Abstract

The association of transferrin receptor expression with cellular proliferation has been studied extensively, but a number of events have not been defined. We therefore assayed receptor on promyelocytic leukemia (HL-60) cells at early times after exposure to a stimulus for proliferation (subculture), as well as agents that either induce differentiation (dimethylsulfoxide [DMSO] ) or inhibit iron uptake (transferrin-gallium). Within 4 h after subculture, we found that a significant increase in total cellular immunoreactive receptor occurred that preceded by 8 h the increase in cell-surface transferrin binding. Automated fluorocytometric analysis of cells in an immunofluorescent assay indicated that increased surface receptor density appeared on cells in the S, G2, and M phases of the cell cycle. DMSO-treated cells proliferated at the same rate as untreated (control) cells for the first 72 h, but as early as 12 h after treatment transferrin receptor was significantly decreased (65% of control cells). Further decreases occurred at later time points until transferrin receptor was undetectable after 7 d, when proliferation had ceased, cells were arrested in G1 phase of the cell cycle, and myeloid differentiation occurred. After exposure to transferrin-gallium, proliferation ceased, but cells exhibited increased surface receptor and were arrested at S phase of the cell cycle without associated myeloid differentiation. We conclude that events preceding cell division provide the regulatory stimulus for the synthesis and subsequent appearance of the transferrin receptor on the cell surface. Additionally, decreased receptor expression may be important in causing cessation of proliferation and/or differentiation. Finally, the way in which gallium salts are currently being investigated as chemotherapeutic agents should be reevaluated in light of our findings concerning transferrin-gallium effects on cellular proliferation.

Authors

C R Chitambar, E J Massey, P A Seligman

Role of complement and polymorphonuclear cells in demethylchlortetracycline-induced phototoxicity in guinea pigs. Inhibition by decomplementation in vivo.

• Text
• PDF

Abstract

In this study, demethylchlortetracycline was used as a prototype of exogenous phototoxic substances. In vitro, exposure of serum containing demethylchlortetracycline to ultraviolet-A irradiation resulted in the diminution of total complement hemolytic activity and C4, C2, C3, and C5 activities. In addition, chemotactic activity for human polymorphonuclear cells was generated, which was thermostable and antigenically related to human C5 but not human C3. In vivo, phototoxic lesions were induced in guinea pigs upon intradermal injections of demethylchlortetracycline solution, followed by ultraviolet-A irradiation. On a scale of 0-3+, the animals developed a maximal response of 2.5 at 20 h. This clinical response was associated with cellular infiltrate in the dermis, consisting of 29 +/- 2% of neutrophils at 24 h. The participation of the polymorphonuclear cells was evaluated in guinea pigs rendered neutropenic by treatment with cyclophosphamide. In these guinea pigs, demethylchlortetracycline and ultraviolet-A induced a maximal response of 0.75 +/- 0.5, which was associated histologically with 1.2 +/- 0.5% neutrophils in the dermis. The role of complement in this process was studied in guinea pigs congenitally deficient in C4, and in guinea pigs decomplemented by treatment with cobra venom factor. In contrast to normal guinea pigs, C4-deficient animals exhibited a maximal reaction of 0.83 +/- 0.16 at 6 h, which subsided within 24 h. Cobra venom factor-treated guinea pigs developed a maximal response of 0.5 at 0.5 and at 6 h. These clinical changes were associated with the development of an increased vascular permeability, as demonstrated by studies using guinea pigs injected intravenously with Evans blue solution. In animals with a normal complement system, there was intense localized bluing at the sites of phototoxic lesion. In contrast, only minimal bluing was observed in decomplemented guinea pigs. These data indicate that a normal number of polymorphonuclear cells and an intact complement system are required for the full development of demethylchlortetracycline-induced phototoxic lesions.

Authors

H W Lim, H Novotny, I Gigli

Endogenous prostacyclin biosynthesis and platelet function during selective inhibition of thromboxane synthase in man.

• Text
• PDF

Abstract

The consequences of inhibiting the metabolism of prostaglandin G2 to thromboxane A2 in man were studied by using an inhibitor of thromboxane synthase, 4-[2-(IH-imidazol-1-yl)ethoxy] benzoic acid hydrochloride (dazoxiben). Single doses of 25, 50, 100, and 200 mg of dazoxiben were administered to healthy volunteers at 2-wk intervals in a randomized, placebo-controlled, double-blind manner. Serum thromboxane B2 and aggregation studies in whole blood and platelet-rich plasma were measured before dosing and at 1, 4, 6, 8, and 24 h after dosing. Both serum thromboxane B2 and the platelet aggregation response to arachidonic acid (1.33 mM) were reversibly inhibited in a dose-dependent manner. Aggregation induced by 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (0.4 and 4.0 microM) in platelet-rich plasma as well as both aggregation and nucleotide release induced by collagen (95 micrograms/ml) in platelet-rich plasma and whole blood were unaltered by dazoxiben. Additional evidence for a platelet-inhibitory effect of the compound was a significant prolongation of the bleeding time at 1 h after administration of the highest dose (200 mg) of dazoxiben. Endogenous prostacyclin biosynthesis was assessed by measurement of the major urinary metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 alpha (PGI-M). PGI-M excretion was increased by dazoxiben; it rose a mean 2.4-fold from predosing control values at 0-6 h after administration of the highest dose studied (200 mg).

Authors

G A FitzGerald, A R Brash, J A Oates, A K Pedersen

Human upper epidermal cytoplasmic antibodies are directed against keratin intermediate filament proteins.

• Text
• PDF

Abstract

Upper cytoplasmic (U-Cyt) antibodies are directed against cytoplasmic antigens found in keratinocytes in the upper layers of the epidermis. Until now, they have been defined by indirect immunofluorescence and are known to occur in the sera of patients with cutaneous diseases such as bullous dermatoses, basal cell carcinomas, and melanomas. An increased incidence of U-Cyt antibodies has also been reported in the sera of patients with noncutaneous diseases, such as pulmonary neoplasms. They have been found in addition in the sera of some normal individuals. In this study we have identified keratin intermediate filaments (KIF) as antigens U-Cyt antibodies are directed against. KIF proteins were prepared, separated by polyacrylamide gel electrophoresis, transblotted to nitro-cellulose strips, and used as substrates for antibody binding. Sera containing U-Cyt antibodies by indirect immunofluorescence also had antibodies that were directed against high molecular weight (65,000, 63,000, 61,500) KIF proteins. When KIF proteins were separated according to their charge and their molecular weight by two-dimensional gel electrophoresis and transblotted, the anti-KIF protein antibodies bound to virtually all charge isomers of the KIF proteins at the respective molecular weight. The antibody titers measured using the transblotting technique were 10 to 160 times higher than those found by indirect immunofluorescence. To determine whether U-Cyt antibodies were directed against KIF, a series of absorption and elution experiments were performed. Absorption of test sera with purified KIF removed both U-Cyt antibodies and anti-KIF protein antibodies. Absorption with another type of intermediate filament derived from fibroblasts, vimentin, did not remove U-Cyt or anti-KIF protein antibodies. Absorbed U-Cyt and anti-KIF protein antibodies were both eluted from the same KIF preparation and shown to bind to U-Cyt antigens by indirect immunofluorescence and KIF proteins by transblotting. Absorption of a serum containing U-Cyt antibodies, anti-nuclear antibodies, and anti-basement membrane zone antibodies with purified KIF resulted in the removal of the U-Cyt antibodies but not the other types of antibody. In addition, all test sera, even those that lacked U-Cyt antibodies, were found to have low-titer antibodies against KIF proteins by the transblotting technique. These data indicate that KIF proteins bear antigens to which U-Cyt antibodies are directed and that low titer antibodies against KIF proteins may be much more common than previously appreciated.

Authors

H Hintner, P M Steinert, T J Lawley

Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves' disease.

• Text
• PDF

Abstract

Studies were carried out with the serum IgG from a mother and her two children who developed neonatal Graves' disease several weeks after birth. The maternal IgG: (a) stimulated the human thyroid in vitro, but maximal stimulation was found only with dilution of the IgG; (b) was very potent in the long-acting thyroid stimulator (LATS)-protector assay, but only when an inhibitor of the system was diluted out; (c) inhibited a standard preparation of LATS in the mouse bioassay; (d) was biphasic in the thyrotropin-binding inhibition (TBI) assay, i.e., enhanced binding at low concentrations of IgG and inhibited binding at high levels. Enhancement in the TBI assay was found only with particulate preparations of human thyroid membranes as receptor and not when that material was solubilized, nor with guinea pig fat cell membranes as receptor. Serial blood samples from the second child were obtained at birth and until 3 mo of age. In the thyroid slice (cyclic AMP) assay system there was a negative dose-response relationship in testing IgG until age 45 d when it became positive, coinciding with the clinical recognition that hyperthyroidism had developed. The data are compatible with a concept that this mother's IgG contained thyroid-stimulating antibody (TSAb) and another moiety that inhibited TSAb through an action on the thyroid cell membrane, thus delaying the onset of hyperthyroidism in the neonate until the inhibiting IgG was metabolically cleared to an ineffective concentration.

Authors

M Zakarija, J M McKenzie, D S Munro

A monoclonal antibody to rhesus erythrocyte band 3 inhibits invasion by malaria (Plasmodium knowlesi) merozoites.

• Text
• PDF

Abstract

Receptors on erythrocytes and malaria parasites mediate specific attachment and junction formation between these cells that lead to invasion of the erythrocytes. We identified monoclonal antibody A9 and its subclone A9D3 that bound to rhesus erythrocytes and blocked invasion of the erythrocytes by Plasmodium knowlesi merozoites. The monoclonal antibodies did not block attachment, the initial step in invasion, although swelling and crenation of the erythrocyte, which normally occur after attachment, were rarely observed in the presence of antibody. The monoclonal antibody immunoprecipitated rhesus erythrocyte band 3. It bound to erythrocytes of another Old World monkey, the kra monkey, but not to erythrocytes of New World monkeys, chimpanzees, or man. Since the antibody did not bind to human erythrocytes, we could test for nonspecific toxicity to the parasite by studying the effect of the ascites and purified antibody on invasion of human erythrocytes. The antibody caused a minimal reduction in invasion of human erythrocytes, a reduction no greater than that seen with an unrelated monoclonal antibody. Further evidence that the inhibition was specific came from study of Fab fragments of A9D3. Column-purified Fab fragments reduced invasion of rhesus erythrocytes without affecting invasion of human erythrocytes. Fab fragments preabsorbed with rhesus erythrocytes did not inhibit invasion. From the above data, we conclude that band 3 is involved in a stage in the invasion process after initial recognition.

Authors

L H Miller, D Hudson, J Rener, D Taylor, T J Hadley, D Zilberstein

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

• Text
• PDF

Abstract

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.

Authors

P A Craven, R Saito, F R DeRubertis

Tissue oxygenation and muscular substrate turnover in two subjects with high hemoglobin oxygen affinity.

• Text
• PDF

Abstract

Oxygen transport to and substrate turnover in leg muscle were studied at rest and during light and heavy upright bicycle exercise in two brothers with a hereditary hemoglobinopathy associated with high oxygen affinity (P50 = 13 mmHg). Femoral venous oxygen tension was below normal and femoral venous oxygen saturation above normal at rest and during exercise. Thus, the arterial-femoral venous oxygen saturation difference was decreased. Despite a compensatory increase in hemoglobin concentration, the arterial-femoral venous oxygen content difference tended to be below normal at heavy exercise. Approximately 25% of the oxygen was delivered via the abnormal hemoglobin at relative heavy exercise. Arterial lactate levels, lactate release, and muscle lactate concentration were not increased at any level of exercise. Glucose, alanine, pyruvate, and glycerol turnover were essentially normal, but the glycogen and creatine phosphate stores were abnormally depleted at the termination of heavy exercise. The exercise electrocardiogram (ECG) was normal, indicating that myocardial oxygenation was adequate. Muscle-surface oxygen pressure fields were normal at rest (not investigated during exercise). It is concluded that the high oxygen affinity of the hemoglobin in our two subjects did not lead to heart or skeletal muscle hypoxia during heavy exercise, as judged from the ECG and from the leg lactate turnover. Despite the lack of evidence for muscle hypoxia, the subjects experienced leg muscle fatigue and the creatine phosphate and glycogen stores were depleted more than normally.

Authors

B Wranne, G Berlin, L Jorfeldt, N Lund

Proximal tubular bicarbonate reabsorption and PCO2 in chronic metabolic alkalosis in the rat.

• Text
• PDF

Abstract

Studies were undertaken to define the pattern of proximal tubular bicarbonate reabsorption and its relation to tubular and capillary PCO2 in rats with chronic metabolic alkalosis (CMA). CMA was induced by administering furosemide to rats ingesting a low electrolyte diet supplemented with NaHCO3 and KHCO3. Proximal tubular bicarbonate reabsorption and PCO2 were measured in CMA rats either 4-7 or 11-14 d after furosemide injection, in order to study a wide range of filtered bicarbonate loads. A group of nine age-matched control animals, fed the same diet but not given furosemide, was studied for comparison. In a third group of controls, the filtered load of bicarbonate was varied over the same range as in the CMA rats by plasma infusion and aortic constriction. The CMA rats had significant alkalemia and hypokalemia (4-7 d: pH 7.58, HCO3 38.3 meq/liter, K+ 2.1 meq/liter; 11-14 d: pH 7.54, HCO3 38.1 meq/liter, K+ 2.5 meq/liter). Nonetheless, proximal bicarbonate reabsorption was not significantly different from that seen in control rats at any given load of filtered bicarbonate (from 250 to 1,300 pmol/min). In both control and CMA rats, 83-85% of the filtered bicarbonate was reabsorbed by the end of the accessible proximal tubule. These observations indicate that proximal bicarbonate reabsorption is determined primarily by the filtered load in chronic metabolic alkalosis. When single nephron glomerular filtration rate (SNGFR) is reduced by volume depletion in the early postfurosemide period, the filtered load and the rate of proximal bicarbonate reabsorption remain at or below control levels, maintaining metabolic alkalosis. In the late postfurosemide period, however, SNGFR returned to control levels in some instances. In these animals, both the filtered load and rate of proximal reabsorption were increased above the highest levels seen in control animals. The PCO2 gradient between the peritubular capillaries and arterial blood (Pc-Art) was significantly higher in CMA than in control, even though the rate of proximal bicarbonate reabsorption did not differ. Thus, proximal bicarbonate reabsorption did not appear to be the primary determinant of Pc-Art PCO2. PCO2 in the early proximal (EP) tubule was significantly higher than in either the late proximal (LP) tubule or peritubular capillaries in both control and CMA rats. The EP-LP PCO2 gradient correlated directly with proximal bicarbonate reabsorption (P less than 0.05). The small elevation in PCO2 in EP may be related to CO2 generated at this site in the process of bicarbonate reabsorption.

Authors

D A Maddox, F J Gennari

Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss.

• Text
• PDF

Abstract

We devised a new method for examining the structural changes that occur in trabecular bone in aging and in osteoporosis. With simultaneous measurement of total perimeter and bone area in thin sections, indirect indices of mean trabecular plate thickness (MTPT) and mean trabecular plate density (MTPD) can be derived, such that trabecular bone volume = MTPD X MTPT. MTPD is an index of the probability that a scanning or test line will intersect a structural element of bone, and is the reciprocal of the mean distance between the midpoints of structural elements, multiplied by pi/2. We applied this method to iliac bone samples from 78 normal subjects, 100 patients with vertebral fracture, and 50 patients with hip fracture. The reduction in trabecular bone volume observed in normal subjects with increasing age was mainly due to a reduction in plate density, with no significant decrease in plate thickness. The further reduction in trabecular bone volume observed in patients with osteoporotic vertebral fracture was mainly due to a further reduction in plate density. There was a relatively smaller reduction in plate thickness that was statistically significant in males but not in females. Only in patients with hip fracture did trabecular thinning contribute substantially to the additional loss of trabecular bone in osteoporosis relative to age. These data indicate that age-related bone loss occurs principally by a process that removes entire structural elements of bone; those that remain are more widely separated and some may undergo compensatory thickening, but most slowly become reduced in thickness. We propose that the process of removal is initiated by increased depth of osteoclastic resorption cavities which leads to focal perforation of trabecular plates; this is followed by progressive enlargement of the perforations with conversion of plates to rods. The resulting structural changes are more severe in osteoporotic patients than in normal subjects, but have been completed in most patients before they develop symptoms.

Authors

A M Parfitt, C H Mathews, A R Villanueva, M Kleerekoper, B Frame, D S Rao

Suppression of in vitro monoclonal human rheumatoid factor synthesis by antiidiotypic antibody. Target cells and molecular requirements.

• Text
• PDF

Abstract

Previous studies have indicated that antiidiotypic antibody can modulate expression of idiotype both in vivo and in vitro. Although the precise mechanisms underlying modulation of idiotype expression by antiidiotype remains unclear, a requirement for intact IgG antiidiotypic antibody has been suggested and T cells appear to play a role in some systems. We have studied peripheral blood mononuclear leukocytes (MNL) from a patient with a B cell lymphoma and a circulating IgMK rheumatoid factor (RF) paraprotein in an effort to delineate mechanisms involved in regulation of idiotype expression by antiidiotypic antibody. 1-10% of MNL from this patient could be cytoplasmically stained with specific F(ab')2 antiidiotypic antibody. MNL from the patient spontaneously synthesized IgM RF in culture that possessed the same idiotype as the circulating IgM RF paraprotein. Production of RF by MNL was suppressed by pretreatment with either intact IgG or the F(ab')2 fragments of antiidiotypic antibody (50% inhibitory concentration was 0.2 and 1.1 micrograms/culture, respectively). In contrast, the Fab' fragment of antiidiotypic antibody was not inhibitory (up to 57 micrograms/culture) despite retaining demonstrable antiidiotype activity. Suppression of RF production was not observed over the same concentration range with the IgG or F(ab')2 fractions of a non-cross-reactive antiidiotypic antibody prepared against another monoclonal IgMK RF paraprotein or with IgG or F(ab')2 fractions prepared from normal rabbit serum. Inhibition of RF production by antiidiotypic antibody did not require T cells. Antiidiotypic antibody decreased intracellular and extracellular levels of idiotype indicating diminished synthesis of idiotype by the patient's B cells. Synthesis of IgM RF by MNL obtained from unrelated donors was not suppressed by the antiidiotypic antibody specific for the patient's paraprotein. The results indicate that (a) antiidiotypic antibody is capable of directly suppressing human B cell release of idiotype, (b) the bivalent antigen-binding fragment (F[ab']2) of antiidiotypic antibody is sufficient for mediating such suppression, (c) an intact Fc portion of antiidiotypic antibody enhances suppression of idiotype, and (d) antiidiotypic antibody inhibits idiotype expression by suppressing synthesis of idiotype.

Authors

W J Koopman, R E Schrohenloher, J C Barton, E C Greenleaf

Metabolism of estradiol in liver cell culture. Differential responses of C-2 and C-16 oxidations to drugs and other chemicals that induce selective species of cytochrome P-450.

• Text
• PDF

Abstract

The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17 beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as the substrate. Oxidation of estradiol by the cultured liver cells was assessed by the release of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative reactions were detectable in the liver cell cultures by this technique. When incubated with a concentration of estradiol substrate close to the Michaelis constant (Km), approximately 45.8 pmol [2-3H]estradiol and 5.0 pmol [16-3H]estradiol/mg protein per minute underwent oxidative metabolism in untreated cells. Total amounts of oxidized product formation after 2 h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively. Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively), whereas no significant change in C-16 oxidation was observed after treatment of the cultures with 3-methylcholanthrene, benzo[a]pyrene, or benz[a]anthracene. The latter chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e., 1.5-2.2-fold increases over control values). The increase in C-2 oxidation after treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that observed for oxidation at C-16. The apparent Km values for these oxidations in control cultures were 23.5 and 30.3 microM for C-2 and C-16 oxidation, respectively; corresponding maximum velocity (Vmax) values were 119 and 11.7 pmol/mg protein per minute, respectively. These data indicate that the C-2 and C-16 oxidations of estradiol take place in cultured avian hepatocytes and that the extent of metabolism at these positions on the hormone molecule can be altered by chemicals, such as drugs and polycyclic aromatic hydrocarbons, which induce distinctive species of cytochrome P-450 in the liver.

Authors

J Schneider, S Sassa, A Kappas

A novel method for generating region-specific monoclonal antibodies to modified proteins. Application to the identification of human glucosylated low density lipoproteins.

• Text
• PDF

Abstract

Modifications of plasma lipoprotein structure and function resulting from in vivo post-translational nonenzymatic glycosylation may play a role in the premature atherosclerosis of patients with diabetes mellitus. This report describes the generation and characterization of six unique murine monoclonal antibodies that bind glucosylated human plasma lipoproteins, but do not react with normal plasma lipoproteins. This was accomplished by immunizing mice with homologous glucosylated low density lipoprotein. In competitive inhibition radioimmunoassays, the dominant epitope recognized by these antibodies on glucosylated low density lipoprotein was identified as glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine. Each of these antibodies was capable of identifying glucitollysine epitopes on all reduced glucosylated proteins studied, including high density lipoprotein, albumin, hemoglobin, and transferrin. These antibodies were also capable of identifying and quantitating glucitollysine residues on the total plasma proteins and isolated lipoproteins of normal and diabetic individuals after reduction of the proteins with NaBH4. Preliminary data suggest that diabetic total plasma proteins and isolated lipoproteins contain at least threefold more immunochemically detectable glucitollysine residues than nondiabetic plasma proteins and lipoproteins. The technique described in this report should allow production of region-specific antibodies to any immunogenic modification of a protein.

Authors

L K Curtiss, J L Witztum

Glomerular prostaglandin and thromboxane synthesis in rat nephrotoxic serum nephritis. Effects on renal hemodynamics.

• Text
• PDF

Abstract

Glomerular arachidonate cyclooxygenation by isolated rat glomeruli was assessed in vitro in antiglomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis by radioimmunoassay for prostaglandins (PG) and thromboxane. After a single intravenous injection of rabbit anti-rat GBM serum, we observed enhancement of glomerular thromboxane B2 (TxB2) synthesis as early as 2 to 3 h with smaller increments in PGF2 alpha, PGE2 and 6-keto-PGF1 alpha synthetic rates. On day 2 of the disease, the glomerular synthesis of TxB2 and, to a lesser extent, PGF2 alpha and PGE2 remained enhanced, whereas on days 8, 11, and 14, TxB2 was the only prostanoid synthesized at increased rates. Glomerular TxB2 synthesis correlated with the presacrifice 24-h protein excretion. 60 min after intravenous infusion of anti-GMB serum, glomerular filtration rate (GFR) decreased (0.66 +/- 0.04 to 0.44 +/- 0.03 ml/min per 100 g, P less than 0.05), without a significant change in renal plasma flow (RPF): 1.97 +/- 0.23 to 1.80 +/- 0.23 ml/min per 100 g) and without a change in glomerular PG synthetic rates. At 2 h, GFR and RPF reached a nadir (0.25 +/- 0.04 and 1.3 +/- 0.1 ml/min per 100 g, respectively) coinciding with a fivefold increment in glomerular TxB2. By 3 h GFR and RPF partially recovered to 0.43 +/- 0.07 and 1.77 +/- 0.20 ml/min per 100 g, respectively, P less than 0.05, despite further increments in TxB2 synthesis. This recovery of GFR and RPF coincided with increments in vasodilatory PG, (PGE2 and PGI2). The thromboxane synthetase inhibitor OKY-1581 markedly inhibited platelet and glomerular TxB2 synthesis and preserved GFR at 1, 2, and 3 h. Another thromboxane synthetase inhibitor, UK-38485, also completely inhibited platelet and glomerular TxB2 synthesis and prevented decrements of GFR at 2 and 3 h. A cyclooxygenase inhibitor, ibuprofen, inhibited platelet TxB2 and PGE2 synthesis and significantly reduced glomerular PGE2 but not TxB2 synthesis. In the ibuprofen-treated rats, the partial recoveries of GFR and RPF at 3 h were attenuated. The in vitro glomerular TxB2 synthesis correlated inversely with the presacrifice GFR and filtration fraction. These observations indicate that in anti-GBM nephritis there is enhanced synthesis of TxA2 and PG in the glomerulus that mediate changes in renal hemodynamics.

Authors

E A Lianos, G A Andres, M J Dunn

Protoporphyrin hepatopathy. Effects of cholic acid ingestion in murine griseofulvin-induced protoporphyria.

• Text
• PDF

Abstract

Short-term effects of cholic acid ingestion on hepatic accumulation, fecal excretion, and blood levels of protoporphyrin were studied in vivo in griseofulvin-induced protoporphyric mice. Experimental mice that received feed with 2% griseofulvin and 0.5% cholic acid were compared with control mice that received feed with 2% griseofulvin for 4 wk. Five mice from each group were assessed each week for liver and blood porphyrin levels. Fecal protoporphyrin was compared weekly in the total pooled output of each population. Mean protoporphyrin levels were significantly lower for liver (P less than 0.0001), erythrocytes (P less than 0.05), and plasma (P less than 0.05), and higher for feces (P less than 0.001) for the mice that were fed cholic acid. Microscopic protoporphyrin deposits, inflammation, necrosis, and dysplasia were more severe in livers of control mice. A second experimental design compared four regimens in the feed given to all mice after 1-wk induction with 2% griseofulvin: (a) 0.5% cholic acid, (b) no adulterant, (c) 2% griseofulvin and 0.5% cholic acid, and (d) 2% griseofulvin. No difference in protoporphyrin removal from livers of mice in groups 1 and 2 was observed after 1 and 2 wk of these regimens. The apparent reduction in hepatic protoporphyrin content in mice of group 3 as compared with group 4 at weeks 2 and 3 was not significant at P less than 0.05. These data suggest that in selected circumstances, hepatic protoporphyrin secretion may be enhanced in protoporphyric disease states by bile salt supplementation.

Authors

M B Poh-Fitzpatrick, J A Sklar, C Goldsman, J H Lefkowitch

Antireceptor antibodies as probes of insulinlike growth factor receptor structure.

• Text
• PDF

Abstract

Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.

Authors

M Kasuga, N Sasaki, C R Kahn, S P Nissley, M M Rechler

Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles.

• Text
• PDF

Abstract

To determine directly the driving forces for bile acid entry into the hepatocyte, the uptake of [3H]taurocholic acid into rat liver plasma membrane vesicles was studied. The membrane preparation contained predominantly right-side-out vesicles, and was highly enriched in plasma membrane marker enzymes. The uptake of taurocholate at equilibrium was inversely related to medium osmolarity, indicating transport into an osmotically sensitive space. In the presence of an inwardly directed sodium gradient (NaCl or sodium gluconate), the initial rate of uptake was rapid and taurocholate was transiently accumulated at a concentration twice that at equilibrium (overshoot). Other inwardly directed cation gradients (K+, Li+, choline+) or the presence of sodium in the absence of a gradient (Na+ equilibrated) resulted in a slower initial uptake rate and did not sustain an overshoot. Bile acids inhibited sodium-dependent taurocholate uptake, whereas bromsulphthalein inhibited both sodium-dependent and sodium-independent uptake and D-glucose had no effect on uptake. Uptake was temperature dependent, with maximal overshoots occurring at 25 degrees C. Imposition of a proton gradient across the vesicle (pHo less than pHi) in the absence of a sodium gradient failed to enhance taurocholate uptake, indicating that double ion exchange (Na+-H+, OH- -anion) is unlikely. Creation of a negative intravesicular potential by altering accompanying anions or by valinomycin-induced K+-diffusion potentials did not enhance taurocholate uptake, suggesting an electroneutral transport mechanism. The kinetics of taurocholate uptake demonstrated saturability with a Michaelis constant at 52 microM and maximum velocity of 4.5 nmol X mg-1 X protein X min-1. These studies provide definitive evidence for a sodium gradient-dependent, carrier-mediated, electrically neutral transport mechanism for hepatic taurocholate uptake. These findings are consistent with a model for bile secretion in which the basolateral enzyme Na+,K+-ATPase provides the driving force for "uphill" bile acid transport by establishing a trans-membrane sodium gradient.

Authors

M C Duffy, B L Blitzer, J L Boyer

Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis.

• Text
• PDF

Abstract

The T cell proliferative response to autologous non-T cells is termed the autologous mixed lymphocyte reaction (AMLR). Recent studies have suggested that the AMLR represents an inducer circuit for the activation of T8+ suppressor/cytotoxic effector cells. Since atopic dermatitis (AD) patients are deficient in T8+ cytolytic T cell function, we investigated the AMLR in AD. When sheep erythrocytes were used to separate T cells from non-T cells, the AMLR was found to be significantly decreased (P less than 0.001) in AD patients (n = 11; delta cpm = 1,550 +/- 393) when compared with normal control subjects (n = 13; delta cpm = 25,819 +/- 4,609). To exclude the possibility that these results were an artifact of the sheep erythrocyte separation, T cells were also separated on a fluorescence-activated cell sorter after treatment of peripheral blood lymphocytes with the OKT3 monoclonal antibody. AD T cells separated by the latter method were also found to have a significantly reduced AMLR response when compared with similarly treated normal T cells. Co-culture studies using cells from AD patients and their HLA identical siblings indicated that the defect resided at the responder T cell level rather than at the stimulator non-T cell level. Co-culture studies revealed no evidence for excessive suppressor cell activity resulting in the decreased AMLR. However, enumeration of T cells reactive with the monoclonal antibody T29, which recognizes a subset of T cells proliferating in the AMLR, demonstrated that AD patients (n = 8; % T29 = 2.5 +/- 0.7) had a significantly decreased (P less than 0.001) number of circulating T29+ T cells when compared with normal controls (n = 8; % T29 = 10.4 +/- 0.8). These studies suggest that a deficiency of T4+ T29+ cells contributes to the deficient AMLR in AD and possibly underlies the abnormalities of T8+ effector cells present in this disease.

Authors

D Y Leung, J A Saryan, R Frankel, M Lareau, R S Geha

Partial characterization and clinical correlation of circulating human immunoglobulins directed against thyrotrophin binding sites in guinea pig fat cell membranes. Development of a direct enzyme immunoassay.

• Text
• PDF

Abstract

To obviate several problems inherent in indirect thyroid-stimulating hormone (TSH) receptor antibody assays, we developed an enzyme-linked immunosorbent assay (ELISA) that measures antibodies binding to guinea pig fat cell membrane, which contain high concentrations of TSH receptors. Solubilized guinea pig fat cell membranes were adsorbed to plastic microtiter plates and served as the solid-phase antigen. Test sera and affinity-purified alkaline phosphatase-conjugated anti-human IgG were co-incubated with membranes, after which p-nitrophenyl phosphate was added. Results were read when a positive control reached a standard color change (OD405nm). Specificity of this assay was demonstrated by the inability of albumin, insulin, TSH subunits, propranolol, or dexamethasone to block binding 30. normal subjects had a mean OD value of 0.080 +/- 0.050 (SD). 23 of 25 untreated Graves' patients had OD values at least 2 SD above the normal mean (Grave's mean +/- SD; 0.46 +/- 0.33, P less than 0.001) and in each case 10(-6) M TSH inhibited the binding by at least 60%, suggesting that the immunoglobulins were directed at the TSH receptor. Seven of 25 serum samples from patients with Hashimoto's disease, seven of 23 serum samples from patients with transient hyperthyroidism (subacute thyroiditis or painless thyrotoxic thyroiditis), and two of 10 samples from patients with thyroid carcinoma had significant elevations in the titers of membrane-directed immunoglobulins. Graves' patients who were treated with ablative therapy at least 6 mo earlier and who were euthyroid when restudied continued to have abnormally elevated membrane-directed immunoglobulins in six of eight samples studied. Further studies involved the substitution of affinity-purified alkaline phosphatase anti-IgM antisera for the anti-IgG antisera routinely used. Seven of 12 serum samples from patients with Graves' disease had significant elevations in binding which in every instance was inhibited by greater than 60% by 10(-6) M TSH. In sum, the present results indicate that (a) we have developed a sensitive, specific, reproducible, convenient ELISA for the measurement both of the total amount of circulating membrane-directed antibodies and of TSH-displaceable membrane-directed immunoglobulins. (b) This ELISA detected significant elevations in TSH-displaceable guinea pig membrane binding in 23 of 25 untreated Graves' patients as well as in approximately 30% of patients with Hashimoto's thyroiditis and subacute thyroiditis. (c) Elevated membrane directed antibodies may continue to be present many months or years after restoration of the euthyroid state. (d) Circulating membrane binding IgM immunoglobulins have been detected in patients with Graves' disease. Further studies using this ELISA should prove useful in a variety of investigative and clinical studies.

Authors

J R Baker Jr, Y G Lukes, R C Smallridge, M Berger, K D Burman

Acute reduction in human platelet alpha 2-adrenoreceptor affinity for agonist by endogenous and exogenous catecholamines.

• Text
• PDF

Abstract

The binding characteristics of l-epinephrine to intact human platelets were assessed under conditions of physiological and pharmacological variations in plasma catecholamine concentration. In competition with the alpha 2-adrenoreceptor antagonist yohimbine, mean platelet receptor affinity for l-epinephrine was decreased 3.4-fold after 2 h of upright posture and exercise. This change in agonist affinity correlated significantly with the increases in plasma epinephrine and norepinephrine that were stimulated by upright posture and exercise. Supine subjects infused with l-norepinephrine or l-epinephrine for 2 h also averaged a 3.3- and 2.7-fold decrease in platelet alpha 2-adrenoreceptor affinity for agonist with no change in receptor number or antagonist affinity. The alpha 2-adrenoreceptor agonist affinity changes were specific for alpha-agonists since they were blocked by phentolamine, and incubation with 10(-5) M isoproterenol produced no change in alpha 2-adrenoreceptor affinity for l-epinephrine. In vitro exposure of intact human platelets to 10(-6) to 10(-10) M l-epinephrine for 2 h produced a concentration-related decrease in alpha 2-adrenoreceptor affinity for agonist. In all three paradigms, average slope factors approached 1.0 as affinity decreased, which is consistent with a heterogeneous receptor population that becomes more homogeneous after agonist exposure. Incubation of platelet-rich plasma with 10(-6) to 10(-8) M l-epinephrine resulted in a dose- and time-related loss of aggregatory response to l-epinephrine; this demonstrates that agonist affinity changes are correlated with changes in receptor sensitivity. These observations demonstrate that physiological variations in plasma catecholamines acutely modulate the intact human platelet alpha 2-adrenoreceptor's affinity for agonist, and can thereby alter the sensitivity of platelets to alpha 2-adrenergic agonist.

Authors

A S Hollister, G A FitzGerald, J H Nadeau, D Robertson

Killing of intracellular Leishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that interferon-gamma is the activating lymphokine.

• Text
• PDF

Abstract

We have found that the crude lymphokines, which prime the human monocyte-derived macrophage to generate H2O2 and exert microbicidal activity against intracellular Leishmania donovani, are rich in interferon (IFN)-gamma (600-3,000 U/ml). To determine the role of this specific lymphocyte product in macrophage activation, lymphokines were pretreated with a monoclonal antibody that neutralizes human IFN-gamma. Antibody exposure completely abolished the capacity of both mitogen- and antigen-stimulated lymphokines to either enhance macrophage H2O2 release or induce leishmanicidal activity. In addition, partially purified and pure recombinant human IFN-gamma were as effective as crude lymphokines in activating macrophages, and 3 d of treatment with 300 U/ml resulted in a seven- to eightfold increase in H2O2 generation and the intracellular killing of both L. donovani promastigotes and amastigotes. The ability of crude lymphokines to induce monocytes and macrophages from a patient with chronic granulomatous disease to kill L. donovani promastigotes was similarly abrogated by anti-IFN-gamma antibody, and could also be achieved by IFN-gamma alone. These results suggest that IFN-gamma is the key macrophage-activating molecule present within human lymphokines, and indicate that IFN-gamma can enhance both the oxygen-dependent and -independent antiprotozoal mechanisms of human mononuclear phagocytes.

Authors

H W Murray, B Y Rubin, C D Rothermel

Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells.

• Text
• PDF

Abstract

The culture media of three cell lines, a human prostate carcinoma (PC3), a rat Leydig cell tumor (Rice-500), and a rat carcinosarcoma (WRC-256), that were derived from tumors associated with humoral hypercalcemia of malignancy (HHM), were examined for stimulation of adenylate cyclase in ROS 17/2.8 osteoblastic cells and for bone resorptive activity in culture. Cells from a nonhypercalcemic variant of the WRC256 tumor served as control. Extracts from three solid human tumors, a lung adenocarcinoma from a patient with HHM and two adenocarcinoma from normocalcemic patients (lung and colon), were also examined for adenylate cyclase stimulation. We found excellent correlation between stimulation of cyclic AMP accumulation in ROS 17/2.8 cells and bone resorbing activity in culture, or production of HHM in vivo. Stimulation of adenylate cyclase by HHM factors was inhibited by the parathyroid hormone competitive inhibitor, [8norleucyl, 18norleucyl, 34tyrosinyl] bovine parathyroid hormone (3-34) amide.

Authors

S B Rodan, K L Insogna, A M Vignery, A F Stewart, A E Broadus, S M D'Souza, D R Bertolini, G R Mundy, G A Rodan

Specific receptors for leukotriene C4 on a smooth muscle cell line.

• Text
• PDF

Abstract

Specific receptors for leukotriene C4 (LTC4) have been identified on an intact smooth muscle cell line, DDT1 MF-2 cells derived from the Syrian hamster vas deferens. Specific [3H]LTC4 binding at a fixed input at 4 degrees C was rapid, reached a plateau at 86% of total binding at 60 min, and was reversible upon addition of excess homoligand. With incremental inputs of radioligand and a constant cell number, specific [3H]LTC4 binding reached a plateau indicative of saturable binding sites. LIGAND analysis of the Scatchard plot demonstrated a single high affinity binding site with a dissociation constant (Kd) of 5 nM. With incremental inputs of unlabeled LTC4, LIGAND analysis of the Scatchard plot demonstrated a single high affinity site with a Kd of 4.4 nM and in some experiments an additional low affinity site with a Kd of 634 nM. The myotonically active structural analogues of LTC4, 5(R),6(S)-LTC4, 11-trans-LTC4, and C1-monoamide-LTC4, competed effectively with radiolabeled LTC4 such that the relative Kd values of these heteroligands were within one log of that of the homoligand. In contrast, the other native sulfidopeptide leukotrienes, leukotriene D4 and leukotriene E4, exhibited relative Kd values that were 2-3 logs less than that of LTC4. Thus, the high affinity receptor on the DDT1 smooth muscle cell line is specific for a single constituent, LTC4, of slow reacting substance of anaphylaxis.

Authors

S Krilis, R A Lewis, E J Corey, K F Austen