Brave new medicine. Presidential address to the American Society for Clinical Investigation, San Diego, California, 2 May 1987.

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Authors

T P Stossel

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

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The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.

Authors

I Kurtz

In vitro activity of nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked 15-hydroxyprostaglandin dehydrogenases in placentas from normotensive and preeclamptic/eclamptic pregnancies.

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Concentrations of prostaglandins E2 and I2 may be decreased in preeclamptic and eclamptic pregnancies. Because these prostaglandins produce vasodilation and inhibit platelet aggregation it has been suggested that a reduction in their biosynthesis might play an important role in the pathogenesis of the hypertension and coagulation abnormalities associated with preeclampsia. Placental tissue is an extremely rich source of several enzymes that catalyze the catabolism of prostaglandins. The present study was initiated to determine whether one of these catabolic enzymes might be increased in preeclamptic/eclamptic pregnancies. The activities of the NAD- and the NADP-linked 15-hydroxyprostaglandin dehydrogenases were measured in 16 preeclamptics (mean diastolic pressure, 108 +/- 13 mmHg) and compared with 16 normotensive controls matched for age (20.8 +/- 5.43 vs. 20.6 +/- 5.16) and gestational week of delivery (34.6 +/- 5.40 vs. 35.0 +/- 5.06). These results indicate that the activity of the placental NAD-linked 15-hydroxyprostaglandin dehydrogenase is elevated in preeclampsia (40.1 +/- 31.3 vs. 14.9 +/- 8.30 mU/g tissue, P less than 0.01). If this increase were also expressed in vivo, its effect on prostaglandin metabolism could be mistaken for impaired prostacyclin biosynthesis unless both the 6-keto- and 6,15-diketo-metabolites of prostacyclin were measured.

Authors

J Jarabak, J D Watkins, M Lindheimer

Characteristics of prostate-derived growth factors for cells of the osteoblast phenotype.

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We examined the characteristics of mitogens extracted from human benign prostatic hyperplasia and prostatic adenocarcinoma tissue. Although mitogens for fetal rat skin fibroblasts as well as for rat calvarial osteoblasts and osterosarcoma cells were found, distinct entities that acted selectively in cells of the osteoblast phenotype could be obtained by sequential reverse-phase high performance liquid chromatography. Two peptides with apparent molecular weights of 10,000 and 13,000 D were derived from hyperplastic tissue, whereas a single moiety of 10,000 D was obtained from malignant tissue. These entities increased cell numbers and alkaline phosphatase activity in osteoblastlike cells consistent with effects on both growth and differentiation. Prostatic peptides did not stimulate adenylate cyclase in osteosarcoma cells. Mitogenic activity selective for osteoblastlike cells was identified in postpubertal but not prepubertal normal prostate. The results demonstrate the existence of osteoblastic growth factors in prostatic tissue whose presence may accompany postpubertal development.

Authors

M Koutsilieris, S A Rabbani, H P Bennett, D Goltzman

Essential fatty acid deficiency depletes rat glomeruli of resident macrophages and inhibits angiotensin II-induced eicosanoid synthesis.

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Essential fatty acid (EFA) deficiency exerts a beneficial effect on immune-mediated glomerulonephritis, preventing both the tissue injury and consequent mortality. Because both macrophages and eicosanoids are thought to play pathogenic roles in glomerulonephritis, and because macrophages play an important role in modulating arachidonate metabolism at sites of renal injury, the effects of EFA deficiency on the population of resident glomerular macrophages and on glomerular eicosanoid generation were examined. EFA deficiency led to a striking reduction in the number of resident glomerular macrophages and a corresponding reduction in the number of resident glomerular Ia+ cells. This phenomenon was not strain-specific, was not due to a decrease in circulating monocytes, was not a function of changes in cell surface labeling characteristics, and was not restricted to a specific subset of glomeruli. In addition, EFA deficiency affected other areas of the renal cortex: a comparable depletion of interstitial macrophages and Ia+ cells was also observed. In conjunction with the decrease in glomerular macrophages seen with the deficiency state, a marked decrease in both basal and angiotensin II-stimulated glomerular eicosanoid production was noted. In contrast to angiotensin II, platelet-activating factor-induced eicosanoid production was not significantly affected by the deficiency state. These changes in glomerular eicosanoid production could not be attributed to changes in glomerular cyclooxygenase or reacylation capacity. Dietary (n-6) fatty acid supplementation, but not (n-3) fatty acid supplementation, reversed both the decrease in glomerular macrophages and the diminished eicosanoid metabolism seen with the deficiency state. Understanding the mechanisms behind the changes in the glomerular microenvironment induced by EFA deficiency may provide a basis for elucidating the protective effect of dietary fatty acid manipulation on immune-mediated glomerulonephritis.

Authors

J B Lefkowith, G Schreiner

Effect of short-term systemic glucocorticoid treatment on human nasal mediator release after antigen challenge.

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The effect of systemic glucocorticoid treatment on early- and late-phase nasal allergic reactions after allergen challenge was determined in a double-blind, cross-over study in 13 allergic individuals. The subjects were pretreated for 2 d before challenge with 60 mg prednisone per day or a matching placebo. A previously described model using repeated nasal lavages for measuring mediator release in vivo was utilized. Symptom scores obtained repeatedly before, during, and after the challenge and the number and timing of sneezes were recorded. The mediators measured were histamine. N-alpha-p-tosyl-L-arginine methyl ester (TAME)-esterase activity, kinins, PGD2, and LTC4/D4. Albumin was also measured as a marker of plasma transudation. Blood samples were taken for determination of total number of white blood cells, differential count, and total blood histamine content. No effect of steroid therapy was found on the appearance of symptoms or any of the mediators, except a reduction in kinins, in the early phase of the allergic reaction. However, in the late phase, the prednisone reduced the number of sneezes (P less than 0.01), as well as the level of histamine (P less than 0.05), TAME-esterase activity (P less than 0.05), kinins (P less than 0.05), and albumin (P less than 0.05). Only low levels of leukotrienes were found in the late phase, but the quantities of these mediators seemed to be decreased by the glucocorticoid treatment (P = 0.06). PGD2 did not increase during the LPR and thus was not affected by glucocorticosteroids. The immediate response to a second challenge 11 h after the first was also evaluated. Whereas the appearance of mediators was enhanced over the initial response to the same challenge dose in placebo-treated subjects, this enhancement was abrogated after prednisone treatment. As this dose of drug is known to be clinically effective in treating hay fever, the present study confirms the earlier findings of others that short-term systemic glucocorticoid treatment inhibits the late phase but not the immediate phase of antigen challenge. Furthermore, secondary enhancement of immediate responses is inhibited. This study shows that glucocorticoids inhibit the generation or release of inflammatory mediators during the late reaction and the physiologic response.

Authors

U Pipkorn, D Proud, L M Lichtenstein, R P Schleimer, S P Peters, N F Adkinson Jr, A Kagey-Sobotka, P S Norman, R M Naclerio

1,25-Dihydroxyvitamin D3 induces collagen binding to the human monocyte line U937.

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Interactions of cells with components of the extracellular matrix can modulate cellular functions. We measured binding of a major matrix protein to U937 cells, a human promonocytic line. Radioiodinated type I or type III human collagen was bound only to U937 cells differentiated to a more mature phenotype with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Binding was observed at 4 degrees C and was saturable; Scatchard analysis of the binding to 1,25-(OH)2D3-pretreated U937 cells indicated a single class of high-affinity binding sites. Preincubation of U937 cells with interferon gamma did not induce collagen binding. Collagen binding did not appear to be dependent on fibronectin binding. Surface proteins of U937 cells were 125I labeled and cell membrane proteins resolved by affinity chromatography on collagen-Sepharose. Major specifically labeled bands of 180, 155, and 125 kD were identified in membrane fractions from 1,25-(OH)2D3-pretreated U937 cells only. 1,25-(OH)2D3 appears to specifically regulate collagen binding to monocyte precursors.

Authors

B S Polla, A M Healy, M Byrne, S M Krane

Role of the Na+/H+ antiporter in rat proximal tubule bicarbonate absorption.

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Amiloride and the more potent amiloride analog, 5-(N-t-butyl) amiloride (t-butylamiloride), were used to examine the role of the Na+/H+ antiporter in bicarbonate absorption in the in vivo microperfused rat proximal convoluted tubule. Bicarbonate absorption was inhibited 29, 46, and 47% by 0.9 mM or 4.3 mM amiloride, or 1 mM t-butylamiloride, respectively. Sensitivity of the Na+/H+ antiporter to these compounds in vivo was examined using fluorescent measurements of intracellular pH with (2', 7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Amiloride and t-butylamiloride were shown to be as potent against the antiporter in vivo as in brush border membrane vesicles. A model of proximal tubule bicarbonate absorption was used to correct for changes in the luminal profiles for pH and inhibitor concentration, and for changes in luminal flow rate in the various series. We conclude that the majority of apical membrane proton secretion involved in transepithelial bicarbonate absorption is mediated by the Na+-dependent, amiloride-sensitive Na+H+ antiporter. However, a second mechanism of proton secretion contributes significantly to bicarbonate absorption. This mechanism is Na+-independent and amiloride-insensitive.

Authors

P A Preisig, H E Ives, E J Cragoe Jr, R J Alpern, F C Rector Jr

Dietary calcium intake and rates of bone loss in women.

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We measured bone mineral density (BMD) at the midradius and lumbar spine in 106 normal women, ages 23-84 yr (61 were postmenopausal). Three to nine measurements (median, four) were made over 2.6 to 6.6 yr (mean, 4.1 yr). The correlation between calcium intake (range, 260-2,035 mg/d) and rate of change in BMD was not significant at the midradius (r = 0.06) or lumbar spine (r = 0.08), even after adjusting for age, menopausal status, and serum estrogen levels by multiple regression analysis. Women in the lower (mean, 501 mg/d) and in the upper (mean, 1,397 mg/d) quartiles of dietary intake had similar rates of change in BMD (%/yr [mean +/- SE], at midradius, -0.78 +/- 0.24 and -0.91 +/- 0.17 for lower and upper quartiles, respectively; at lumbar spine, -1.06 +/- 0.24 and 0.98 +/- 0.24). These data do not support the hypothesis that insufficient dietary calcium is a major cause of bone loss in women.

Authors

B L Riggs, H W Wahner, L J Melton 3rd, L S Richelson, H L Judd, W M O'Fallon

Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

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The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane.

Authors

L Baud, E J Goetzl, C H Koo

Weight reduction increases adipose tissue lipoprotein lipase responsiveness in obese women.

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Lipoprotein lipase was measured in gluteal adipose tissue from nine obese (90.6 +/- 2.7 kg) women fasting and after the intravenous infusion of insulin and glucose before, immediately after, and 3 mo subsequent to a 14.0 +/- 1.8% (mean +/- SEM) weight reduction. Fasting adipose tissue lipoprotein lipase activity (ATLPL) decreased from 5.3 to 2.3 nEq FFA/10(6) cells per min (P less than 0.02) immediately after weight reduction, yet after weight maintenance, higher levels were again found (6.1 nEq FFA/10(6) cells per min). Although responsiveness of ATLPL to 40 mU/m2 per min of insulin infusion over 6 h was absent before weight loss, increases were seen immediately after weight loss (delta 0.8, P = 0.05) and more so (delta 7.7, P less than 0.01) after 3 mo. Moreover, whereas before weight loss the ATLPL response to ingested mixed meals (delta 0.9) was minimal, in the maintained reduced-obese state a marked increase was seen (delta 12.6, P = 0.02). Thus, because ATLPL is important to lipid filling in adipose tissue, the maintenance of high levels of fasting ATLPL and the increase in enzyme responsiveness in the reduced-obese state could play an important role in the resumption of the obese state, which so commonly follows weight reduction.

Authors

R H Eckel, T J Yost

Defective antigen presentation and novel structural properties of DR1 from an HLA haplotype associated with 21-hydroxylase deficiency.

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We have segregated DR1+ individuals into two categories according to whether or not their class II+ cells stimulated T lymphocyte clones specific for or restricted to DR1. In a majority of cases (87%), failure to stimulate was a property of cells having the B14;DR1 haplotype and/or nonclassical 21-hydroxylase deficiency. Absence of clonal proliferation could not be explained by release of an intercellular suppressor factor or by stimulator cell absorption of interleukin 2. Homozygous cells inheriting both stimulatory (DR1n) and nonstimulatory (DR1x) haplotypes did not successfully mediate clonal expansion, implying that a trans acting factor operates intracellularly to modify both DR1 alleles or their products. Other DR alleles did not appear to be affected as evidence by normal proliferative responses of T lymphocyte clones restricted to DR2 or DR7 and stimulated by DR1x,2 and DR1x,7 cells, respectively. By two-dimensional gel analysis, we have further identified a 50-kD surface glycoprotein contained in anti-DR immunoprecipitates of DR1x, but not DR1n or non-DR1 cellular lysates. This 50-kD structure had antigenic and peptide identity to DR alpha and beta chains but was resistant to dissociation under conditions that normally separate DR alpha and beta (8 M urea plus 5% 2-mercaptoethanol); boiling in sodium dodecyl sulfate was required to segregate the component polypeptides of the 50-kD heterodimer. We postulate that a product of a novel combinatorial association between constitutive chains of DR may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and a restricting element. We further propose that gene abnormalities within the class III region of a haplotype associated with nonclassical 21-hydroxylase deficiency may extend into the DR subregion of the major histocompatibility complex with consequent aberrations in DR1 presentation.

Authors

J E Davis, R R Rich, M Van, H V Le, M S Pollack, R G Cook

Recombinant interferon gamma augments phagocyte superoxide production and X-chronic granulomatous disease gene expression in X-linked variant chronic granulomatous disease.

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We examined the potential of interferon gamma (IFN-gamma) to ameliorate the physiologic defect of chronic granulomatous disease (CGD) by studying its effects on CGD phagocyte superoxide generation, NADPH oxidase kinetics, cytochrome b559 content, and expression of X-CGD (the gene for the X-linked disease). Granulocytes and macrophages from three patients in two kindreds with "variant" X-linked CGD (i.e., with very low, but detectable, baseline superoxide-generating activity) responded to IFN-gamma with enhanced nitroblue tetrazolium reduction and two- to eightfold increases in superoxide generation. IFN-gamma did not augment the respiratory burst activity of phagocytes from patients with "classic" CGD (i.e., no detectable baseline superoxide generation) or autosomal variant CGD. Incubation of a responding patient's granulocytes with IFN-gamma nearly doubled the maximal velocity for the NADPH oxidase, but did not change its abnormal Michaelis constant. Although the interferon-treated CGD granulocytes produced superoxide at a rate 40% of normal, the cytochrome b spectrum remained undetectable. IFN-gamma treatment of cultured monocytes from an IFN-gamma-responsive CGD patient increased the steady state level of RNA transcripts from the X-CGD gene from barely detectable up to approximately 5% of normal.

Authors

R A Ezekowitz, S H Orkin, P E Newburger

Purification of a human liver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats.

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Among characterized forms of liver microsomal cytochromes P-450 in rats are four related isozymes (P-450f-i) notable for their lack of inducibility. Immunoblot analyses demonstrated that human livers microsomes contained several proteins related to these rat P-450s. A human liver P-450, termed HLx, was purified and found by immunochemical assays to resemble rat P-450g. Analysis of the NH2-terminal amino acid sequence of HLx indicates that it is related to rat P-450s f-i and human liver P-450MP. A monoclonal antibody was used to measure the amounts of HLx in 21 human liver specimens. No correlation between the levels of HLx protein in these specimens and the patients' environmental histories was observed. However, statistical analysis of the data suggests that the distribution of HLx is at least bimodal. We conclude that HLx is a member of a family of human liver P-450s that resembles in its structure, and possibly in its distribution, several liver P-450s found in other animals.

Authors

S A Wrighton, P E Thomas, P Willis, S L Maines, P B Watkins, W Levin, P S Guzelian

Hemophilia B (factor IXSeattle 2) due to a single nucleotide deletion in the gene for factor IX.

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To understand the molecular basis for hemophilia B in patients with little or no circulating Factor IX antigen, a patient who had less than 0.2% circulating Factor IX antigen (Factor IXSeattle 2) was selected for analysis of his Factor IX gene. Genomic DNA fragments from the abnormal gene were cloned into bacteriophage lambda vectors and recombinant phage were identified using radiolabeled genomic probes obtained from the normal Factor IX gene. The exons and flanking regions of the abnormal gene were sequenced by the dideoxy chain-termination method and this sequence was compared with that of the normal gene. Only one significant difference was observed, the deletion of a single adenine nucleotide in exon V. This resulted in a frameshift that converted an aspartic acid at position 85 in the protein to a valine and the formation of a stop signal at position 86. These data indicate that the gene for Factor IXSeattle 2 codes for an 85 residue polypeptide that terminates after the first epidermal growth factor domain. Thus, the putative Factor IXSeattle 2 polypeptide lacks the second epidermal growth factor domain, the activation peptide, and the catalytic domain present in the normal protein. This provides an explanation for the coagulation disorder in this patient and represents the first report of a single nucleotide deletion and frameshift resulting in hemophilia B.

Authors

B G Schach, S Yoshitake, E W Davie

Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man.

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We used monoclonal antibodies and complementary DNAs (cDNAs) to glucocorticoid-inducible liver cytochromes P-450 in rats (P-450p) and in man (HLp) to search for related cytochromes in intestinal mucosa. In rat enterocytes, we found two dexamethasone-inducible proteins related to the steroid-inducible liver cytochromes P-450. Induction of these proteins in enterocytes was associated with increases in the amount of a P-450p-related messenger RNA and of erythromycin demethylase, an activity highly characteristic of P-450p and HLp. Similar studies on human jejunal enterocytes revealed a microsomal protein indistinguishable from HLp on immunoblots and an abundance of RNA hybridizing with HLp cDNA. In human enterocytes the specific concentration of the HLp-related cytochrome (measured immunochemically or as erythromycin demethylase activity) was similar to that found in human liver and could account for all of the CO-binding hemo-protein detected. We conclude that the intestinal mucosa contains prominent form(s) of cytochromes P-450 similar to liver cytochrome P-450p in their structure, function, and some regulatory characteristics.

Authors

P B Watkins, S A Wrighton, E G Schuetz, D T Molowa, P S Guzelian

Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats.

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We have examined the effect of chronic (4 wk) hyperglycemia on insulin secretion in vivo in an awake, unstressed rat model. Three groups of animals were examined: control, partial (90%) pancreatectomy, and partial pancreatectomy plus phlorizin, in order to normalize plasma glucose levels. Insulin secretion in response to arginine (2 mM), hyperglycemia (+100 mg/dl), and arginine plus hyperglycemia was evaluated. In diabetic compared with control animals three specific alterations were observed: (a) a deficient insulin response, in both first and second phases, to hyperglycemia; (b) an augmented insulin response to the potentiating effect of arginine under basal glycemic conditions; and (c) an inability of hyperglycemia to augment the potentiating effect of arginine above that observed under basal glycemic conditions. Normalization of the plasma glucose profile by phlorizin treatment in diabetic rats completely corrected all three beta cell abnormalities. These results indicate that chronic hyperglycemia can lead to a defect in in vivo insulin secretion which is reversible when normoglycemia is restored.

Authors

L Rossetti, G I Shulman, W Zawalich, R A DeFronzo

Effects of angiotensin-converting enzyme inhibition on altered renal hemodynamics induced by low protein diet in the rat.

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We assessed the role of angiotensin II in mediating the alterations in renal hemodynamics known to result from low protein feeding to normal rats by examining the effect of the angiotensin-converting enzyme (ACE) inhibitor captopril. 2 wk of low protein (6% casein) diet resulted in decreased glomerular filtration rate (normal protein [NP], 1.82 +/- 0.17 vs. low protein [LP], 0.76 +/- 0.01 ml/min; P less than 0.05) and renal plasma flow (NP, 6.7 +/- 0.2 vs. LP, 3.3 +/- 0.3 ml/min; P less than 0.05); renal vascular resistance rose (NP, 8.7 +/- 0.4 vs. LP, 19.8 +/- 1.4 dyn . s per cm5; P less than 0.05). These changes were accompanied by a significant decrease in plasma renin activity (NP, 7.0 +/- 0.7 vs. LP, 4.4 +/- 0.8 ng A I/ml per h; P less than 0.05), plasma aldosterone concentration (NP, 7.0 +/- 0.6 vs. LP, 4.1 +/- 0.7 ng/dl; P less than 0.05), and urinary PGE2 excretion (NP, 3,120 +/- 511 vs. LP, 648 +/- 95 pg/mgCr; P less than 0.05); by contrast renal renin content was significantly increased (NP, 2,587 +/- 273 vs. LP, 7,032 +/- 654 ng A I/mg protein; P less than 0.05). Treatment with captopril (30 mg/kg per d) raised glomerular filtration rate (GFR; LP + capt, 1.6 +/- 0.2 ml/min) and renal plasma flow (RPF; LP + capt, 6.7 +/- 0.7 ml/min), and reduced renal vascular resistance (LP + capt, 9.2 +/- 0.5 dyn/s per cm5) in low protein-fed animals. These values were not different from those measured in untreated and captopril-treated rats fed a normal (23%) protein diet. There were no changes in systemic mean arterial pressure in any group of rats. These data provide evidence that intrarenal angiotensin II mediates the changes in intrarenal hemodynamics induced by protein deprivation. The effects of low protein feeding may be partly potentiated by the reduction in PGE2 synthesis. However, the normalization of GFR and RPF in view of only modest increases in PGE2 excretion after captopril (LP, 648 +/- 95 vs. LP + capt, 1,131 +/- 82 pg/mgCr; P less than 0.05) suggests that if PGE2 is involved in these changes, it plays a permissive but not essential role in the increased renovascular resistance.

Authors

E Fernández-Repollet, E Tapia, M Martínez-Maldonado

Body composition, not body weight, is related to cardiovascular disease risk factors and sex hormone levels in men.

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To clarify the independent relationships of obesity and overweight to cardiovascular disease risk factors and sex steroid levels, three age-matched groups of men were studied: (i) 8 normal weight men, less than 15% body fat, by hydrostatic weighing; (ii) 16 overweight, obese men, greater than 25% body fat and 135-160% of ideal body weight (IBW); and (iii) 8 overweight, lean men, 135-160% IBW, but less than 15% fat. Diastolic blood pressure was significantly greater for the obese (mean +/- SEM, 82 +/- 2 mmHg) than the normal (71 +/- 2) and overweight lean (72 +/- 2) groups, as were low density lipoprotein levels (131 +/- 9 vs. 98 + 11 and 98 + 14 mg/dl), the ratio of high density lipoprotein to total cholesterol (0.207 +/- 0.01 vs. 0.308 +/- 0.03 and 0.302 +/- 0.03), fasting plasma insulin (22 +/- 3 vs. 12 +/- 1 and 13 +/- 2 microU/ml), and the estradiol/testosterone ratio (0.076 +/- 0.01 vs. 0.042 +/- 0.02 and 0.052 +/- 0.02); P less than 0.05. Estradiol was 25% greater for the overweight lean group (40 +/- 5 pg/ml) than the obese (30 +/- 3 pg/ml) and normal groups (29 +/- 2 pg/ml), P = 0.08, whereas total testosterone was significantly lower in the obese (499 +/- 33 ng/dl) compared with the normal and overweight, lean groups (759 +/- 98 and 797 +/- 82 ng/dl). Estradiol was uncorrelated with risk factors and the estradiol/testosterone ratio appeared to be a function of the reduced testosterone levels in obesity, not independently correlated with lipid levels after adjustment for body fat content. Furthermore, no risk factors were significantly different between the normal and overweight lean groups. We conclude that (a) body composition, rather than body weight per se, is associated with increased cardiovascular disease risk factors; and (b) sex steroid alterations are related to body composition and are not an independent cardiovascular disease risk factor.

Authors

K R Segal, A Dunaif, B Gutin, J Albu, A Nyman, F X Pi-Sunyer

Retrovirus-induced feline pure red cell aplasia. Hematopoietic progenitors are infected with feline leukemia virus and erythroid burst-forming cells are uniquely sensitive to heterologous complement.

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Feline leukemia virus subgroup C/Sarma (FeLV-C) induces pure red cell aplasia (PRCA) in cats. Just before the onset of anemia, erythroid colony-forming cells (CFU-E) become undetectable in marrow culture, yet normal frequencies of erythroid burst-forming cells (BFU-E)- and granulocyte-macrophage colony-forming cells (CFU-GM) persist. To determine if erythroid progenitors were uniquely infected with retrovirus, marrow mononuclear cells from cats viremic with FeLV-C were labeled with monoclonal antibodies to gp70 and then analyzed with a fluorescence-activated cell sorter. Both erythroid and granulocyte-macrophage progenitors were among cells sorting positively, suggesting that infection of BFU-E alone did not result in PRCA. The results were confirmed by complement (C') lysis studies using baby rabbit or guinea pig sera as sources of C'. These studies also suggested that BFU-E from cats with PRCA were unusually sensitive to C' alone, without the addition of antibody. In further studies, we demonstrated that C' activation was via the classical pathway and that C' sensitivity was unique to BFU-E and not a property of CFU-E, CFU-GM, or progenitors that were capable of giving rise to BFU-E in suspension culture. As BFU-E from cats viremic with FeLV-A/Glasgow-1 or the Rickard strain of feline leukemia virus were not sensitive to C', this finding may relate to the pathogenesis of feline PRCA. We hypothesize that, in cats viremic with FeLV-C, the abnormal C' sensitivity of BFU-E leads to the absence of CFU-E and anemia.

Authors

J L Abkowitz, R D Holly, C K Grant

Regulation of gastric acid secretion by neurotensin in man. Evidence against a hormonal role.

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The present study was designed to evaluate neurotensin as a hormonal regulator of gastric acid secretion in man. After a fat-rich meal, the strongest known stimulus of neurotensin release, plasma neurotensin-like immunoreactivity (NTLI) was elevated from 7.6 +/- 1.9 to 15.5 +/- 2.5 pM. Plasma NTLI was measured with antiserum L170, which requires the biologically active carboxyl-terminal hexapeptide of the neurotensin molecule for recognition and does not crossreact significantly with any known natural catabolite in human plasma. Intravenous infusion of neurotensin at 25 pmol X kg-1 h-1 resulted in a plasma level of 14.7 +/- 2.1 pM, similar to the maximal physiological level observed after the fat-rich meal. Intravenous infusion of neurotensin at 25 pmol X kg-1 h-1 during 2 h, however, failed to significantly inhibit peptone meal-stimulated gastric acid secretion measured by intragastric titration. The 2-h acid output to peptone was 40.8 +/- 6.2 and 41.3 +/- 6.9 mmol during the vehicle and the neurotensin infusion, respectively. Intravenous infusion of neurotensin at 100 or 400 pmol X kg-1 h-1 did not affect acid output, whereas at 1,600 pmol X kg-1 h-1, which resulted in a plasma neurotensin concentration of 725 +/- 80 pM, significantly reduced peptone meal-stimulated gastric acid secretion. The neurotensin-induced inhibition of acid output was independent of the hormone gastrin. The present results provide evidence against a hormonal role for neurotensin in the regulation of meal-stimulated gastric acid secretion in man.

Authors

M H Mogard, V Maxwell, B Sytnik, J H Walsh

Role of sodium in thyroid hormone uptake by rat skeletal muscle.

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Abstract

Whether Na+ movement through the plasma membrane plays a role in thyroid hormone uptake was investigated in intact rat soleus muscles. After preincubation for 120 min at 37 degrees C in modified Krebs-Ringer bicarbonate containing 140 or 5 mM Na+ plus choline or lithium to maintain osmolarity, muscles were incubated with 50 pM [125I]triiodo-L-thyronine (T3) or [125I]L-thyroxine (T4) for 60 min. T3 uptake was decreased when extracellular Na+ was replaced by either choline or lithium, the amount of decrease corresponding to the specific (or saturable) uptake component. Monensin, an ionophore that stimulates Na+ entry, increased T3 uptake at 140 mM Na+ but not at 5 mM Na+. Amiloride, a Na+/H+ exchange inhibitor, had no effect on T3 uptake under basal conditions or when Na+ was replaced by choline, but reversed the action of lithium. Ouabain, an inhibitor of Na+/K+ ATPase, reduced specific T3 uptake. T4 uptake was unaffected by low extracellular Na+. These results are consistent with a major role of Na+ movement in T3 uptake by skeletal muscle, but not in T4 uptake, and suggest an involvement of membrane pumps in this process.

Authors

M Centanni, J Robbins

Long-term effect of insulin on glucose transport and insulin binding in cultured adipocytes from normal and obese humans with and without non-insulin-dependent diabetes.

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Abstract

We have tested the hypothesis that in vitro exposure of insulin-resistant adipocytes with insulin results in improved insulin action. A primary culture system of adipocytes from obese subjects with or without non-insulin-dependent diabetes mellitus (NIDDM) and nonobese control subjects has been developed. The adipocytes when cultured in serum-free medium do not lose their original characteristics in regard to insulin binding and glucose transport. The adipocytes from three groups were incubated with insulin (0, 10(-10) M, and 10(-7) M) for 24 h at 37 degrees C, receptor-bound insulin was dissociated, and basal and insulin (1 X 10(-11)-10(-7) M)-stimulated glucose transport and 125I-insulin binding were determined. The 24-h insulin exposure of adipocytes from control subjects decreased basal and insulin-stimulated glucose transport. The effects of 1 X 10(-7) M insulin were more pronounced than 1 X 10(-10) M insulin. Similarly, insulin exposure decreased insulin sensitivity and responsiveness of cultured adipocytes from obese and NIDDM patients. The insulin-induced reduction in insulin sensitivity and responsiveness for glucose transport in three groups were due to alterations at insulin binding and postbinding levels. In conclusion, insulin induces insulin resistance in control adipocytes and further worsens the insulin resistance of adipocytes from obese and NIDDM subjects. For insulin to improve the insulin resistance of adipocytes from NIDDM patients, either more prolonged in vitro insulin exposure and/or other hormonal factors might be required.

Authors

M K Sinha, L G Taylor, W J Pories, E G Flickinger, D Meelheim, S Atkinson, N S Sehgal, J F Caro

Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells.

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Abstract

The extracellular matrix secreted by cultured bovine smooth muscle cells (BSMC) contains an endothelial type plasminogen activator (PA) inhibitor. When PA is incubated with the matrix, a high molecular weight complex containing a truncated PA inhibitor is released into the supernatant. The inhibitor also dissociates from the matrix by treatment with glycine, pH 2.7, in its intact, functionally active, 45-kD form, whereas treatment of the matrix with thrombin results in the release of a cleaved, inactive, 41 kD PA inhibitor. Bowes melanoma cells but not smooth muscle cells cultured on BSMC matrices decrease available matrix associated PA inhibitor. PA inhibitor incorporated into the extracellular matrix may serve an important role in the regulation of plasminogen activator mediated matrix degradation.

Authors

B S Knudsen, P C Harpel, R L Nachman

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

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Abstract

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production.

Authors

J H Jackson, I U Schraufstatter, P A Hyslop, K Vosbeck, R Sauerheber, S A Weitzman, C G Cochrane

Effect of fetal adrenalectomy on catecholamine release and physiologic adaptation at birth in sheep.

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Abstract

Plasma catecholamine levels increase dramatically at birth. To determine the contribution of adrenal catecholamine secretion to the surge in catecholamines at birth and the role in newborn adaptation, we performed surgical adrenalectomy or sham operation on near-term ovine fetuses. After recovery in utero, the animals were delivered and supported by mechanical ventilation. Plasma catecholamine levels, heart rate, blood pressure, cardiac output, pulmonary function, surfactant secretion, and release of free fatty acids (FFA) and glucose were compared in control and adrenalectomized animals. Plasma epinephrine increased rapidly at birth in controls but was undetectable in adrenalectomized animals. Norepinephrine levels were not statistically different. Heart rate, blood pressure, cardiac output and contractility increased abruptly after cord cutting in controls but did not increase in adrenalectomized animals. Lung compliance, pulmonary function, surfactant pool size, glucose and FFA levels were significantly decreased in adrenalectomized animals. These results suggest that adrenal epinephrine secretion is vital to many of the adaptive events at birth.

Authors

J Padbury, Y Agata, J Ludlow, M Ikegami, B Baylen, J Humme

Effects of polymorphonuclear leukocyte transmigration on the barrier function of cultured intestinal epithelial monolayers.

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Abstract

We describe a model to study the effects of polymorphonuclear leukocyte (PMN) transmigration on the intestinal epithelial barrier. Human PMN were induced to transmigrate across high resistance monolayers of a cultured human intestinal epithelial cell line (T84 cells) by chemotactic gradients produced by formyl methionyl leucyl phenylalanine (FMLP). With maximal transmigration monolayer resistance decreased by 48 +/- 12.6% in 15 min and by 83 +/- 1.6% in 60 min. This response was dependent on the size of the FMLP gradient and the density of PMN transmigration. The decrease in resistance correlated with number of PMN migrating across monolayers, and was accompanied by increases in flux of paracellular tracers. Macromolecular tracer studies localized the leak sites to foci at which PMN impaled the epithelium. Removal of the chemotactic gradient led to restoration of baseline resistance within 18 h. PMN transmigration across intestinal epithelial monolayers occurs via intercellular occluding junctions and may be associated with a reversible increase in epithelial permeability.

Authors

S Nash, J Stafford, J L Madara

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage.

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Abstract

Human alveolar macrophages release chemotactic activity for neutrophils, providing a role for alveolar macrophages in regulating inflammation in the lung. As alveolar macrophages produce large amounts of leukotriene B4 (LTB4), a chemotactically active lipoxygenase product of arachidonic acid, we investigated the contribution of LTB4 to the total neutrophil chemotactic activity produced by these cells. Normal human alveolar macrophages were recovered by bronchoalveolar lavage from healthy volunteers and incubated either with the calcium ionophore A23187 for 1 h, or with opsonized zymosan particles or latex beads for 3 h. Nordihydroguaretic acid (NDGA), a relatively specific lipoxygenase inhibitor, blocked the release of neutrophil chemotactic activity after all three stimuli in a dose-dependent manner. This correlated with blockade of LTB4 production as measured by high performance liquid chromatography using freshly isolated alveolar macrophages, as well as blockade of [3H]LTB4 production by macrophages prelabeled with [3H]arachidonate. Molecular sieve chromatography using Sephadex G-50 confirmed that essentially all of the chemotactic activity in the stimulated macrophage supernatants co-eluted with authentic [3H]LTB4, and that NDGA completely blocked the chemotactic activity in the eluting fractions. Readdition of authentic LTB4 (1 X 10(-7) M) to the NDGA-blocked macrophage supernatants restored the chemotactic activity in the supernatants. The macrophage supernatants did not contain platelet-activating factor-like activity, as measured by the stimulation of [3H]serotonin release from rabbit platelets, and by high performance liquid chromatography. NDGA did not change the protein-secretion profiles of fresh alveolar macrophages, or of macrophages prelabeled with [35S]methionine. The complement (C) components C5adesarg were not detected in any of the supernatants by radioimmunoassay. Concentration of the supernatants by positive pressure filtration (5,000-D membrane) did not augment chemotactic activity in the stimulated supernatants or uncover chemotactic activity in the NDGA-blocked supernatants. As with the 3-h studies, when alveolar macrophages were incubated overnight with opsonized zymosan, all of the increase in chemotactic activity could also be blocked by NDGA. These data indicate that LTB4 is the predominant neutrophil chemotactic factor secreted by the normal resident human alveolar macrophage in response to two major types of stimuli, calcium fluxes across the cell membrane and the phagocytosis of opsonized particulates.

Authors

T R Martin, G Raugi, T L Merritt, W R Henderson Jr

Demonstration of 2-hydroxybenzoylglycine as a drug binding inhibitor in newborn infants.

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Abstract

Newborn infants have drug binding defects that share similarities to those of uremic subjects. Since 2-hydroxybenzoylglycine has been chemically defined to be a major drug binding inhibitor in uremia, a search for the presence of a similar compound in the sera of newborn infants was made. An organic substance that has the characteristics of 2-hydroxybenzoylglycine as supported by the retardation factor values on thin-layer chromatograms, retention times of high performance liquid chromatograms, fluorescence emission spectra, and mass spectrum has been demonstrated to be present in the majority of the neonatal sera studied. A strong positive correlation between the levels of the binding inhibitor and the extent of binding defects for nafcillin has been observed. The substance could effectively reduce the total bilirubin concentration when added to the cord sera specimens. It is concluded that 2-hydroxybenzoylglycine plays an important role in drug binding defects observed in the newborn, and the inhibitor may also play a part in the precipitation of bilirubin-induced neurotoxicity in neonates when the substance is abnormally elevated.

Authors

B Suh, S J Wadsworth, D M Lichtenwalner

Metabolism and placental transfer of 125I-proinsulin and 125I-tyrosylated C-peptide in the pregnant rhesus monkey.

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Abstract

125I-Proinsulin or 125I-tyrosylated-C-peptide (125I-tyr-CP) was administered to pregnant Rhesus monkeys by bolus followed by constant infusion to examine placental transfer of these peptides. At the end of each infusion, fetuses were exsanguinated in situ via the umbilical vein. The bolus-constant infusion technique produced a steady state in maternal plasma of immunoprecipitable label, measured using excess insulin or C-peptide antiserum. In animals infused with 125I-proinsulin, analysis of umbilical venous plasma revealed no apparent transfer to the fetus of immunoprecipitable label. In animals infused with 125I-tyr-CP, 3-13% of the umbilical venous plasma radioactivity was immunoprecipitable, representing 1.4-5.8% of the immunoprecipitable radioactivity in maternal plasma at delivery. Gel filtration chromatography of umbilical venous plasma revealed that the immunoprecipitated moiety was a fragment of 125I-tyr-CP. Analysis of maternal plasma showed that the predominant peak of radioactivity represented intact C-peptide. A peak corresponding to the fetal immunoprecipitable peak was also present. Analysis of simultaneous maternal arterial and uterine vein plasma samples showed that degradation of 125I-tyr-CP occurred across the uterus. Studies in one nonpregnant and three postpartum animals indicated that pregnancy increased the rate of metabolism of 125I-tyr-CP. When 125I-tyr-CP was incubated with trophoblastic cells in culture, degradation to a species corresponding on gel filtration to the immunoprecipitable fetal metabolite was found. We conclude that proinsulin, like insulin, does not traverse the placenta. Immunoreactive fragments of C-peptide do cross, however, and pregnancy alters the metabolism of 125I-tyr-CP, probably owing to placental degradation.

Authors

P A Gruppuso, J B Susa, P Sehgal, B Frank, R Schwartz

Gray platelet syndrome. Demonstration of alpha granule membranes that can fuse with the cell surface.

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Abstract

Platelets from patients with the gray platelet syndrome have decreased recognizable alpha granules and are markedly deficient in some alpha-granule secretory proteins. Using immunocytochemical techniques with antibodies to an alpha-granule membrane protein, GMP-140, we identified the membranes of intracellular vesicles in gray platelets as alpha-granule membranes. Gray platelets contained normal amounts of GMP-140 as measured by electroimmunoassay. The activation of gray platelets with thrombin caused GMP-140 to be redistributed to the plasma membrane surface, as in normal platelets. In agreement with previous studies, an endogenously synthesized secretory protein, platelet factor 4, was undetectable in gray platelets. However, the alpha-granule proteins albumin and IgG, which are thought to be derived from endocytosis of plasma proteins into megakaryocytes, were present in substantial quantities and were secreted efficiently from gray platelets. Therefore, the fundamental defect in the gray platelet syndrome may be in the targeting of endogenously synthesized secretory proteins to developing alpha granules in megakaryocytes.

Authors

J P Rosa, J N George, D F Bainton, A T Nurden, J P Caen, R P McEver

Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.

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Abstract

We recently reported that in healthy men, changes in the production rate (PR) of 1,25-dihydroxyvitamin D [1,25-(OH)2D] accounted for the 80% increase and the 30% decrease in its serum concentration that was induced by restriction and supplementation, respectively, of dietary phosphorus. These changes in PR and serum concentration of 1,25-(OH)2D could be mediated by changes in serum concentrations of phosphorus that occur after the morning fasting period. To examine this hypothesis, we measured serum concentrations of phosphorus in blood drawn at hourly intervals for 24 h in six healthy men in whom dietary phosphorus was initially maintained at 1,500 mg/70 kg body weight per day for 9 d, then restricted to 500 mg/d (coupled with orally administered aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. When dietary phosphorus was normal, the serum concentration of phosphorus exhibited the normal circadian rhythm: a rapid decrease in early morning to a nadir at 1100, followed by an increase to plateau at 1600 h and a further increase to an acrophase (peak) at 0030 h. The variation in serum levels of phosphorus can be described as the sum of sinusoidal functions with periodicities of 24 and 12 h. Phosphorus restriction for 10 d induced a 40% reduction in the 24-h mean serum level of phosphorus, abolished the early afternoon rise in its serum level (i.e., the 12-h periodic component of the time series), and delayed the acrophase by 3 h to 0330 h. Phosphorus supplementation for 10 d induced a 14% increase in the 24-h mean serum level of phosphorus but no significant change in its morning fasting level, exaggerated the early afternoon rise in serum phosphorus, and advanced the acrophase by 9 h to 1530 h. The changes in the PR of 1,25-(OH)2D induced by restriction and supplementation of dietary phosphorus varied inversely and significantly with those induced in the 24-h mean serum level of phosphorus (R = -0.88, P less than 0.001). These data demonstrate that in healthy men, dietary phosphorus is an important determinant of the serum concentration of phosphorus throughout most of the day. The data suggest that diet-induced changes in serum levels of phosphorus mediate the changes in PR and serum concentration of 1,25(OH)2D.

Authors

A A Portale, B P Halloran, R C Morris Jr

Analysis of HLA-D micropolymorphism by a simple procedure: RNA oligonucleotide hybridization.

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Abstract

Recent progress in the molecular genetics of HLA class II antigens has revealed the existence of multiple loci and of a large degree of polymorphism, with more individual alleles than was expected. An accurate detection and analysis of this extensive polymorphism is essential for optimal HLA typing for transplantation and for a reevaluation of HLA-disease association. Because of the limitations of the current typing methods, including restriction fragment length polymorphisms, we have proposed a DNA typing procedure based on hybridization with loci- and allele-specific oligonucleotides. Here we present a much simpler way of analyzing class II micropolymorphism down to the level of single nucleotide differences. RNA oligonucleotide typing (ROT) relies on RNA dot blots and requires 10-20 ml of blood. It is shown that with appropriate oligonucleotide probes, ROT can reliably and unambiguously identify any polymorphism at any of the HLA loci, including new alleles, not identified with previous methods. This illustrates the importance of oligonucleotide typing to optimize HLA matching, in particular for transplantation involving unrelated donors.

Authors

C Ucla, J J van Rood, J Gorski, B Mach

Effect of adrenalectomy on transport in the rat medullary thick ascending limb.

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Abstract

Previous studies in adrenalectomized (Adx) rats suggest that aldosterone may regulate ion transport in the ascending portion of Helen's loop. In order to examine directly the effect of adrenalectomy on transport, medullary thick ascending limb (Mtal) segments were isolated from Adx, Adx replaced with aldosterone (Adx + Ald, 0.5 micrograms X 100 g X body wt X d), and control Sprague-Dawley rats. Both net sodium and net chloride fluxes were significantly less in the Mtal segments from Adx rats compared with those in the control or Adx + Ald group. Physiologic levels of exogenous aldosterone increased net sodium chloride flux toward control values in the Adx + Ald group. Net potassium flux was not different among the three groups. We conclude that adrenalectomy impairs reabsorptive NaCl but not K transport in the Mtal, and that aldosterone restores this process. This reabsorptive defect may contribute to the urinary concentrating and diluting abnormality associated with adrenal insufficiency.

Authors

J Work, R L Jamison

Myocardial energy production and consumption remain balanced during positive inotropic stimulation when coronary flow is restricted to basal rates in rabbit heart.

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Abstract

The effect on myocardial energy balance of increasing oxygen demand without altering basal myocardial perfusion rate was assessed in isolated, isovolumic, retrograde blood perfused rabbit hearts. Myocardial energy requirements were increased with paired stimulation. The capacity of rapid paired stimulation to increase mechanical energy consumption was demonstrated in the presence of increased perfusion with the rate X pressure product and oxygen consumption increasing 86 and 148%, respectively, compared with control values. In contrast, rapid paired stimulation under constant, basal flow conditions did not alter the rate X pressure product, while oxygen extraction and consumption increased only 40% relative to control. Myocardial ATP, creatine-phosphate, and lactate content were identical under control and constant flow-paired stimulation conditions. The results of this study indicate that no detectable energy imbalance was produced by rapid paired stimulation with flow held constant at basal rates. These results suggest that the myocardium does not increase mechanical energy expenditure in response to inotropic or rate stimulation in the presence of restricted flow reserve and are inconsistent with the concept of "demand-induced" or "relative" myocardial ischemia.

Authors

R C Marshall, W W Nash, M M Bersohn, G A Wong

Transient elevation of messenger RNA encoding gastrin-releasing peptide, a putative pulmonary growth factor in human fetal lung.

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Abstract

Gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Previously we have reported the cloning of the messenger RNAs (mRNAs) and gene that encode human GRP. We now report that GRP mRNAs are markedly elevated in human fetal lung during the canalicular phase of pulmonary development (from approximately 16 to 30 wk gestation). By RNA blot and in situ hybridization analyses, GRP mRNAs were first detectable in fetal lung at 9-10 wk, plateaued at levels 25-fold higher than in adult lungs from 16 to approximately 30 wk and then declined to near adult levels by 34 wk gestation. By contrast, GRP peptide levels remain elevated until several months after birth. Consistent with this, in situ hybridization and immunohistochemical studies showed that GRP mRNA and peptide consistently colocalized in early gestation lung but that in neonatal lung, many cells that contained GRP peptide no longer contained GRP mRNA. The transient expression of high levels of GRP mRNAs during an approximately 12-wk phase of fetal lung development suggests that the secretion of GRP or its COOH-terminal peptides from pulmonary neuroendocrine cells may play a role in normal lung development.

Authors

E R Spindel, M E Sunday, H Hofler, H J Wolfe, J F Habener, W W Chin

Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells.

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Abstract

Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear.

Authors

O D Ratnoff, M M Emanuelson, N P Ziats

Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (MkMk). Strain differences indicate receptor heterogeneity and two pathways for invasion.

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Abstract

To determine the ligands on erythrocytes for invasion by Plasmodium falciparum, we tested invasion into MkMk erythrocytes that lack glycophorins A and B and enzyme-treated erythrocytes by parasites that differ in their requirement for erythrocyte sialic acid. The 7G8 strain invaded MkMk erythrocytes and neuraminidase-treated normal erythrocytes with greater than 50% the efficiency of normal erythrocytes. In contrast, the Camp strain invaded MkMk erythrocytes at 20% of control and neuraminidase-treated normal erythrocytes at only 1.8% of control. Invasion of MkMk erythrocytes by 7G8 parasites was unaffected by treatment with neuraminidase but was markedly reduced by treatment with trypsin. In contrast, invasion of MkMk cells by Camp parasites was markedly reduced by neuraminidase but was unaffected by trypsin. We conclude that the 7G8 and Camp strains differ in ligand requirements for invasion and that 7G8 requires a trypsin sensitive ligand distinct from glycophorins A and B.

Authors

T J Hadley, F W Klotz, G Pasvol, J D Haynes, M H McGinniss, Y Okubo, L H Miller

Induction of the skeletal alpha-actin gene in alpha 1-adrenoceptor-mediated hypertrophy of rat cardiac myocytes.

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Abstract

Myocardial hypertrophy in vivo is associated with reexpression of contractile protein isogenes characteristic of fetal and neonatal development. The molecular signals for hypertrophy and isogene switching are unknown. We studied alpha (sarcomeric)-actin messenger RNA (mRNA) expression in cultured cardiac myocytes from the neonatal rat. In the cultured cells, as in the adult heart in vivo, expression of cardiac alpha-actin (cACT) predominated over that of skeletal alpha-actin (sACT) mRNA, the fetal/neonatal isoform. alpha 1-Adrenergic receptor stimulation induced hypertrophy of these cells, increasing total RNA and cytoskeletal actin mRNA by 1.8-fold over control, and total alpha-actin mRNA by 4.3 fold. This disproportionate increase in total alpha-actin mRNA was produced by a preferential induction of sACT mRNA, which increased by 10.6-fold over control versus only 2.6-fold for cACT mRNA. The alpha 1-adrenoceptor is the first identified molecular mediator of early developmental isogene reexpression in cardiac myocyte hypertrophy.

Authors

N H Bishopric, P C Simpson, C P Ordahl

Molecular studies of ceruloplasmin deficiency in Wilson's disease.

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Abstract

Deficiency of serum ceruloplasmin is a characteristic biochemical abnormality of Wilson's disease, although the mechanism of this finding is unknown. Ceruloplasmin messenger RNA (mRNA) levels were therefore examined in five patients with Wilson's disease and five controls with other types of hepatic disease. Northern and dot blot hybridizations showed that detectable ceruloplasmin mRNA was present in all of the patients with Wilson's disease, including one patient with no detectable serum ceruloplasmin. However, the ceruloplasmin mRNA levels in the Wilson's disease patients were only 33% that of controls (P less than 0.001). In contrast, albumin mRNA levels in the Wilson's disease patients averaged 161% that of controls. In an attempt to better delineate the level of gene expression responsible for this decrease in ceruloplasmin mRNA, the nuclear run-on assay was used to analyze transcriptional rates. The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that the diminished serum ceruloplasmin levels in patients with Wilson's disease are due at least in part to a decrease in ceruloplasmin gene transcription.

Authors

M J Czaja, F R Weiner, S J Schwarzenberg, I Sternlieb, I H Scheinberg, D H Van Thiel, N F LaRusso, M A Giambrone, R Kirschner, M L Koschinsky

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

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Abstract

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.

Authors

P Seto, H Hirayu, R P Magnusson, J Gestautas, L Portmann, L J DeGroot, B Rapoport

Rearrangement of the genes for the beta and gamma chains of the T cell receptor is rarely observed in adult B cell lymphoma and chronic lymphocytic leukemia.

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Abstract

We determined the configuration of the genes for the beta (T beta) and gamma (T gamma) chains of the T cell receptor in DNA from 100 consecutive cases of B cell lymphoma and B cell chronic lymphocytic leukemia (B-CLL), and compared the findings with those in 18 T cell neoplasms. In 7 of the 100 B cell specimens, a single nongermline band was detected after digestion with the restriction enzyme BamHI, but the rearrangement could be confirmed with a second restriction enzyme in only two. The B cell fragments were small in size and of limited size diversity when compared with the T cell cases, and germline bands of equal intensity were present. A rearrangement of the T gamma gene was never seen in a B cell sample. In contrast, T cell specimens usually rearranged both alleles of T beta (15 of 18), the rearrangement could be confirmed with a second restriction enzyme (17 of 18), both alleles of the first constant region gene segment of T beta always underwent either rearrangement or deletion, and the T gamma gene was also rearranged or deleted (17 of 18). We conclude that ordered rearrangement of the T cell receptor is a rare event in B cell lymphoma and B-CLL. T cell receptor gene studies allow B and T cell lymphomas to be distinguished from each other and from common acute lymphoblastic leukemia antigen-positive non-T, non-B acute lymphoblastic leukemia.

Authors

A C Aisenberg, B M Wilkes, J O Jacobson