Diseases of abnormal protein glycosylation: an emerging area.

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S Kornfeld

Correction

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Correction

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Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas.

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Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue.

Authors

T Saito, T Endo, A Kawaguchi, M Ikeda, R Katoh, A Kawaoi, A Muramatsu, T Onaya

Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity.

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Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.

Authors

Y Zhang, H Weiler-Guettler, J Chen, O Wilhelm, Y Deng, F Qiu, K Nakagawa, M Klevesath, S Wilhelm, H Böhrer, M Nakagawa, H Graeff, E Martin, D M Stern, R D Rosenberg, R Ziegler, P P Nawroth

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

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The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

Authors

R Evers, M Kool, L van Deemter, H Janssen, J Calafat, L C Oomen, C C Paulusma, R P Oude Elferink, F Baas, A H Schinkel, P Borst

Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy.

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Carbonic anhydrase II (CAII) deficiency in humans is associated with a syndrome of renal tubular acidosis, osteopetrosis, and cerebral calcification. A strain of mice of CAII deficiency due to a point mutation also manifests renal tubular acidosis. We report here that retrograde injection of cationic liposome complexed with a CAII chimeric gene, using a cytomegalovirus (CMV) promoter/enhancer as an expression cassette to drive human CAII cDNA, into the renal pelvis of CAII-deficient mice results in expression of CAII in the kidney. The levels of both the CAII gene and its corresponding mRNA were highest by day 3 after treatment, diminishing thereafter, but remaining detectable by 1 mo. After gene therapy, CAII-deficient mice restored the ability to acidify urine after oral administration of ammonium chloride. The ability to acidify urine was maintained at 3 wk after gene therapy, and was eventually lost by 6 wk. Immunohistochemistry studies using anti-CAII antibodies showed that CAII was expressed in tubular cells of the outer medulla and corticomedullary junction. The gene therapy was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. To our knowledge, this is the first successful gene therapy of a genetic renal disease. Our results demonstrate the potential of gene therapy as a novel treatment for hereditary renal tubular defects.

Authors

L W Lai, D M Chan, R P Erickson, S J Hsu, Y H Lien

Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.

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Physical forces activate apoptosis and gene expression, but the mechanism is unknown. For this purpose, adult myocytes were stretched in an equibiaxial stretch apparatus and the magnitude of cell death was examined 4 and 24 h later. The possibility of stretch-mediated activation of p53 and p53-dependent genes was evaluated at 30 min, 2, 4, 8, and 24 h. Myocyte apoptosis increased by 4.4- and 7.6-fold at 4 and 24 h after stretch. p53 binding to the promoter of angiotensinogen, AT1 receptor, and Bax also increased. Expression of angiotensinogen, AT1 receptor, p53, and Bax increased and Bcl-2 decreased in stretched myocytes. The changes in AT1 receptor, p53, Bax, and Bcl-2 became more apparent with the duration of stretch. Angiotensin II concentration in the medium increased at 10 min, reaching maximal levels at 1 and 20 h. The AT1 blocker, losartan, abolished apoptosis in stretched myocytes. Myocyte volume was not influenced by stretch. In conclusion, stretch-mediated release of angiotensin II is coupled with apoptosis and the activation of p53 which may be responsible for the prolonged upregulation of the local renin-angiotensin system and the increased susceptibility of myocytes to undergo apoptosis.

Authors

A Leri, P P Claudio, Q Li, X Wang, K Reiss, S Wang, A Malhotra, J Kajstura, P Anversa

Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

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Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis.

Authors

P L Majano, C García-Monzón, M López-Cabrera, E Lara-Pezzi, E Fernández-Ruiz, C García-Iglesias, M J Borque, R Moreno-Otero

Caloric restriction reverses hepatic insulin resistance in aging rats by decreasing visceral fat.

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Hyperinsulinemia and increased visceral/abdominal fat (VF) are common features of human aging. To examine the relationships among VF, peripheral, and hepatic insulin sensitivity, we studied 4- and 18-mo-old male Sprague-Dawley rats (n = 42) fed ad libitum (4 AL and 18 AL) or moderately calorie restricted (18 CR) up to 18 mo of age. Total fat mass (FM) and VF were decreased in 18 CR to approximately one-third of that of 18 AL (P < 0.001), while lean body mass (LBM) was unchanged. Most important, 18 CR had more FM (65+/-6 vs. 45+/-6 g) but less VF (7.8+/-0.6 vs. 12.3+/-3.3 g) compared with 4 AL (P < 0.01 for both). Thus, the effects of variable VF on HIS could be assessed, independent of FM and age. Marked hepatic insulin resistance ensued with aging (18 AL) and CR restored hepatic insulin sensitivity to the levels of young rats, while peripheral insulin sensitivity remained unchanged (by insulin clamp of 18 mU/kg/min). In fact, the rates of insulin infusion required to maintain basal hepatic glucose production in the presence of pancreatic clamp were 0.75+/-0.10, 1.41+/-0.13, and 0.51+/-0.12 mU/kg . min, in 4 AL, 18 AL, and 18 CR, respectively (P < 0.01 between all groups), and in 18 CR rats infused with insulin at similar rates as in the 18 AL (1.4 mU/kg/min) hepatic glucose production was decreased by 32% (P < 0. 005). Furthermore, when 18 CR rats were fed AL for 14 d, VF rapidly and selectively increased and severe hepatic insulin resistance was induced. We propose that in this animal model the age-associated decrease in hepatic (rather than peripheral) insulin action is the major determinant of fasting hyperinsulinemia and that increased visceral adiposity plays the major role in inducing hepatic insulin resistance. Thus, interventions designed to prevent the accumulation of VF are likely to represent an effective mean to improve carbohydrate metabolism in aging.

Authors

N Barzilai, S Banerjee, M Hawkins, W Chen, L Rossetti

Synovium as a source of increased amino-terminal parathyroid hormone-related protein expression in rheumatoid arthritis. A possible role for locally produced parathyroid hormone-related protein in the pathogenesis of rheumatoid arthritis.

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Proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 (IL-1), mediate the joint destruction that characterizes rheumatoid arthritis (RA). Previous studies have shown that parathyroid hormone-related protein (PTHrP) is a member of the cascade of proinflammatory cytokines induced in parenchymal organs during lethal endotoxemia. To test the hypothesis that NH2-terminal PTHrP, a potent bone resorbing agent, could also be a member of the synovial cascade of tissue-destructive cytokines whose expression is induced in RA, PTHrP expression was examined in synovium and synoviocytes obtained from patients with RA and osteoarthritis (OA). PTHrP production, as determined by measurement of immunoreactive PTHrP(1-86) in tissue explant supernatants, was increased 10-fold in RA versus OA synovial tissue. Synovial lining cells and fibroblast-like cells within the pannus expressed both PTHrP and the PTH/PTHrP receptor, findings that were confirmed by in vitro studies of cultured synoviocytes. TNF-alpha and IL-1beta stimulated PTHrP expression in synoviocytes, while dexamethasone and interferon-gamma, agents with some therapeutic efficacy in the treatment of RA, inhibited PTHrP release. Treatment of synoviocytes with PTHrP(1-34) stimulated IL-6 secretion. These results suggest that proinflammatory cytokine-stimulated production of NH2-terminal PTHrP by synovial tissue directly invading cartilage and bone in RA may mediate joint destruction through direct effects on cartilage or bone, or, indirectly, via the induction of mediators of bone resorption in the tumor-like synovium.

Authors

J L Funk, L A Cordaro, H Wei, J B Benjamin, D E Yocum

Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis.

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Tissue factor (TF), the initiating cell surface receptor of the coagulation cascade, plays important roles in embryogenesis, angiogenesis, and tumor cell metastasis. It is controversial whether proteolytic function of TF complexed with its serine protease ligand VIIa is required for metastatic tumor dissemination. We show here in a model for TF-dependent experimental hematogenous metastasis, that TF supports metastasis by both proteolytic activity of the TF-VIIa complex and currently undefined functions of the cytoplasmic domain. We demonstrate that ligand binding of VIIa to TF is required for metastasis. Antimetastatic properties of covalently inactivated VIIa provide evidence that ligand binding is insufficient per se to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process. Ala or Asp mutations of cytoplasmic serine residues were introduced to preclude or mimic phosphorylation. In vivo analysis of these mutants suggests that local protease generation on the tumor cell surface does not serve simply to activate the cytoplasmic domain of TF by serine phosphorylation. Thus, extracellular functions of the catalytically active TF-VIIa complex cooperate with specific functions of the TF cytoplasmic domain to support the complex process of hematogenous tumor cell dissemination.

Authors

B M Mueller, W Ruf

HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium.

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Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.

Authors

HS Taylor, A Arici, D Olive, P Igarashi

Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin.

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To probe the physiological role of calsequestrin in excitation-contraction coupling, transgenic mice overexpressing cardiac calsequestrin were developed. Transgenic mice exhibited 10-fold higher levels of calsequestrin in myocardium and survived into adulthood, but had severe cardiac hypertrophy, with a twofold increase in heart mass and cell size. In whole cell-clamped transgenic myocytes, Ca2+ channel- gated Ca2+ release from the sarcoplasmic reticulum was strongly suppressed, the frequency of occurrence of spontaneous or Ca2+ current-triggered "Ca2+ sparks" was reduced, and the spark perimeter was less defined. In sharp contrast, caffeine-induced Ca2+ transients and the resultant Na+-Ca2+ exchanger currents were increased 10-fold in transgenic myocytes, directly implicating calsequestrin as the source of the contractile-dependent pool of Ca2+. Interestingly, the proteins involved in the Ca2+-release cascade (ryanodine receptor, junctin, and triadin) were downregulated, whereas Ca2+-uptake proteins (Ca2+-ATPase and phospholamban) were unchanged or slightly increased. The parallel increase in the pool of releasable Ca2+ with overexpression of calsequestrin and subsequent impairment of physiological Ca2+ release mechanism show for the first time that calsequestrin is both a storage and a regulatory protein in the cardiac muscle Ca2+-signaling cascade. Cardiac hypertrophy in these mice may provide a novel model to investigate the molecular determinants of heart failure.

Authors

L R Jones, Y J Suzuki, W Wang, Y M Kobayashi, V Ramesh, C Franzini-Armstrong, L Cleemann, M Morad

Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function.

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Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy.

Authors

L H O'Connor, L C Erway, C A Vogler, W S Sly, A Nicholes, J Grubb, S W Holmberg, B Levy, M S Sands

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.

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This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.

Authors

G Thurston, J W McLean, M Rizen, P Baluk, A Haskell, T J Murphy, D Hanahan, D M McDonald

Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy.

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Phosphomannose isomerase (PMI) deficiency is the cause of a new type of carbohydrate-deficient glycoprotein syndrome (CDGS). The disorder is caused by mutations in the PMI1 gene. The clinical phenotype is characterized by protein-losing enteropathy, while neurological manifestations prevailing in other types of CDGS are absent. Using standard diagnostic procedures, the disorder is indistinguishable from CDGS type Ia (phosphomannomutase deficiency). Daily oral mannose administration is a successful therapy for this new type of CDG syndrome classified as CDGS type Ib.

Authors

R Niehues, M Hasilik, G Alton, C Körner, M Schiebe-Sukumar, H G Koch, K P Zimmer, R Wu, E Harms, K Reiter, K von Figura, H H Freeze, H K Harms, T Marquardt

Exendin(9-39)amide is an antagonist of glucagon-like peptide-1(7-36)amide in humans.

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The gastrointestinal hormone, glucagon-like peptide-1(7-36)amide (GLP-1) is released after a meal. The potency of synthetic GLP-1 in stimulating insulin secretion and in inhibiting glucagon secretion indicates the putative physiological function of GLP-1. In vitro, the nonmammalian peptide, exendin(9-39)amide [ex(9-39)NH2], is a specific and competitive antagonist of GLP-1. This in vivo study examined the efficacy of ex(9-39)NH2 as an antagonist of exogenous GLP-1 and the physiological role of endogenous GLP-1. Six healthy volunteers underwent 10 experiments in random order. In each experiment, a 30-min period of euglycemia was followed by an intravenous infusion of glucose for 150 min that established a stable hyperglycemia of 8 mmol/liter. There was a concomitant intravenous infusion of one of the following: (1) saline, (2) GLP-1 (for 60 min at 0.3 pmol . kg-1 . min-1 that established physiological postprandial plasma levels, and for another 60 min at 0.9 pmol . kg-1 . min-1 to induce supraphysiological plasma levels), (3-5) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + GLP-1, (6-8) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + saline, (9 and 10) GIP (glucose-dependent insulinotropic peptide; for 60 min at 0.8 pmol . kg-1 . min-1, with saline or ex(9-39)NH2 at 300 pmol . kg-1 . min-1). Each volunteer received each of these concomitant infusions on separate days. ex(9-39)NH2 dose-dependently reduced the insulinotropic action of GLP-1 with the inhibitory effect declining with increasing doses of GLP-1. ex(9-39)NH2 at 300 pmol . kg-1 . min-1 blocked the insulinotropic effect of physiological doses of GLP-1 and completely antagonized the glucagonostatic effect at both doses of GLP-1. Given alone, this load of ex(9-39)NH2 increased plasma glucagon levels during euglycemia and hyperglycemia. It had no effect on plasma levels of insulin during euglycemia but decreased plasma insulin during hyperglycemia. ex(9-39)NH2 did not alter GIP-stimulated insulin secretion. These data indicate that in humans, ex(9-39)NH2 is a potent GLP-1 antagonist without any agonistic properties. The pancreatic A cell is under a tonic inhibitory control of GLP-1. At hyperglycemia, the B cell is under a tonic stimulatory control of GLP-1.

Authors

J Schirra, K Sturm, P Leicht, R Arnold, B Göke, M Katschinski

Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth.

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In the search for the existence of adrenergic regulation of the autocrine/paracrine function of the white adipose tissue, it was observed that conditioned media from isolated adipocytes or dialysates obtained by in situ microdialysis of human subcutaneous adipose tissue increased spreading and proliferation of 3T3F442A preadipocytes. These effects were amplified when an alpha2-adrenergic agonist was present during the obtention of conditioned media and microdialysates. This alpha2-adrenergic-dependent trophic activity was completely abolished by pretreatment of the conditioned media or microdialysates with the lysophospholipase, phospholipase B. Among the different lysophospholipids tested only lysophosphatidic acid (LPA) was able to induce spreading and proliferation of 3T3F442A preadipocytes. Moreover, previous chronic treatment of 3T3F442A preadipocytes with LPA which led to a specific desensitization of LPA responsiveness, abolished the alpha2-adrenergic-dependent trophic activities of the conditioned media and microdialysates. Finally, alpha2-adrenergic stimulation led to a rapid, sustained, and pertussis toxin-dependent release of [32P]LPA from [32P]-labeled adipocytes. Based upon these results it was proposed that in vitro and in situ stimulation of adipocyte alpha2-adrenergic receptors provokes the extracellular release of LPA leading, in turn, to regulation of preadipocyte growth.

Authors

P Valet, C Pagès, O Jeanneton, D Daviaud, P Barbe, M Record, J S Saulnier-Blache, M Lafontan

Excess corticotropin releasing hormone-binding protein in the hypothalamic-pituitary-adrenal axis in transgenic mice.

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Corticotropin-releasing hormone (CRH) is the primary hypothalamic releasing factor that mediates the mammalian stress response. The CRH-binding protein (CRH-BP) is secreted from corticotropes, the pituitary CRH target cells, suggesting that the CRH-BP may modulate hypothalamic-pituitary-adrenal (HPA) axis activity by preventing CRH receptor stimulation. Transgenic mice were generated that constitutively express elevated levels of CRH-BP in the anterior pituitary gland. RNA and protein analyses confirmed the elevation of pituitary CRH-BP. Basal plasma concentrations of corticosterone and adrenocorticotropin hormone (ACTH) are unchanged, and a normal pattern of increased corticosterone and ACTH was observed after restraint stress. However, CRH and vasopressin (AVP) mRNA levels in the transgenic mice are increased by 82 and 35%, respectively, to compensate for the excess CRH-BP, consistent with the idea that CRH-BP levels are important for homeostasis. The transgenic mice exhibit increased activity in standard behavioral tests, and an altered circadian pattern of food intake which may be due to transgene expression in the brain. Alterations in CRH and AVP in response to elevated pituitary CRH-BP clearly demonstrate that regulation of CRH-BP is important in the function of the HPA axis.

Authors

H L Burrows, M Nakajima, J S Lesh, K A Goosens, L C Samuelson, A Inui, S A Camper, A F Seasholtz

Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus. Deficient expression of the T cell receptor zeta chain.

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Cellular immunity aberrations in patients with SLE are underscored by the abnormal early Ag receptor-mediated lymphocyte signal transduction pathway. To further characterize the T cell receptor (TCR)/CD3-initiated signaling defects, we studied 22 patients with SLE, 12 patients with other systemic rheumatic diseases, and 14 normal donors. The early (1 min) TCR/CD3-mediated tyrosine phosphorylation of cellular proteins with a molecular size between 36 and 64 kD was increased in 15 of 21 SLE patients, compared to normal or disease control subjects. The deficiency or absence of a band with a molecular size of approximately 16 kD in the immunoblots of SLE patients led us to investigate the expression of the TCRzeta chain. In immunoblots using anti-zeta antibodies we found that 10 of 22 lupus patients tested lacked the expression of TCRzeta, which was always present in control subjects (P < 0.001). Flow cytometric studies using permeabilized cells confirmed the deficiency or absence of the TCRzeta chain in lupus T cells. Using Northern blots we found that for eight patients tested, the TCRzeta mRNA was missing in three, decreased in three, and apparently normal in two patients (P < 0.003), but was always present in control subjects. Reverse transcriptase-PCR verified Northern blot results. We conclude that TCRzeta chain expression is either decreased or absent in the majority of patients with SLE, but not in patients with other systemic rheumatic diseases, regardless of disease activity, treatment status, or clinical manifestations. The previously described increases in TCR-initiated Ca2+ responses and the herein described increases in TCR-induced protein tyrosine phosphorylation and deficient TCRzeta expression may represent intrinsic defects modulating lupus T cell function.

Authors

S N Liossis, X Z Ding, G J Dennis, G C Tsokos

Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model.

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Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.

Authors

C C Silliman, N F Voelkel, J D Allard, D J Elzi, R M Tuder, J L Johnson, D R Ambruso

Altered wound healing in mice lacking a functional osteopontin gene (spp1).

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Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)- containing glycoprotein encoded by the gene secreted phosphoprotein 1 (spp1). spp1 is expressed during embryogenesis, wound healing, and tumorigenesis; however, its in vivo functions are not well understood. Therefore, OPN null mutant mice were generated by targeted mutagenesis in embryonic stem cells. In OPN mutant mice, embryogenesis occurred normally, and mice were fertile. Since OPN shares receptors with vitronectin (VN), we tested for compensation by creating mice lacking both OPN and VN. The double mutants were also viable, suggesting that other RGD-containing ligands replace the embryonic loss of both proteins. We tested the healing of OPN mutants after skin incisions, where spp1 was upregulated as early as 6 h after wounding. Although the tensile properties of the wounds were unchanged, ultrastructural analysis showed a significantly decreased level of debridement, greater disorganization of matrix, and an alteration of collagen fibrillogenesis leading to small diameter collagen fibrils in the OPN mutant mice. These data indicate a role for OPN in tissue remodeling in vivo, and suggest physiological functions during matrix reorganization after injury.

Authors

L Liaw, D E Birk, C B Ballas, J S Whitsitt, J M Davidson, B L Hogan

Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus.

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The mechanism that regulates the synthesis of nitric oxide synthase (NOS), a key enzyme responsible for NO production in the myenteric plexus, remains unknown. We investigated the roles of the vagal nerve and nicotinic synapses in the mediation of NOS synthesis in the gastric myenteric plexus in rats. Truncal vagotomy and administration of hexamethonium significantly reduced nonadrenergic, noncholinergic relaxation, the catalytic activity of NOS, the number of NOS-immunoreactive cells, and the density of NOS-immunoreactive bands and NOS mRNA bands obtained from gastric tissue. These results suggest that NOS expression in the gastric myenteric plexus is controlled by the vagal nerve and nicotinic synapses. We also investigated if stimulation of the nicotinic receptor increases neuronal NOS (nNOS) expression in cultured gastric myenteric ganglia. Incubation of cultured gastric myenteric ganglia with the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP, 10(-10)-10(-7) M), for 24 h significantly increased the number of nNOS-immunoreactive cells and the density of immunoreactive nNOS bands and nNOS mRNA bands. nNOS mRNA expression stimulated by DMPP was antagonized by a protein kinase C antagonist, a phospholipase C inhibitor, and an intracellular Ca2+ chelator. We concluded that activation of the nicotinic receptor stimulates a Ca2+-dependent protein kinase C pathway, which in turn, upregulates nNOS mRNA expression and nNOS synthesis in the gastric myenteric plexus.

Authors

K Nakamura, T Takahashi, M Taniuchi, C X Hsu, C Owyang

A nonsense mutation in the carboxyl-terminal domain of type X collagen causes haploinsufficiency in schmid metaphyseal chondrodysplasia.

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Abstract

Type X collagen is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggested a critical role for this type X collagen domain, but since no direct analysis of cartilage has been conducted in SMCD patients, the mechanisms of type X collagen dysfunction remain controversial. To resolve this problem, we obtained SMCD growth plate cartilage, determined the type X collagen mutation, and analyzed the expression of mutant and normal type X collagen mRNA and protein. The mutation was a single nucleotide substitution that changed the Tyr632 codon (TAC) to a stop codon (TAA). However, analysis of the expression of the normal and mutant allele transcripts in growth plate cartilage by reverse transcription PCR, restriction enzyme mapping, and a single nucleotide primer extension assay, demonstrated that only normal mRNA was present. The lack of mutant mRNA is most likely the result of nonsense-mediated mRNA decay, a common fate for transcripts carrying premature termination mutations. Furthermore, no mutant protein was detected by immunoblotting cartilage extracts. Our data indicates that a functionally null allele leading to type X collagen haploinsufficiency is the molecular basis of SMCD in this patient.

Authors

D Chan, Y M Weng, H K Graham, D O Sillence, J F Bateman

Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes.

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Abstract

The mechanisms involved in host clearance of symptomatic mucocutaneous herpes simplex virus (HSV) infection are unclear. We studied the functional properties of bulk cultures of skin-infiltrating lymphocytes from normal skin and serial biopsies of recurrent genital HSV-2 lesions, and compared HSV-specific and NK responses with viral clearance. HSV-specific CD4+ or CD8+ T cells were rarely detected in lymphocytes cultured from normal skin. The total lymphocyte count and HSV-specific and NK-like effector cell activities were markedly higher in cultures derived from lesional skin. HSV-specific CD4+ proliferative responses and NK-like cytotoxic responses were present at all stages of herpetic lesions, including biopsies early in the disease course. In contrast, cytotoxic T lymphocyte activity was generally low among cells derived from early culture-positive lesions, and increased during lesion evolution. Viral clearance from the lesion site was associated with a high level of local cytolytic activity towards HSV-infected cells. The phenotypes of cells with HSV-specific cytotoxic responses varied between patients, having CD4+ and CD8+ components. Immunotherapeutic approaches to HSV should be directed at improving in vivo cytolytic activity to HSV.

Authors

D M Koelle, C M Posavad, G R Barnum, M L Johnson, J M Frank, L Corey

Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

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Abstract

In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis.

Authors

Z Bata-Csorgo, K D Cooper, K M Ting, J J Voorhees, C Hammerberg

Bidirectional modulation of insulin action by amino acids.

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Abstract

Amino acids have been shown to stimulate protein synthesis, inhibit proteolysis, and decrease whole-body and forearm glucose disposal. Using cultured hepatoma and myotube cells, we demonstrate that amino acids act as novel signaling elements in insulin target tissues. Exposure of cells to high physiologic concentrations of amino acids activates intermediates important in the initiation of protein synthesis, including p70 S6 kinase and PHAS-I, in synergy with insulin. This stimulatory effect is largely due to branched chain amino acids, particularly leucine, and can be reproduced by its transamination product, ketoisocaproic acid. Concurrently, amino acids inhibit early steps in insulin action critical for glucose transport and inhibition of gluconeogenesis, including decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, decreased binding of grb 2 and the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2, and a marked inhibition of insulin-stimulated phosphatidylinositol 3-kinase. Taken together, these data support the hypothesis that amino acids act as specific positive signals for maintenance of protein stores, while inhibiting other actions of insulin at multiple levels. This bidirectional modulation of insulin action indicates crosstalk between hormonal and nutritional signals and demonstrates a novel mechanism by which nutritional factors contribute to insulin resistance.

Authors

M E Patti, E Brambilla, L Luzi, E J Landaker, C R Kahn

Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats.

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Abstract

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

Authors

F Cosentino, S Patton, L V d'Uscio, E R Werner, G Werner-Felmayer, P Moreau, T Malinski, T F Lüscher