Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

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Abstract

Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

Authors

M K Sinha, I Opentanova, J P Ohannesian, J W Kolaczynski, M L Heiman, J Hale, G W Becker, R R Bowsher, T W Stephens, J F Caro

Human neutrophils employ chlorine gas as an oxidant during phagocytosis.

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Abstract

Reactive oxidants generated by phagocytes are of central importance in host defenses, tumor surveillance, and inflammation. One important pathway involves the generation of potent halogenating agents by the myeloperoxidase-hydrogen peroxide-chloride system. The chlorinating intermediate in these reactions is generally believed to be HOCl or its conjugate base, ClO-. However, HOCl is also in equilibrium with Cl2, raising the possibility that Cl2 executes oxidation/ halogenation reactions that have previously been attributed to HOCl/ClO-. In this study gas chromatography-mass spectrometric analysis of head space gas revealed that the complete myeloperoxidase-hydrogen peroxide-chloride system generated Cl2. In vitro studies demonstrated that chlorination of the aromatic ring of free L-tyrosine was mediated by Cl2 and not by HOCl/ClO-. Thus, 3-chlorotyrosine serves as a specific marker for Cl2-dependent oxidation of free L-tyrosine. Phagocytosis of L-tyrosine encapsulated in immunoglobulin- and complement-coated sheep red blood cells resulted in the generation of 3-chlorotyrosine. Moreover, activation of human neutrophils adherent to a L-tyrosine coated glass surface also stimulated 3-chlorotyrosine formation. Thus, in two independent models of phagocytosis human neutrophils convert L-tyrosine to 3-chlorotyrosine, indicating that a Cl2-like oxidant is generated in the phagolysosome. In both models, synthesis of 3-chlorotyrosine was inhibited by heme poisons and the peroxide scavenger catalase, implicating the myeloperoxidase-hydrogen peroxide system in the reaction. Collectively, these results demonstrate that myeloperoxidase generates Cl2 and that human neutrophils use an oxidant with characteristics identical to those of Cl2 during phagocytosis. Moreover, our observations suggest that phagocytes exploit the chlorinating properties of Cl2 to execute oxidative and cytotoxic reactions at sites of inflammation and vascular disease.

Authors

S L Hazen, F F Hsu, D M Mueller, J R Crowley, J W Heinecke

HIV infection--induced posttranslational modification of T cell signaling molecules associated with disease progression.

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Abstract

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.

Authors

I Stefanová, M W Saville, C Peters, F R Cleghorn, D Schwartz, D J Venzon, K J Weinhold, N Jack, C Bartholomew, W A Blattner, R Yarchoan, J B Bolen, I D Horak

An intrinsic adrenergic system in mammalian heart.

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Abstract

We have identified a previously undescribed intrinsic cardiac adrenergic (ICA) cell type in rodent and human heart. Northern and Western blot analyses demonstrated that ICA cell isolates contain mRNA and protein of enzymes involved in catecholamine biosynthesis. Radioenzymatic catecholamine assays also revealed that the catecholamine profile of adult rat ICA cell isolates differed from that of sympathetic neurons. Unlike sympathetic neuronal cells, isolated ICA cells have abundant clear vesicles on electron microscopy. Endogenous norepinephrine and epinephrine constitutively released by ICA cells in vitro affect the spontaneous beating rate of neonatal rat cardiac myocytes in culture. Finally, ICA cells could be identified in human fetal hearts at a developmental stage before sympathetic innervation of the heart has been documented to occur. These findings support the concept that these cells constitute an ICA signaling system capable of participating in cardiac regulation that appears to be independent of sympathetic innervation.

Authors

M H Huang, D S Friend, M E Sunday, K Singh, K Haley, K F Austen, R A Kelly, T W Smith

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

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Abstract

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Authors

P J French, J H van Doorninck, R H Peters, E Verbeek, N A Ameen, C R Marino, H R de Jonge, J Bijman, B J Scholte

Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

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Abstract

To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.

Authors

L Lavaissiere, S Jia, M Nishiyama, S de la Monte, A M Stern, J R Wands, P A Friedman

Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice.

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Abstract

Oral administration of self-antigens has been proposed as a therapy to prevent and treat autoimmune diseases. Here we report that oral treatment with insulin prevents virus-induced insulin-dependent diabetes mellitus (IDDM) in a transgenic (tg) mouse model. Such mice express the viral nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter in their pancreatic beta cells and < 2% spontaneously develop diabetes. However, 2 mo after challenge with LCMV, IDDM occurs in > 95% of tg mice but not in controls. Oral treatment with 1 mg of insulin twice per week for 2 mo starting either 1 wk before or 10 d after initiating LCMV infection prevents IDDM in > 50% of the tg mice (observation time 8 mo). Thus, insulin therapy is effective in preventing progression to overt IDDM in prediabetic tg mice with ongoing islet infiltration. Oral administration of insulin does not affect the generation of LCMV-NP-specific anti-self cytotoxic T lymphocytes nor the infiltration of lymphocytes into the pancreas. However, less beta cells are destroyed in insulin-treated mice, upregulation of MHC class I and II molecules does not occur, and antiviral (self) cytotoxic T lymphocytes are not found in the islets, events present in tg mice developing IDDM. The majority of lymphocytes in the islets of insulin-treated tg mice without IDDM produces IL-4, IL-10, and TGF-beta. In contrast, lymphocytes from islets of tg mice developing IDDM mainly make gamma-IFN.

Authors

M G von Herrath, T Dyrberg, M B Oldstone

RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

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Abstract

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

Authors

P J Gruber, S W Kubalak, T Pexieder, H M Sucov, R M Evans, K R Chien

Selective expansion of specific T cell receptors in the inflamed colon of Crohn's disease.

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Abstract

To identify disease-specific T cell changes that occur in Crohn's disease (CD), the T cell receptor BV repertoires of lamina propria lymphocytes (LPL) isolated from both the inflamed and "disease-inactive" colons of seven CD patients were compared by the quantitative PCR and DNA sequence analysis. It was observed that the BV repertoires of LPL isolated from the disease-active and disease-inactive parts of the colon from the same individual were very different. Furthermore, nearly all of the differences occurred in CD4+ LPL, with very few differences in the CD8+ population of LPL. Although the pattern of BV segments that was increased in disease-active tissue relative to disease-inactive tissue was different for all seven CD patients, there were several BV segments that increased uniformly in the disease-active tissue of all seven individuals. CDR3 length analysis and DNA sequencing of these BV segments revealed that in six of the seven CD patients there was a striking degree of oligoclonality that was absent from disease-inactive tissue of the same individual. These observations suggest that at least some of the inflammation in CD is the result of responses by CD4+ T cells to specific antigens. The isolation of such inflammation-specific CD4+ T cells may make it possible to identify the antigens that are responsible for the inflammatory process in CD and provide a better understanding of its pathogenesis.

Authors

B Gulwani-Akolkar, P N Akolkar, A Minassian, R Pergolizzi, M McKinley, G Mullin, S Fisher, J Silver

Gene mapping of familial autosomal dominant dilated cardiomyopathy to chromosome 10q21-23.

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Abstract

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial disorder, accounting for 60% of all cardiomyopathies. In 20-30% of cases, familial inheritance can be demonstrated; an autosomal dominant transmission is the usual type of inheritance pattern identified. Previously, genetic heterogeneity was demonstrated in familial autosomal dominant dilated cardiomyopathy (FDCM). Gene localization to chromosome 1 (1p1-1q1 and 1q32), chromosome 3 (3p25-3p22), and chromosome 9 (9q13-9q22) has recently been identified. We report one family with 26 members (12 affected) with familial autosomal dominant dilated cardiomyopathy in which linkage to chromosome 10 at the 10q21-q23 locus is identified. Using short tandem repeat polymorphism (STR) markers with heterozygosity > 70%, 169 markers (50% of the genome) were used before linkage was found to markers D10S605 and D10S201 with a pairwise LOD score = 3.91, theta = 0, penetrance = 100% for both markers. Linkage to 1p1-1q1, 1q32, 3p25-3p22, and 9q13-9q22 was excluded. We conclude that a new locus for pure autosomal dominant FDCM exists, and that this gene is localized to a 9 cM region of 10q21-10q23. The search for the disease causing gene and the responsible mutation(s) is ongoing.

Authors

K R Bowles, R Gajarski, P Porter, V Goytia, L Bachinski, R Roberts, R Pignatelli, J A Towbin