Antibiotic proteins of human neutrophils.

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Authors

J K Spitznagel

Expression of human bone-related proteins in the hematopoietic microenvironment.

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Given the intimate relationship between bone and bone marrow, we hypothesized that the human bone marrow may function as a source (or reservoir) of bone-forming progenitor cells. We observed a population of cells within the bone marrow which produce bone-specific or bone-related proteins. The production of these proteins was developmentally regulated in human long-term bone marrow cell cultures; the bone protein-producing cells (BPPC) are observed under serum-free, short-term culture conditions, respond to bone-related and not hematopoietic growth factors, and are derived from a population of low-density, nonadherent, My10-negative (or low My10 density), marrow cells (My10 is an antigen found on most hematopoietic progenitor cells). Cultivation of marrow-derived BPPC in secondary, serum-containing cultures results in their differentiation into osteoblastlike cells. At this stage of development, BPPC produce an extracellular matrix which incorporates both bone-related proteins and radiolabeled calcium. Human bone marrow BPPC thus represent a newly described cell phenotype important to both bone and hematopoietic cell biology.

Authors

M W Long, J L Williams, K G Mann

Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor.

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Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammatory responses. A cysteine-rich, highly glycosylated 30-kD TNF-binding protein (TNF-BP) purified from urine may have a role in regulation because it protects in vitro against the biological effects of TNF. The cytotoxic effect of TNF on the fibrosarcoma cell line WEHI 164 was inhibited by 50% at a 10-fold excess of TNF-BP. The binding of TNF to the receptor was partially reversed after the addition of TNF-BP. Results from biosynthetic labeling of cells with 35S-cysteine followed by immunoprecipitation with anti-TNF-BP indicated that TNF-BP is formed and released at the cell surface by cleavage because no corresponding cellular polypeptide was observed. A cellular 60-kD polypeptide, which was immunoprecipitated with anti-TNF-BP, may correspond to the transmembrane TNF-receptor molecule and be the precursor of TNF-BP. Thus, TNF-BP appears to be a soluble form of a transmembrane TNF-receptor. Moreover our results demonstrate that the production of TNF-BP is increased when the TNF receptor is downregulated in cells by treatment with TNF or by activation of protein kinase C with phorbol esters. TNF-BP may be an important agent that blocks harmful effects of TNF, and, therefore, useful in clinical applications.

Authors

M Lantz, U Gullberg, E Nilsson, I Olsson

Whole-body lipolysis and triglyceride-fatty acid cycling in cachectic patients with esophageal cancer.

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Whole-body lipolytic rates and the rate of triglyceride-fatty acid cycling (reesterification of fatty acids released during lipolysis) were measured with stable isotopic tracers in the basal state and during beta-adrenergic blockade with propranolol infusion in five cachectic patients with squamous cell carcinoma of the esophagus, five cachectic cancer-free, nutritionally-matched control patients, and 10 healthy volunteers. Resting energy expenditure and plasma catecholamines were normal in all three groups. The basal rate of glycerol appearance in blood in the patients with cancer (2.96 +/- 0.45 mumol.kg-1.min-1) was similar to that in the nutritionally matched controls (3.07 +/- 0.28 mumol.kg-1.min-1), but 48% greater than in the normal-weight volunteers (2.00 +/- 0.16 mumol.kg-1.min-1) (P = 0.028). The antilipolytic effect of propranolol and the rate of triglyceride-fatty acid cycling in the patients with cancer were also similar in the cachectic control group and approximately 50% greater than in the normal-weight volunteers, but the differences were not statistically significant because of the variability in the data. We conclude that the increase in lipolysis and triglyceride-fatty acid cycling in "unstressed" cachectic patients with esophageal cancer is due to alterations in their nutritional status rather than the presence of tumor itself. Increased beta-adrenergic activity may be an important contributor to the stimulation of lipolysis.

Authors

S Klein, R R Wolfe

Glucocorticoids inhibit neurogenic plasma extravasation and prevent virus-potentiated extravasation in the rat trachea.

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Capsaicin increases the permeability of blood vessels in the rat tracheal mucosa through a mechanism involving the release of tachykinins from sensory nerves. This capsaicin-induced increase in vascular permeability is potentiated by viral infections of the respiratory tract. The present study was done to determine whether this "neurogenic plasma extravasation" can be inhibited by glucocorticoids, to learn the time course of this inhibition, and to determine whether glucocorticoids can prevent the potentiating effect of viral respiratory infections on neurogenic plasma extravasation. Groups of pathogen-free F344 rats were treated with dexamethasone for 2 or 8 h (4 mg/kg i.p.) or 48 or 120 h (0.5-4 mg/kg per d i.p.). Another group of rats was treated with dexamethasone for 120 h following the intranasal inoculation of Sendai virus. The magnitude of plasma extravasation produced by capsaicin or substance P was assessed after this treatment by using Monastral blue pigment and Evans blue dye as intravascular tracers. We found that dexamethasone reduced, in a dose-dependent fashion, the magnitude of plasma extravasation produced in the rat trachea by capsaicin and substance P. Significant inhibition was produced by a dose of dexamethasone as small as 0.5 mg/kg i.p. The effect of dexamethasone had a latency of several hours and reached a maximum after 2 d of treatment. Furthermore, dexamethasone prevented the potentiation of neurogenic plasma extravasation usually present after 5 d of Sendai virus respiratory infection.

Authors

G Piedimonte, D M McDonald, J A Nadel

Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda.

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Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.

Authors

J R Garey, L M Harrison, K F Franklin, K M Metcalf, E S Radisky, J P Kushner

Skeletal muscle metabolism is a major determinant of resting energy expenditure.

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Energy expenditure varies among people, independent of body size and composition, and persons with a "low" metabolic rate seem to be at higher risk of gaining weight. To assess the importance of skeletal muscle metabolism as a determinant of metabolic rate, 24-h energy expenditure, basal metabolic rate (BMR), and sleeping metabolic rate (SMR) were measured by indirect calorimetry in 14 subjects (7 males, 7 females; 30 +/- 6 yr [mean +/- SD]; 79.1 +/- 17.3 kg; 22 +/- 7% body fat), and compared to forearm oxygen uptake. Values of energy expenditure were adjusted for individual differences in fat-free mass, fat mass, age, and sex. Adjusted BMR and SMR, expressed as deviations from predicted values, correlated with forearm resting oxygen uptake (ml O2/liter forearm) (r = 0.72, P less than 0.005 and r = 0.53, P = 0.05, respectively). These findings suggest that differences in resting muscle metabolism account for part of the variance in metabolic rate among individuals and may play a role in the pathogenesis of obesity.

Authors

F Zurlo, K Larson, C Bogardus, E Ravussin

Immunogenicity in animals of a polysaccharide-protein conjugate vaccine against type III group B Streptococcus.

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The native capsular polysaccharide of type III group B Streptococcus elicits a specific antibody response in only 60% of nonimmune human subjects. To enhance the immunogenicity of this polysaccharide, we coupled the type III polysaccharide to tetanus toxoid. Prior to coupling, aldehyde groups were introduced on the polysaccharide by controlled periodate oxidation, resulting in the conversion of 25% of the sialic acid residues of the polysaccharide to residues of the 8-carbon analogue of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. Tetanus toxoid was conjugated to the polysaccharide by reductive amination, via the free aldehyde groups present on the partially oxidized sialic acid residues. Rabbits vaccinated with the conjugate vaccine produced IgG antibodies that reacted with the native type III group B streptococcal polysaccharide (3/3 rabbits), while rabbits immunized with the unconjugated type III polysaccharide failed to respond (0/3 rabbits). Sera from animals receiving conjugate vaccine opsonized type III group B streptococci for phagocytic killing by human peripheral blood leukocytes, and protected mice against lethal challenge with live type III group B streptococci. The results suggest that this method of conjugation to a carrier protein may be a useful strategy to improve the immunogenicity of the type III group B Streptococcus polysaccharide in human subjects.

Authors

M R Wessels, L C Paoletti, D L Kasper, J L DiFabio, F Michon, K Holme, H J Jennings

Role of cytochrome P-450 in reperfusion injury of the rabbit lung.

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Reactive oxygen species are a major cause of damage occurring in ischemic tissue after reperfusion. During reperfusion transitional metals such as iron are required for reactive oxygen species to mediate their major toxic effects. Xanthine oxidase is an important source of reactive oxygen species during ischemia-reperfusion injury, but not in all organs or species. Because cytochrome P-450 enzymes are an important pulmonary source of superoxide anion (O2-.) generation under basal conditions and during hyperoxia, and provide iron catalysts necessary for hydroxyl radical (.OH) formation and propagation of lipid peroxidation, we postulated that cytochrome P-450 might have a potential role in mediating ischemia-reperfusion injury. In this report, we explored the role of cytochrome P-450 enzymes in a rabbit model of reperfusion lung injury. The P-450 inhibitors 8-methoxypsoralen, piperonyl butoxide, and cimetidine markedly decreased lung edema from transvascular fluid flux. Cimetidine prevented the reperfusion-related increase in lung microvascular permeability, as measured by movement of 125I-albumin from the vascular space into lung water and alveolar fluid. P-450 inhibitors also prevented the increase in lung tissue levels of thiobarbituric acid reactive products in the model. P-450 inhibitors did not block enhanced O2-. generation by ischemic reperfused lungs, measured by in vivo reduction of succinylated ferricytochrome c in lung perfusate, but did prevent the increase in non-protein-bound low molecular weight chelates of iron after reperfusion. Thus, cytochrome P-450 enzymes are not likely a major source of enhanced O2-. generation, but serve as an important source of iron in mediating oxidant injury to the rabbit lung during reperfusion. These results suggest an important role of cytochrome P-450 in reperfusion injury to the lung and suggest potential new therapies for the disorder.

Authors

G K Bysani, T P Kennedy, N Ky, N V Rao, C A Blaze, J R Hoidal

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

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Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.

Authors

J H Segal, A S Pollock

Noncoordinate regulation of alpha-1 adrenoreceptor coupling and reexpression of alpha skeletal actin in myocardial infarction-induced left ventricular failure in rats.

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To determine the effects of myocardial infarction-induced left ventricular failure on the regulation of surface alpha-1 adrenoreceptors and signal transduction, large infarcts were produced in rats and the animals killed seven days later. After the documentation of impaired left ventricular pump performance, radioligand binding studies of the alpha-1 adrenoreceptor, norepinephrine-stimulated phosphoinositol turnover, and ADP ribosylation of 41 kD substrate by pertussis toxin were examined in the hypertrophying unaffected myocardium. Moreover, the expression of sarcomeric actin isoforms was analyzed by Northern blots and hybridization with specific oligonucleotide probes. Alpha-1 adrenoreceptor density was found not to be altered in membranes obtained from the spared left ventricular tissue, whereas phosphoinositol turnover was increased 3.1-fold in the viable myocytes of infarcted hearts. Furthermore, pertussis toxin substrate was augmented 2.5-fold in membranes prepared from the surviving left ventricular myocardium. Finally, an upregulation of the skeletal actin isoform was detected in the tissue of the failing left ventricle. In conclusion, the possibility is raised that in the presence of severe myocardial dysfunction and ongoing reactive hypertrophy, effector pathways linked to the alpha-1 adrenoreceptor may stimulate the myocyte hypertrophic response which would tend to normalize cardiac hemodynamics. The reexpression of alpha skeletal actin may be a molecular indicator of the persistance of an overload on the myocardium.

Authors

L G Meggs, J Tillotson, H Huang, E H Sonnenblick, J M Capasso, P Anversa

Transforming growth factor-beta activity in sheep lung lymph during the development of pulmonary hypertension.

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Chronic pulmonary hypertension is associated with extensive structural remodeling of the pulmonary arterial bed. The structural changes in the arterial walls include increased production of extracellular matrix components and smooth muscle cell hypertrophy, changes that have been similarly induced by transforming growth factor-beta (TGF-beta) in culture. In the present study, experiments were performed to determine whether TGF-beta is present in sheep lung lymph, and whether TGF-beta levels were altered in an animal model of chronic pulmonary hypertension induced by continuous air embolization. Several standard biological assays for TGF-beta activity were used for these determinations including soft agar assays, inhibition of epithelial cell proliferation, and a TGF-beta-specific radioreceptor assay. In each case, control lung lymph contained high concentrations of TGF-beta (100 ng/ml) which required transient acidification for detection. Samples of lung lymph from hypertensive sheep showed a transient and early two- to threefold increase in concentrations of latent TGF-beta. This activity could be partially blocked by TGF-beta antibodies. These studies indicate that sheep lung lymph contains TGF-beta and that the level of TGF-beta increases early during the development of pulmonary hypertension. Thus, TGF-beta may contribute to the development of the structural changes in the pulmonary arteries that occur during the onset of chronic pulmonary hypertension.

Authors

E A Perkett, R M Lyons, H L Moses, K L Brigham, B Meyrick

A mutation in the gene for type III procollagen (COL3A1) in a family with aortic aneurysms.

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Experiments were carried out to test the hypothesis that familial aortic aneurysms, either thoracic or abdominal, are caused by mutations in the gene for type III procollagen (COL3A1) similar to mutations in the same gene that have been shown to cause rupture of aorta and other disastrous consequences in the rare genetic disorder known as Ehlers-Danlos syndrome type IV. A family was identified through a 37-yr-old female captain in the United States Air Force who was scrutinized only because many of her direct blood relatives had died of ruptured aortic aneurysms. The woman was heterozygous for a single-base mutation that converted the codon for glycine 619 of the alpha 1(III) chain of type III procollagen to a codon for arginine. Studies on cultured skin fibroblasts demonstrated the mutation caused synthesis of type III procollagen that had a decreased temperature for thermal unfolding of the protein. The same mutation was identified in DNA extracted from pathologic specimens from her mother who had died at the age of 34 and a maternal aunt who died at the age of 55 of aortic aneurysms. Examination of DNA from samples of saliva revealed that the woman's daughter, her son, a brother, and an aunt also had the mutation. The results demonstrated that mutations in the type III procollagen gene can cause familial aortic aneurysms and that DNA tests for such mutations can identify individuals at risk for aneurysms.

Authors

S Kontusaari, G Tromp, H Kuivaniemi, A M Romanic, D J Prockop

Mechanism of acid-induced release of secretin in rats. Presence of a secretin-releasing peptide.

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In fasting rats, intraduodenal infusion of dilute hydrochloric acid results in significant increases in both pancreatic exocrine secretion and plasma concentration of secretin. To test the hypothesis that acid-induced release of secretin is mediated by a secretin-releasing factor (S-RF), anesthetized rats were prepared with pyloric ligation, duodenal and jejunal cannulas, and pancreatic duct cannulas. Donor rats were infused intraduodenally with 0.01 N HCl, 0.15 M NaCl, or a combination of 0.01 N HCl and 0.05 N NaHCO3 at 0.3 ml/min for 1.5 h, and the perfusates were collected via jejunal cannulas. The perfusates with pH adjusted to 6.0 were concentrated threefold and infused into the duodena of recipient rats. The concentrate of acid perfusate (CAP) significantly increased both pancreatic volume flow and bicarbonate output and plasma concentration of secretin, whereas concentrates of the saline perfusate (CSP) or the perfusate of a combination of 0.01 N HCl and 0.05 N NaHCO3 (CABP) did not influence pancreatic secretion or plasma concentration of secretin. The increased pancreatic secretion by CAP was attributed to increased circulating secretin because when secretin was immunoneutralized by a rabbit antisecretin serum, CAP-stimulated pancreatic secretion was abolished. The bioactivity of CAP was trypsin-sensitive and heat stable. The active substance in CAP had a molecular weight of less than 5,000 and greater than 1,000, as determined by ultrafiltration and bioassay. In conclusion, dilute HCl releases an S-RF into the upper small intestinal lumen to stimulate release of secretin. This substance, with molecular weight of less than 5,000, is heat stable and trypsin sensitive. Thus, the acid-stimulated release of secretin is mediated by a secretin-releasing peptide in the upper small intestinal lumen.

Authors

P Li, K Y Lee, T M Chang, W Y Chey

Potent toxicity of 2-chlorodeoxyadenosine toward human monocytes in vitro and in vivo. A novel approach to immunosuppressive therapy.

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Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA damage and the toxicity of CldAdo or dAdo toward monocytes were blocked by deoxycytidine, but not by inhibitors of poly(ADP-ribose) polymerase. A partial decrease in RNA synthesis and a gradual decline of cellular NAD were early biochemical events associated with monocyte DNA damage. Low CldAdo concentrations (5-20 nM) inhibited monocyte phagocytosis and reduced the release of interleukin 6. Higher CldAdo concentrations led to a dose- and time-dependent loss of monocyte viability. Circulating monocytes disappeared within 1 wk in patients with cutaneous T cell lymphoma or with rheumatoid arthritis during continuous CldAdo infusion. The marked sensitivity of human monocyte function and survival to CldAdo in vitro, together with the monocyte depletion in patients receiving CldAdo chemotherapy, suggests that CldAdo or other dAdo analogues offer a novel therapeutic strategy for chronic inflammatory and autoimmune diseases characterized by inappropriate monocyte deployment or function.

Authors

C J Carrera, C Terai, M Lotz, J G Curd, L D Piro, E Beutler, D A Carson

Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.

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Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.

Authors

V M Kähäri, Y Q Chen, M W Su, F Ramirez, J Uitto

Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development.

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Mononuclear phagocytes are developmentally and functionally complex cells that play critical roles in extracellular matrix remodeling. We hypothesized that differentiated mononuclear phagocytes, typified by alveolar macrophages, use a spectrum of metalloproteinases to degrade various matrix macromolecules. To test this hypothesis, we have evaluated synthesis and secretion of four metalloproteinases (interstitial collagenase, stromelysin, 72-kD type IV collagenase, and 92-kD type IV collagenase) by human mononuclear phagocytes with regard to (a) the effect of cellular differentiation, (b) regulation of secretion, and (c) comparisons/contrasts with a prototype metalloproteinase-secretory cell, the human fibroblast. We found that regulated secretion of greater quantities and a wider spectrum of metalloenzymes correlated with a more differentiated cellular phenotype. As extreme examples, the 92-kD type IV collagenase was released by peripheral blood monocytes and uninduced U937 monocyte-like cells, whereas stromelysin was secreted only by lipopolysaccharide-stimulated alveolar macrophages. Macrophage production of interstitial collagenase, stromelysin, and 72-kD type IV collagenase was approximately 20%, 10%, and 1-2%, respectively, of that from equal numbers of fibroblasts; secretion of the 92-kD type IV collagenase was not shared by fibroblasts. This work confirms the potential of macrophages to directly degrade extracellular matrix via secreted metalloproteinases in a manner that differs both qualitatively and quantitatively from that of fibroblasts. Moreover, varying regulation of metalloenzyme synthesis, evidenced by distinct patterns of basal and stimulated secretion during differentiation, can be studied at a molecular level in this model system.

Authors

H G Welgus, E J Campbell, J D Cury, A Z Eisen, R M Senior, S M Wilhelm, G I Goldberg

Elevated insulin receptor content in human breast cancer.

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The growth of breast cancer cells is under the regulation of hormones, growth factors, and their receptors. In the present study, we have employed a new, sensitive, and specific radioimmunoassay for the direct measurement of insulin receptors in surgical specimens of breast cancers. In 159 specimens the insulin receptor content was 6.15 +/- 3.69 ng/0.1 mg protein. This value was more than sixfold higher than the mean value found in both 27 normal breast tissues obtained at total mastectomy (0.95 + 0.68, P less than 0.001) and in six normal specimens obtained from reduction mammoplasty (0.84 +/- 0.78, P less than 0.001). The insulin receptor content in breast cancer tissues was also higher than in any normal tissue investigated including liver (Pezzino, V., V. Papa, V. Trischitta, A. Brunetti, P.A. Goodman, M.K. Treutelaar, J.A. Williams, B.A. Maddux, R. Vigneri, and I.D. Goldfine, 1989. Am. J. Physiol. 257:E451-457). The insulin receptor in breast cancer retained its ability to both bind insulin and undergo insulin-induced tyrosine kinase activation. Immunostaining of the specimens revealed that the insulin receptor was present in malignant epithelial cells, but was not detected in stromal and inflammatory cells. Univariant analysis revealed that the insulin receptor content of the tumors correlated positively with tumor size (P = 0.014), histological grading (P = 0.030), and the estrogen receptor content (P = 0.035). There were no significant correlations between insulin receptor content and the age, body weight, menopausal status, and nodal involvement of the patients. These studies indicate, therefore, that the insulin receptor content is increased in breast cancers and raise the possibility that the insulin receptor may have a role in the biology of these tumors.

Authors

V Papa, V Pezzino, A Costantino, A Belfiore, D Giuffrida, L Frittitta, G B Vannelli, R Brand, I D Goldfine, R Vigneri

Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria.

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Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

Authors

M H Delfau, C Picat, F W de Rooij, K Hamer, M Bogard, J H Wilson, J C Deybach, Y Nordmann, B Grandchamp

Transcriptional regulation of c-jun gene expression by arabinofuranosylcytosine in human myeloid leukemia cells.

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Previous studies have demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces terminal differentiation of human myeloid leukemia cells. Other studies have shown that the c-jun protooncogene is expressed during phorbol ester-induced myeloid differentiation. This work examines the effects of ara-C on c-jun gene expression in human KG-1 myeloid leukemia cells. The results demonstrate that c-jun transcripts are undetectable in uninduced KG-1 cells and that ara-C induces expression of this gene in a concentration- and time-dependent manner. Ara-C treatment was also associated with increases in c-jun transcripts in U-937, THP-1, and HL-60 myeloid leukemia cells. Furthermore, transcriptional run-on analysis has demonstrated that exposure to ara-C increases the rate of c-jun gene transcription. The results also demonstrate that while inhibition of protein synthesis superinduces c-jun mRNA levels in phorbol ester-treated KG-1 cells, cycloheximide had no effect on the induction of c-jun transcripts during ara-C treatment. Moreover, the half-life of c-jun transcripts in ara-C-treated KG-1 cells was 42 min. These findings suggest that the increase in c-jun mRNA observed during ara-C treatment is regulated by a transcriptional mechanism, and that c-jun may be involved in the induction of differentiation and regulation of gene expression by ara-C.

Authors

S M Kharbanda, M L Sherman, D W Kufe

Structure and distribution of an Alu-type deletion mutation in Sandhoff disease.

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Sandhoff disease is a recessively inherited lysosomal storage disease resulting from a deficiency of beta-hexosaminidase activity. The enzyme occurs in two major forms, beta-hexosaminidase A, composed of an alpha- and beta-subunit and beta-hexosaminidase B, composed of two beta-subunits. Both isozyme activities are deficient in Sandhoff disease, owing to mutations of the HEXB gene encoding the common beta-subunit. We have cloned and fully characterized a deletion at the HEXB gene from fibroblasts of a patient with the infantile form of Sandhoff disease. The deletion removes approximately 16 kb of DNA including the HEXB promoter, exons 1-5 and part of intron 5. It most likely arose from recombination between two Alu sequences, with the breakpoints occurring at the midpoint between the left and right arms in each case and regenerating an intact Alu element in the deletion sequence. The deletion allele accounts for 27% of the Sandhoff mutant alleles we analyzed. Two cell lines were shown to be homozygous for the deletion and both had the infantile form of the disease. Four additional patients were compound heterozygotes with other mutations, all of whom displayed a different clinical phenotype. Finally, the mutant allele was present in different ethnic backgrounds, suggesting that it may have been subject to genetic drift.

Authors

K Neote, B McInnes, D J Mahuran, R A Gravel

Angiotensin and thromboxane in the enhanced renal adrenergic nerve sensitivity of acute renal failure.

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The roles of intrarenal angiotensin (A) and thromboxane (TX) in the vascular hypersensitivity to renal nerve stimulation (RNS) and paradoxical vasoconstriction to renal perfusion pressure (RPP) reduction in the autoregulatory range in 1 wk norepinephrine (NE)-induced acute renal failure (ARF) in rats were investigated. Renal blood flow (RBF) responses were determined before and during intrarenal infusion of an AII and TXA2 antagonist. Saralasin or SQ29548 alone partially corrected the slopes of RBF to RNS and RPP reduction in NE-ARF rats (P less than 0.02). Saralasin + SQ29548 normalized the RBF response to RNS. While combined saralasin + SQ29548 eliminated the vasoconstriction to RPP reduction, similar to the effect of renal denervation, appropriate vasodilatation was not restored. Renal vein norepinephrine efflux during RNS was disproportionately increased in NE-ARF (P less than 0.001) and was suppressed by saralasin + SQ29548 infusion (P less than 0.005). It is concluded that the enhanced sensitivity to RNS and paradoxical vasoconstriction to RPP reduction in 1 wk NE-ARF kidneys are the result of intrarenal TX and AII acceleration of neurotransmitter release to adrenergic nerve activity.

Authors

J B Robinette, J D Conger

Physiological measurements of luminal stirring in the dog and human small bowel.

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The resistance to absorption resulting from poor stirring of luminal contents (RLum) is considered to be equivalent to an unstirred layer of greater than 600 microns in the human small intestine. We measured RLum in the jejunum of conscious dogs by assessing the absorption rate of two rapidly absorbed probes, glucose, and [14C]warfarin. When RLum was expressed as an unstirred layer, the maximal thickness of the unstirred layer (assuming negligible epithelial cell resistance) was only approximately 35 and 50 microns for perfusion rates of 26 and 5 ml/min, respectively. Maximal unstirred layer thickness for the human jejunum, calculated from previous studies of glucose absorption, yielded a mean value of only 40 microns (range: 23 to 65 microns). Since epithelial resistance appears to be negligible during absorption of low concentrations of glucose, the maximal unstirred layer of 40 microns should be close to the true value for glucose in the human small intestine. We conclude that the unstirred layer for rapidly absorbed compounds in dogs and man are less than one-tenth of previously reported values, but this layer still may remain the rate limiting step in absorption of rapidly transported compounds.

Authors

M D Levitt, J K Furne, A Strocchi, B W Anderson, D G Levitt

Relationship between proliferation and cell cycle-dependent Ca2+ influx induced by a combination of thyrotropin and insulin-like growth factor-I in rat thyroid cells.

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Abstract

The mechanism of cell proliferation by a combination of thyroid-stimulating hormone (TSH) and insulin-like growth factor-I (IGF-I) was studied in rat thyroid (FRTL-5) cells. IGF-I stimulated an approximately 3.5-fold increase in the rate of Ca2+ influx sustained for at least 6 h in TSH-pretreated cells but not in quiescent cells. The significant cell proliferation was observed when TSH-primed cells were incubated with IGF-I for 24 h but not for 12 h. IGF-I stimulated the rate of Ca2+ influx in a dose-dependent manner that was similar to that for induction of DNA synthesis. Both Ca2+ influx and DNA synthesis observed in response to IGF-I in TSH-primed cells were inhibited by cobalt. In addition, the stimulations of Ca2+ influx and DNA synthesis by IGF-I were dependent on extracellular Ca2+ in TSH-pretreated cells. When TSH-primed cells were pretreated with pertussis toxin, both IGF-I-induced Ca2+ influx and DNA synthesis were abolished. However, pertussis toxin did not block the priming action of TSH or forskolin. When calcium entry was induced by Bay K8644, it stimulated cell growth in TSH-primed cells but not in quiescent cells. Moreover, cobalt and lanthanum inhibited DNA synthesis even when added several hours after the addition of Bay K8644 but not when added 24 h after the growth factor in TSH-primed cells. These findings suggest that at least two important mechanisms may work in response to IGF-I only in the TSH-primed G1 phase of the cell cycle: first, IGF-I can activate directly or indirectly the Ca2+ channel via a pertussis toxin-sensitive substrate in TSH-primed cells; and second, a long lasting calcium entry by IGF-I may be a cell cycle-dependent mitogenic signal.

Authors

K Takada, N Amino, H Tada, K Miyai

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan.

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Abstract

The capacity of lipopolysaccharide (LPS), zymosan, and calcium ionophore A23187 to induce neutrophil chemotactic activity (NCA), leukotriene B4 (LTB4), and neutrophil attractant/activation protein (NAP-1) release from human alveolar macrophages (AM) retrieved from normal nonsmokers was evaluated. LPS induced a dose-dependent release of LTB4 that began by 1 h, 4.0 +/- 3.2 ng/10(6) viable AM; peaked at 3 h, 24.7 +/- 13.5 ng/10(6) viable AM; and decreased by 24 h, 1.2 +/- 1.0 ng/10(6) viable AM (n = 8). Quantities of LTB4 in cell-free supernatants of AM stimulated with LPS were determined by reverse-phase high-performance liquid chromatography and corresponded well with results obtained by radioimmunoassay. By contrast, NAP-1 release began approximately 3-5 h after stimulation of AM with LPS, 197 +/- 192 ng/ml, and peaked at 24 h, 790 +/- 124 ng/ml. Release of NAP-1 was stimulus specific because A23187 evoked the release of LTB4 but not NAP-1, whereas LPS and zymosan induced the release of both LTB4 and NAP-1. The appearance of neutrophil chemotactic activity in supernatants of AM challenged with LPS for 3 h could be explained completely by the quantities of LTB4 present. After stimulation with LPS or zymosan for 24 h, AM had metabolized almost all generated LTB4. Preincubation of AM with nordihydroguiaretic acid (10(-4) M) completely abolished the appearance of NCA, LTB4, and NAP-1 in supernatants of AM challenged with LPS. Therefore, LPS and zymosan particles were potent stimuli of the sequential release of LTB4 and NAP-1 from AM.

Authors

J A Rankin, I Sylvester, S Smith, T Yoshimura, E J Leonard

Ibuprofen prevents oxidant lung injury and in vitro lipid peroxidation by chelating iron.

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Abstract

Because ibuprofen protects from septic lung injury, we studied the effect of ibuprofen in oxidant lung injury from phosgene. Lungs from rabbits exposed to 2,000 ppm-min phosgene were perfused with Krebs-Henseleit buffer at 50 ml/min for 60 min. Phosgene caused no increase in lung generation of cyclooxygenase metabolites and no elevation in pulmonary arterial pressure, but markedly increased transvascular fluid flux (delta W = 31 +/- 5 phosgene vs. 8 +/- 1 g unexposed, P less than 0.001), permeability to albumin (125I-HSA) lung leak index 0.274 +/- 0.035 phosgene vs. 0.019 +/- 0.001 unexposed, P less than 0.01; 125I-HSA lavage leak index 0.352 +/- 0.073 phosgene vs. 0.008 +/- 0.001 unexposed, P less than 0.01), and lung malondialdehyde (50 +/- 7 phosgene vs. 24 +/- 0.7 mumol/g dry lung unexposed, P less than 0.01). Ibuprofen protected lungs from phosgene (delta W = 10 +/- 2 g; lung leak index 0.095 +/- 0.013; lavage leak index 0.052 +/- 0.013; and malondialdehyde 16 +/- 3 mumol/g dry lung, P less than 0.01). Because iron-treated ibuprofen failed to protect, we studied the effect of ibuprofen in several iron-mediated reactions in vitro. Ibuprofen attenuated generation of .OH by a Fenton reaction and peroxidation of arachidonic acid by FeCl3 and ascorbate. Ibuprofen also formed iron chelates that lack the free coordination site required for iron to be reactive. Thus, ibuprofen may prevent iron-mediated generation of oxidants or iron-mediated lipid peroxidation after phosgene exposure. This suggests a new mechanism for ibuprofen's action.

Authors

T P Kennedy, N V Rao, W Noah, J R Michael, M H Jafri Jr, G H Gurtner, J R Hoidal

Inactivation of beef brain alpha-ketoglutarate dehydrogenase complex by valproic acid and valproic acid metabolites. Possible mechanism of anticonvulsant and toxic actions.

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Abstract

The anticonvulsant valproic acid (VPA, 2-n-propylpentanoic acid) causes inhibition of the citric acid cycle and elevations of central nervous system (CNS) gamma-aminobutyric acid (GABA) levels, which correlates with anticonvulsant action. No unifying mechanism for these actions of VPA has won general acceptance. alpha-Ketoglutarate dehydrogenase complex (KDHC) is a critical control enzyme in the CNS. We hypothesized that VPA may be an inhibitor of this enzyme since decreased KDHC activity would reduce substrate flux through the citric acid cycle and may increase flux into GABA synthesis. To test this hypothesis, inhibition of purified beef brain KDHC by VPA and its metabolites 2-n-propylpent-2-enoic acid (delta 2,3 VPE) and their coenzyme A (CoA) derivatives were studied. Preincubation of the NADH-reduced enzyme with delta 2,3 VPE, VPA-CoA, and delta 2,3 VPE-CoA caused time-dependent inactivation, reversible by addition of CoA. Under steady-state conditions, delta 2,3 VPE and VPA-CoA were competitive inhibitors of KDHC and delta 2,3 VPE-CoA was a mixed inhibitor. These observations have implications for the molecular mechanisms of VPA action. VPA derivatives cause inactivation and inhibition of KDHC, which may explain the anticonvulsant and some toxic actions of VPA.

Authors

A S Luder, J K Parks, F Frerman, W D Parker Jr

Pathogenesis of hyperadrenergic orthostatic hypotension. Evidence of disordered venous innervation exclusively in the lower limbs.

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Abstract

The pathogenesis of hyperadrenergic orthostatic hypotension was studied in eight patients. Correction of the abnormal orthostatic changes by an inflated pressure suit (MAST) confirmed previous evidence of excessive gravitational pooling of blood in the leg veins. Intravenous L-norepinephrine infusion raised diastolic blood pressure in the same relationship to the infusion-induced increments in plasma norepinephrine concentrations as in normal subjects, indicating normal arteriolar responses. Contractile responses of the veins to infused L-norepinephrine were measured with a linear variable differential transformer (LVDT). The venous responses of hand veins in the patients fell within the 95% confidence limits of the responses of normal hand veins, as did the responses of foot veins in the seven normal subjects. However, foot veins of the patients with hyperadrenergic orthostatic hypotension, and both hand and foot veins of patients with "diffuse" autonomic failure, were supersensitive to norepinephrine, as reflected by a steeper slope of the regression of log (norepinephrine infusion rate) on percentage reduction in venous distensibility, and a significantly lower ED50 (i.e., norepinephrine infusion rate that induced 50% reduction in venous distensibility). The findings suggest anatomical or functional postganglionic denervation of lower limb veins causing excessive gravitational blood pooling with consequent orthostatic hypotension in these patients.

Authors

D H Streeten

Cephalosporin-induced alteration in hepatic glutathione redox state. A potential mechanism for inhibition of hepatic reduction of vitamin K1,2,3-epoxide in the rat.

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Abstract

Hypoprothrombinemia is a serious adverse effect of antimicrobial therapy that occurs after administration of some second- and third-generation cephalosporins which contain the methyltetrazole-thiol (MTT) group. Previous studies have shown that in vitro MTT directly inhibits microsomal gamma-carboxylation of a synthetic pentapeptide. Since MTT is a thiocarbamide, a type of compound that can increase oxidation of glutathione, the present studies were carried out to determine whether alterations in hepatic glutathione redox state might interfere with vitamin K metabolism. Dose-related increases in biliary efflux and hepatic concentration of oxidized glutathione (GSSG) occurred after intravenous administration of MTT or MTT-containing antibiotics to rats. This finding suggested that these compounds could alter the hepatic glutathione redox state in vivo. Microsomal reduction of vitamin K epoxide occurred in the presence of 100 microM dithiothreitol (DTT), but was inhibited by preincubation with GSSG at concentrations as low as 10 microM. At higher concentrations of DTT (1.0 mM) inhibition by GSSG persisted, but higher concentrations were required, suggesting that the thiol/disulfide ratio, rather than the absolute concentration of GSSG was important. By contrast, GSSG did not effect microsomal gamma-carboxylation of a pentapeptide, using either vitamin K1 or its hydroquinone as a cofactor. These findings suggest a novel mechanism for the hypoprothrombinemia occurring after administration of MTT-containing antibiotics.

Authors

M C Mitchell, A Mallat, J J Lipsky

Beta-adrenoceptor expression in human fat cells from different regions.

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Abstract

The expression of beta-adrenoceptors (BAR) was investigated in abdominal and gluteal fat cells of 32 nonobese men and women using radioligand binding and RNA excess solution hybridization. In both sexes the number of BAR binding sites was about twice as high in abdominal as in gluteal fat cells (P less than 0.01). Northern blot analysis of total RNA from adipose tissue showed hybridization of the BAR1 probe to an mRNA species of about 2.5 kb and of the BAR2 probe to an mRNA species of approximately 2.2 kb. The steady-state mRNA levels of BAR 1 and BAR 2 were also about twice as high in abdominal as in gluteal adipocytes of men and women (P less than 0.01). In abdominal fat cells the mRNA levels were approximately 45 and 30 molecules/cell for BAR1 and BAR2, respectively. There were no regional or sex variations in BAR 1 and BAR 2 mRNA stability. The apparent half-life of mRNA for both receptor subtypes was approximately 6 h in both regions. The mRNA levels for beta actin did not differ between the two regions in either sex. Thus, differences in expression of the genes encoding for BAR 1 and BAR 2 can explain why abdominal fat cells have more BAR than gluteal fat cells. This variation in gene expression may be a molecular mechanism underlying the well known regional differences in catecholamine-induced lipolysis activity between central and peripheral adipose tissue.

Authors

P Arner, L Hellström, H Wahrenberg, M Brönnegård

Pearson's marrow-pancreas syndrome. A multisystem mitochondrial disorder in infancy.

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Abstract

Pearson's marrow-pancreas syndrome (McKusick No. 26056) is a fatal disorder of hitherto unknown etiology involving the hematopoietic system, exocrine pancreas, liver, and kidneys. The observation of high lactate/pyruvate molar ratios in plasma and abnormal oxidative phosphorylation in lymphocytes led us to postulate that Pearson's syndrome belongs to the group of mitochondrial cytopathies. Since rearrangements of the mitochondrial genome between direct DNA repeats were consistently found in all tissues tested, our results show that this disease is in fact a multisystem mitochondrial disorder, as suggested by the clinical course of the patients. Based on these observations, we would suggest giving consideration to the hypothesis of a defect of oxidative phosphorylation in elucidating the origin of other syndromes, especially those associated with an abnormal oxidoreduction status in plasma.

Authors

A Rötig, V Cormier, S Blanche, J P Bonnefont, F Ledeist, N Romero, J Schmitz, P Rustin, A Fischer, J M Saudubray

Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferase.

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Abstract

Hepatitis C virus (HCV) is the predominant etiologic agent of posttransfusion non-A, non-B hepatitis, characterized by undulating elevation of alanine aminotransferase (ALT) and chronic liver disease. A commercial enzyme-linked immunosorbent assay detected antibodies to HCV (anti-HCV) in 11 specimens among 101 nontransfusable plasma units obtained from asymptomatic, volunteer blood donors with elevated levels' of ALT. Using a combined reverse-transcription polymerase chain reaction (RT-PCR) assay developed by us, HCV RNA was detected in 0.6 ml of plasma from 8 of 11 (73%) of the anti-HCV-positive but in none of the 90 anti-HCV-negative specimens. The relatively low concentration of HCV RNA could be detected in the remaining three anti-HCV-positive specimens when 2.4 ml of plasma was analyzed. The plasma concentration of virions was estimated to range from 10(2) to 5 x 10(7)/ml. Direct sequencing performed on the PCR-amplified HCV cDNAs (210 base pairs) from three specimens revealed heterogeneity between 2.5 and 8.6% at the nucleotide level and less than 4% at the amino acid level. Our findings demonstrate that RT-PCR can be performed with 2.4 ml of plasma, providing an assay for the direct detection of HCV RNA and confirming the existence of an asymptomatic carrier state for HCV infection in the apparently healthy anti-HCV-positive donors.

Authors

P P Ulrich, J M Romeo, P K Lane, I Kelly, L J Daniel, G N Vyas

Reduced beta-cell glucose transporter in new onset diabetic BB rats.

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Abstract

Previous studies from our laboratories have suggested a defect in glucose transport in islets isolated from BB rats on the first day of overt diabetes. To quantitate by immunostaining the glucose transporter of beta-cells (GLUT-2) before and at the onset of autoimmune diabetes we employed an antibody to its COOH-terminal octapeptide. On the first day of overt diabetes, defined as the day the daily blood glucose first reached 200 mg/dl, the volume density ratio of GLUT-2-positive to insulin-positive beta-cells was only 0.48 +/- 0.06, compared to 0.91 +/- 0.02 in age-matched nondiabetic diabetes-resistant controls (P less than 0.001). In age-matched nondiabetic diabetes-prone rats, most of which would have become diabetic, the ratio was 0.85 +/- 0.02, also less than the controls (P less than 0.05). Protein A-gold labeling of GLUT-2 in beta-cells of day 1 diabetic rats revealed 2.17 +/- 0.16 gold particles per micrometer length of microvillar plasma membranes compared to 3.91 +/- 0.14 in controls (P less than 0.001) and 2.87 +/- 0.24 in the nondiabetic diabetes-prone rats (P less than 0.02). Reduction in GLUT-2 correlates temporally with and may contribute to the loss of glucose-stimulated insulin secretion that precedes profound beta-cell depletion of autoimmune diabetes.

Authors

L Orci, R H Unger, M Ravazzola, A Ogawa, I Komiya, D Baetens, H F Lodish, B Thorens

A novel X-linked combined immunodeficiency disease.

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Abstract

A novel X-linked combined immunodeficiency disease was found in five living males in an extended family in the United States. The age of the affected males ranged from 2.5 to 34 yr. The most prominent clinical abnormalities were a paucity of lymphoid tissue; recurrent sinusitis, otitis media, bronchitis, and pneumonia; severe varicella; and chronic papillomavirus infections. The principal immunologic features of the disorder were normal concentrations of serum immunoglobulins but restricted formation of IgG antibodies to immunogens; normal numbers of B cells and NK cells but decreased numbers of CD4+ and CD8+ T lymphocytes, particularly the CD45RA+ subpopulations; diminished proliferative responses of blood T cells to allogeneic cells, mitogens and antigens; and decreased production of IL-2 by mitogen stimulated blood lymphocytes. Thus, affected males in this family carry an abnormal gene on their X chromosome that results in a combined immunodeficiency that is distinct from previously reported disorders.

Authors

E G Brooks, F C Schmalstieg, D P Wirt, H M Rosenblatt, L T Adkins, D P Lookingbill, H E Rudloff, T A Rakusan, A S Goldman

Epidermal growth factor regulates the in vitro sensitivity of human ovarian carcinoma cells to cisplatin.

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Abstract

Cisplatin (DDP) is the most effective drug for the treatment of human ovarian cancer, but the mechanisms that determine sensitivity to the cytotoxic action of DDP are not well understood. Treatment of two human ovarian carcinoma cell lines with epidermal growth factor (EGF) simultaneously increased sensitivity to DDP and caused a persistent change in morphology in the absence of any mitogenic effect. Sensitization to DDP was shown to be dependent on both EGF concentration and EGF receptor number in C127 mouse fibroblasts expressing the human EGF receptor after transfection with a pBPV plasmid construct containing the human EGF receptor gene under control of the transferrin receptor 3'-inducible regulator. Sensitization of human ovarian carcinoma cells to DDP was not blocked by inhibition of protein synthesis. EGF did not enhance sensitivity to DDP or alter morphology in DDP-resistant human ovarian carcinoma cells despite the presence of functional EGF receptors on these cells. These results showed that elements of the signal transduction pathway activated by EGF determined cellular sensitivity to DDP, and that a DDP-resistant phenotype is associated with a defect in this signal transduction pathway.

Authors

R D Christen, D K Hom, D C Porter, P A Andrews, C L MacLeod, L Hafstrom, S B Howell

Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo.

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Abstract

Whether parenchymal or nonparenchymal liver cells play a predominant role in the pathophysiology of hepatic fibrosis has not been firmly established in vivo. We have addressed this question by quantitating the relative abundance of specific mRNAs for collagen types I, III, and IV, and laminin in purified populations of hepatocytes, sinusoidal endothelial cells, and lipocytes from normal and fibrotic rat liver. In normal liver, type I collagen gene expression was minimal in all cell types; mRNA for types III and IV collagen were apparent in endothelial cells and lipocytes, but not in hepatocytes. Laminin mRNA was present in all cell types. Induction of fibrogenesis by either bile duct ligation or carbon tetrachloride administration was associated with a substantial increase in mRNA for types I and III collagen in nonparenchymal cells. Lipocytes from fibrotic animals exhibited a greater than 30-fold increase in type I collagen mRNA relative to normal lipocytes, and greater than 40-fold relative to hepatocytes. Type III collagen mRNA reached 5 times that in normal lipocytes and greater than 120 times that in hepatocytes. Endothelial cells exhibited an isolated increase in type I collagen mRNA, reaching five times that in normal liver. Type IV collagen and laminin gene expression were not significantly increased in nonparenchymal cells during fibrogenesis; in fact, mRNA for type IV collagen and laminin decreased by up to 50% in endothelial cells. Despite the pronounced changes that occurred in matrix gene expression in nonparenchymal cells during fibrogenesis, no change was noted in hepatocytes. We conclude that nonparenchymal liver cells, particularly lipocytes, are important effectors of hepatic fibrosis in vivo.

Authors

J J Maher, R F McGuire

Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell.

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Abstract

In this study experiments were performed to correlate the rate of digestive enzyme secretion to morphologic observations of the apical cytoskeleton using dispersed rat pancreatic acini with various concentrations of caerulein. Caerulein at concentrations of 10 pM to 0.1 nM stimulated increasing rates of secretion of amylase, a digestive enzyme. Greater concentrations of caerulein caused progressively less amylase secretion. Transmission electron microscopy demonstrated several characteristics of the apical cytoskeleton in untreated acini that were altered with the "inhibitory" concentrations of caerulein. In control acini and acini stimulated with concentrations of caerulein up to 0.1 nM, the micrographs reveal an apical actin network extending into microvilli, an intermediate filament band, and electron-dense structures contained in both the actin filament network and the intermediate filament band. With concentrations of caerulein greater than 0.1 nM, these structures were progressively ablated. The findings with respect to the actin filament network were confirmed with light microscopic observations of dispersed acini stained with rhodamine-phalloidin. These results indicate that caerulein has marked morphologic effects on the pancreatic acinar cell cytoskeleton and that the cytoskeletal changes may modulate the secretory response.

Authors

M S O'Konski, S J Pandol

Mapping of a functional autoimmune epitope on the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.

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Abstract

The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta 2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the beta 1-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the beta 1 receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the beta 1-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications.

Authors

Y Magnusson, S Marullo, S Hoyer, F Waagstein, B Andersson, A Vahlne, J G Guillet, A D Strosberg, A Hjalmarson, J Hoebeke

A phosphatase activity present in peripheral blood myeloid cells of chronic myelogenous leukemia patients but not normal individuals alters nuclear protein binding to transcriptional enhancers of interferon-inducible genes.

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Abstract

Cytoplasmic protein from peripheral blood myeloid cells of chronic myelogenous leukemia (CML) patients altered the electrophoretic mobility of complexes formed between nuclear proteins and interferon-inducible transcriptional enhancers. Immature myeloid marrow cells (blasts and promyelocytes) have a higher level of this activity than do mature myeloid marrow cells (bands and polys). This activity, which is not detectable in the peripheral blood cells of normal individuals, is at least 50-fold higher in CML marrow blasts and promyelocytes than that found in marrow blasts and promyelocytes of normal individuals. This activity was inhibited by in vivo incubation of immature myeloid cells with the phosphatase inhibitor, sodium orthovanadate (0.2 mM), and by adding orthovanadate (20 mM) directly to cytoplasmic proteins of myeloid cells. Interferon-alpha (1,000 U/ml) reduced the effects of the CML myeloid cell cytoplasmic protein on the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that a unique phosphatase may be involved in the abnormalities in CML which are modulated by interferon-alpha.

Authors

D C Seong, S Sims, E Johnson, O M Howard, B Reiter, J Hester, M Talpaz, H Kantarjian, A Deisseroth

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

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Abstract

Expression of myelin basic protein (MBP) in mice is regulated in a cell- and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d), young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-protein complexes with the MBP regulatory region. Brain extracts from young adult and adult mice showed enhanced binding activities with the sequences supporting transcriptional activation in glial cells. Deletion analysis of the proximal activating sequence located at position -14 to -50 with respect to the RNA initiation site resulted in identification of a small region, located between nucleotides -14 to -37, which is required for formation of the complexes. Southwestern assay revealed a major 39-kD protein from young adult brain extract that recognizes the sequences between nucleotides -14 to -37. An additional minor 37-kD protein, derived from young adult brain extract, was also found to be associated with this proximal activating region. Of particular interest is the observation that the minor 37-kD protein became more abundant in the extract derived from adult brain, whereas the major 39-kD protein became less abundant. The possible role of these proteins in cell/stage-specific transcription of MBP is discussed.

Authors

M S Lashgari, K Devine-Beach, S Haas, K Khalili

Mechanism of Pneumocystis carinii attachment to cultured rat alveolar macrophages.

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Abstract

Pneumocystis carinii (PC) pneumonia begins as an intra-alveolar process resulting in injury to the alveolar epithelium with subsequent invasion of the lung interstitium. The clearance of PC organisms from the alveolar space is a critical function of alveolar macrophages (AM), the resident alveolar phagocytic cells. In this study the mechanism of PC attachment to AM was determined using 51Cr-labeled organisms, with PC attachment reaching a maximum of 18.9 +/- 2.5% after 4 h. Attachment was significantly decreased by preincubation of the AM with a monoclonal anti-fibronectin antibody directed against the cell attachment site of fibronectin (from 17.8 +/- 2.2% to 8.3 +/- 1.0%, P less than 0.01), or by addition of the fibronectin cell binding site analogue Arg-Gly-Asp-Ser (RGDS) (from 18.1 +/- 2.3% to 2.9 +/- 0.8%, P less than 0.01). An anti-fibronectin monoclonal antibody directed against the heparin binding domain of fibronectin had no effect on PC attachment. Addition of the specific calcium ion chelating agent EGTA to the culture media similarly decreased attachment from 16.9 +/- 2.0% to 5.1 +/- 1.1% (P less than 0.01). Fibronectin-mediated attachment of PC to AM did not result in phagocytosis of the organisms by the AM as determined by chemiluminescence measurements. Therefore, the data indicate that PC attachment to AM is a calcium-dependent process mediated by the cell binding domain of fibronectin which does not trigger a phagocytic response by the AM.

Authors

S T Pottratz, W J Martin 2nd

CD4-Pseudomonas exotoxin conjugates delay but do not fully inhibit human immunodeficiency virus replication in lymphocytes in vitro.

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Abstract

The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120). This glycoprotein is expressed on the surface membrane of cells infected with HIV. It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV. We demonstrate that CD4-Pseudomonas exotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells. However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures. Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes. These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.

Authors

H Tsubota, G Winkler, H M Meade, A Jakubowski, D W Thomas, N L Letvin

Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells.

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Abstract

Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. Recent evidence suggests that vasoconstrictive substances, such as angiotensin II (AII), may function as a vascular smooth muscle growth promoting substance. To explore the role of the counterregulatory hormone, atrial natriuretic polypeptide (ANP) in this process, we examined the effect of ANP (alpha-rat ANP [1-28]) on the growth characteristics of cultured rat aortic smooth muscle (RASM) cells. ANP (10(-7) M) significantly suppressed the proliferative effect of 1% and 5% serum as measured by 3H-thymidine incorporation and cell number, confirming ANP as an antimitogenic factor. In quiescent RASM cells, ANP (10(-7), 10(-6) M) significantly suppressed the basal incorporations of 3H-uridine and leucine by 50 and 30%, respectively. ANP (10(-7), 10(-6) M) also suppressed AII-induced RNA and protein syntheses (by 30-40%) with the concomitant reduction of the cell size. Furthermore, ANP also significantly attenuated the increase of 3H-uridine and leucine incorporations caused by transforming growth factor-beta (4 x 10(-11), 4 x 10(-10) M), a potent hypertrophic factor. These results indicate that ANP possesses an antihypertrophic action on vascular smooth muscle cells. Down-regulation of protein kinase C by 24-h treatment with phorbol 12,13-dibutyrate did not inhibit ANP-induced suppression on 3H-uridine incorporation. Based on the observation that ANP was more potent than a ring-deleted analogue of ANP on inhibiting 3H-uridine incorporation, we conclude that the ANP's inhibitory effect is primarily mediated via the activation of a guanylate cyclase-linked ANP receptor(s). Indeed 8-bromo cGMP mimicked the antihypertrophic action of ANP. Accordingly, we speculate that in addition to its vasorelaxant and natriuretic effects, the antihypertrophic action of ANP observed in the present study may serve as an additional compensatory mechanism of ANP in hypertension.

Authors

H Itoh, R E Pratt, V J Dzau

Evidence that diffusion limitation determines oxygen uptake kinetics during exercise in humans.

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Abstract

To determine the role of arterial O2 content on the mechanism of muscle O2 utilization, we studied the effect of 2, 11, and 20% carboxyhemoglobin (COHb) on O2 uptake (VO2), and CO2 output (VCO2) kinetics in response to 6 min of constant moderate- and heavy-intensity cycle exercise in 10 subjects. Increased COHb did not affect resting heart rate, VO2 or VCO2. Also, the COHb did not affect the asymptotic VO2 in response to exercise. However, VO2 and VCO2 kinetics were affected differently. The time constant (TC) of VO2 significantly increased with increased COHb for both moderate and heavy work intensities. VO2 TC was positively correlated with blood lactate. In contrast, VCO2 TC was negatively correlated with increased COHb for the moderate but unchanged for the heavy work intensity. The gas exchange ratio reflected a smaller increase in CO2 stores and faster VCO2 kinetics relative to VO2 with increased COHb. These changes can be explained by compensatory cardiac output (heart rate) increase in response to reduced arterial O2 content. The selective slowing of VO2 kinetics, with decreased blood O2 content and increased cardiac output, suggests that O2 is diffusion limited at the levels of exercise studied.

Authors

A Koike, K Wasserman, D K McKenzie, S Zanconato, D Weiler-Ravell

Renal vasoconstriction caused by short-term cholesterol feeding is corrected by thromboxane antagonist or probucol.

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Abstract

Recent studies indicate that short-term cholesterol feeding causes vascular hyperreactivity and/or increased tone in certain vascular beds. The present study in rats examined the effect of 3 wk of cholesterol-supplemented diet (CSD) on renal hemodynamics. We tested the hypothesis that LDL oxidized in vivo is causally related to increased renal vascular tone by adding the antioxidant drug probucol to the CSD (CSD + P). Micropuncture of surface nephrons in the CSD rats demonstrated that single nephron glomerular filtration rate (SNGFR) and single nephron afferent plasma flow (QA) were markedly lower than in normal rats, whereas glomerular capillary pressure (PGC), afferent arteriolar resistance (RA), and single nephron filtration fraction (SNFF) were higher. In the CSD + P animals, almost all of these hemodynamic abnormalities were absent. TXB2 and PGE2 were increased in proximal tubule fluid and urine in the CSD rats, but normal in the CSD + P group. Infusion of a TXA2 receptor antagonist into the suprarenal aorta of CSD rats caused a rapid return to normal of RBF (renal blood flow), GFR (glomerular filtration rate), SNGFR, QA, RA, PGC, and Kf (ultrafiltration coefficient). Our observations demonstrate that cholesterol feeding leads to renal vasoconstriction, which appears to be mediated largely by increased TXA2 production. The fact that probucol prevented the hemodynamic abnormalities as well as the increased TX production is consistent with the hypothesis that LDL oxidized in vivo initiates events leading to TX mediated vasoconstriction.

Authors

R Kaplan, H S Aynedjian, D Schlondorff, N Bank

Arginine-glycine-aspartic acid- and fibrinogen gamma-chain carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wall shear rates. An effect dissociable from interference with adhesive protein binding.

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Abstract

Arg-Gly-Asp (RGD)- and fibrinogen gamma-chain carboxyterminal (GQQHHLGGAKQAGDV) peptides inhibit fibrinogen, fibronectin (Fn), vitronectin, and von Willebrand factor (vWF) binding to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). GP IIb-IIIa, vWF, and Fn are essential for normal platelet adherence to subendothelium. We added peptides to normal citrated whole blood before perfusion over human umbilical artery subendothelium and evaluated platelet adherence morphometrically at high (2,600 s-1) and low (800 s-1) wall shear rates. We also examined the effects of the peptides on platelet adhesion to collagen in a static system. At the high wall shear rate, RGDS and GQQHHLGGAKQAGDV caused dose-dependent reduction in the surface coverage with spread and adherent platelets. Amino acid transposition and conservative substitutions of RGD peptides and the AGDV peptide significantly inhibited platelet adherence at 2,600 s-1. By contrast, the modified RGD peptides and AGDV do not affect adhesive protein binding to platelets. None of the native or modified RGD- or fibrinogen gamma-chain peptides significantly inhibited either platelet adherence to subendothelium at 800 s-1 or platelet adhesion to collagen. Our findings demonstrate that peptides that interfere with adhesive protein binding to GP IIb-IIIa inhibit platelet adherence to vascular subendothelium with flowing blood only at high wall shear rates. Platelet adherence to subendothelium at high wall shear rates appears to be mediated by different recognition specificities from those required for fluid-phase adhesive protein binding or static platelet adhesion.

Authors

J B Lawrence, W S Kramer, L P McKeown, S B Williams, H R Gralnick

A substitution at a non-glycine position in the triple-helical domain of pro alpha 2(I) collagen chains present in an individual with a variant of the Marfan syndrome.

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Abstract

A substitution for a highly conserved non-glycine residue in the triple-helical domain of the pro alpha 2(I) collagen molecule was found in an individual with a variant of the Marfan syndrome. A single base change resulted in substitution of arginine618 by glutamine at the Y position of a Gly-X-Y repeat, and is responsible for the decreased migration in SDS-polyacrylamide gels of some pro alpha 2(I) chains of type I collagen synthesized by dermal fibroblasts from this individual. Family studies suggest that this substitution was inherited from the individual's father who also produces abnormally migrating pro alpha 2(I) collagen chains and shares some of the abnormal skeletal features. This single base change creates a new Bsu36 I (Sau I, Mst II) restriction site detectable in genomic DNA by Southern blot analysis when probed with a COL1A2 fragment. The analysis of 52 control individuals (103 chromosomes) was negative for the new Bsu36 I site, suggesting that the substitution is not a common polymorphism.

Authors

C L Phillips, A W Shrago-Howe, S R Pinnell, R J Wenstrup

Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease.

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Abstract

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein (heavy chain) and a 22-kD polypeptide (light chain), is an essential component of the phagocyte NADPH-oxidase responsible for superoxide generation. Cytochrome b is absent in two subgroups of chronic granulomatous disease (CGD), an inherited disorder characterized by the lack of oxidase activity. Mutations in the cytochrome heavy chain gene, encoded by the CYBB locus in Xp21.1, result in the X-linked form of CGD. A rare subgroup of autosomal recessive CGD also lacks cytochrome b (A- CGD), but the genetic defect has not previously been identified. In order to search for possible mutations in the cytochrome light chain locus, CYBA, the structure of this gene was characterized. The CYBA locus was localized to 16q24, and the approximately 600-bp open reading frame determined to be encoded by six exons that span approximately 8.5 kb. Three unrelated patients with A- CGD were studied for evidence of mutations in the light chain gene. One patient, whose parents were first cousins, was homozygous for a large deletion that removed all but the extreme 5' coding sequence of the gene. The other two patients had a grossly normal light chain transcript on Northern blot of mononuclear cell RNA. The light chain transcript was amplified by the polymerase chain reaction and sequenced. One patient was a compound heterozygote for two alleles containing point mutations in the open reading frame that predict a frame shift and a nonconservative amino acid replacement, respectively. The second patient, whose parents were second cousins, was homozygous for a different single-base substitution resulting in another nonconservative amino acid change. These results indicate that A- CGD can results from defects in the gene encoding the 22-kD light chain of the phagocyte cytochrome b.

Authors

M C Dinauer, E A Pierce, G A Bruns, J T Curnutte, S H Orkin

Cholesterol synthesis is required for cutaneous barrier function in mice.

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Abstract

Previous studies have shown that topical acetone treatment results in the removal of stratum corneum lipids and disruption of the permeability barrier. This disruption stimulates epidermal lipid synthesis which is associated with the rapid restoration of stratum corneum lipids and barrier function. The aim of this study was to determine the role of cutaneous cholesterol synthesis in the barrier recovery. Here we show that topical lovastatin, a competitive inhibitor of HMG CoA reductase, inhibits cholesterol synthesis. After acetone disruption of the barrier, the normal rapid return of cholesterol to the stratum corneum and recovery of barrier function is impaired in animals treated topically with lovastatin. When lovastatin animals are simultaneously treated topically with either mevalonate, the immediate product of HMG CoA reductase, or cholesterol, the final end product of the pathway, the recovery of the barrier is normalized. Lovastatin resulted in the delayed secretion and abnormal appearance of lamellar bodies. These results provide the first evidence demonstrating that cholesterol synthesis is required for the maintenance of barrier structure and function and suggests a crucial role for cholesterol synthesis in allowing for terrestrial existence.

Authors

K R Feingold, M Q Man, G K Menon, S S Cho, B E Brown, P M Elias

Insulin regulates apolipoprotein B turnover and phosphorylation in rat hepatocytes.

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Abstract

Our laboratory has previously shown that insulin inhibits the secretion of newly-synthesized and immunoreactive apo B from rat hepatocytes. We have also shown that apo B is secreted as a phosphoprotein and that phosphorylation is increased in hypoinsulinemic nonketotic diabetes. The present studies were conducted to determine whether the ability of insulin to inhibit apo B secretion is related to alterations in apo B turnover and whether insulin itself affects apo B phosphorylation. Pulse-chase studies with [35S]methionine in primary cultures of hepatocytes from normal rats in the absence and presence of insulin show that the secretion of apo B100 and apo B48 are inhibited by insulin and that this inhibition may be due in part to enhanced intracellular degradation. In addition, there is a second intracellular apo B48 pool which is not insulin regulated or degraded. In experiments in which hepatocytes were incubated with [32P]orthophosphate, insulin decreased 32P incorporation into apo B100 (42%) with only small effects on apo B48 (11%). The small insulin effect on apo B48 may relate to an insulin-insensitive apo B48 intracellular pool. These studies show that insulin can affect the intracellular turnover, secretion, degradation, and phosphorylation of apo B and emphasize the differential regulation of apo B100 and apo B48 with regard to these parameters in rat liver.

Authors

T K Jackson, A I Salhanick, J Elovson, M L Deichman, J M Amatruda

Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

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Abstract

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, alpha-N-acetylgalctosaminidase (alpha-GalNAc). The recent isolation and expression of the full-length cDNA encoding alpha-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal alpha-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the alpha-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the alpha-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an alpha-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity.

Authors

A M Wang, D Schindler, R Desnick

L-tryptophan implicated in human eosinophilia-myalgia syndrome causes fasciitis and perimyositis in the Lewis rat.

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Abstract

Tryptophan-associated eosinophilia-myalgia syndrome (L-TRP-EMS) is a newly described syndrome which occurred in epidemic fashion in the United States in the summer and fall of 1989. Epidemiologic data has linked the syndrome to intake of L-tryptophan (L-TRP) from one specific manufacturer, but the precise etiologic compound(s) must be established by replication of the syndrome in an appropriate animal model. In this study, implicated L-TRP, United States Pharmacopeia (USP) grade L-TRP, or vehicle was administered by gavage in a blinded fashion for 38 d to female Lewis rats at doses comparable with those ingested by patients who developed the eosinophilia-myalgia syndrome. Animals receiving implicated L-TRP, but not those receiving USP grade L-TRP or vehicle, developed histologic signs consistent with fasciitis and perimyositis, specific pathologic features of human L-TRP-EMS. Peripheral blood eosinophilia was not observed. Hypothalamic corticotropin releasing hormone mRNA levels were lower and plasma corticosterone levels tended to be lower in the animals that received implicated L-TRP. Plasma L-kynurenine was higher in both L-TRP-treated groups compared to the vehicle-treated animals. The female Lewis rat is known to be susceptible to a wide variety of inflammatory diseases. Identification of specific inflammatory changes in this rat following exposure to implicated L-TRP indicates that this animal model will be important in subsequent investigations into the etiology, pathogenesis, and treatment of human L-TRP-EMS.

Authors

L J Crofford, J I Rader, M C Dalakas, R H Hill Jr, S W Page, L L Needham, L S Brady, M P Heyes, R L Wilder, P W Gold

Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction.

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Abstract

Hepatitis C virus (HCV) RNA was detected in the sera of patients with non-A, non-B chronic liver disease by polymerase chain reaction (PCR). RNA was extracted from the serum, reverse transcribed to cDNA, and amplified by PCR. With this method, 30 patients with non-A, non-B chronic liver disease and 10 healthy subjects were tested. HCV RNA was detected in 13 of 16 (81%) anti-HCV-positive patients and also in 7 of 14 (50%) anti-HCV-negative patients, but in none of 10 anti-HCV-negative healthy subjects. Specificity of this method was confirmed by direct sequencing of amplified cDNA segment. The nucleotide sequences (37 nucleotides) obtained from 15 patients showed only 68-78% homology compared with the prototype HCV nucleotide sequence. In addition, of 15 nucleotide sequences, there were 12 different types. But the translated amino acid sequences (12 amino acids) showed 83-100% homology compared with the prototype HCV amino acid sequence. These data suggest the majority of anti-HCV-positive patients are carriers of HCV. But to detect all the viremic patients, the anti-HCV antibody testing may be insufficient. Direct detection of HCV RNA may be useful in the study of virus replication and its association with various liver diseases.

Authors

N Kato, O Yokosuka, M Omata, K Hosoda, M Ohto

Role of endothelium-derived nitric oxide in the bleeding tendency of uremia.

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Abstract

Endothelium-derived relaxing factor, now identified as nitric oxide (NO), is a labile humoral agent formed by vascular endothelial cells from L-arginine. NO mediates the action of substances that induce endothelium-dependent relaxation and plays a role in regulating blood pressure. In this study we investigated whether NO is involved in the pathogenesis of the bleeding tendency associated with renal failure. Rats with extensive surgical ablation of renal mass develop renal insufficiency due to progressive glomerulosclerosis. Like uremic humans, rats with renal mass reduction and uremia have a bleeding tendency that manifests itself by a prolonged bleeding time. We found that N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO formation from L-arginine, completely normalized bleeding time when given to uremic rats. L-NMMA injection also increased ex vivo platelet adhesion but did not affect ex vivo platelet aggregation induced by adenosine diphosphate, arachidonic acid, and calcium ionophore A23187. The shortening effect of L-NMMA on bleeding time was completely reversed by giving the animals the NO precursor L-arginine, but not D-arginine, which is not a precursor of NO. It thus appears that NO is a mediator of the bleeding tendency of uremia.

Authors

G Remuzzi, N Perico, C Zoja, D Corna, D Macconi, G Viganò