Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha v beta3 integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta3 is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta3 integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta3 or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P<0.01) and 18.4 +/- 0.9 days (P<0.01), respectively. XT-199, a chemical blocker of the alpha v beta3 receptor also prolonged survival to 19.5 +/- 1.3 days (P<0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta1 antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta1 receptors as well as through beta3 receptors in vivo. Our data demonstrate that the beta3 receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta3 for cellular entry within an organism.
Animals ; Animals, Newborn ; Antibodies, Monoclonal/therapeutic use ; Antigens, CD29/metabolism ; Hantaan virus/*metabolism/pathogenicity ; Hemorrhagic Fever with Renal Syndrome/mortality/*virology ; Imidazoles/pharmacology ; Integrin alphaV/metabolism ; Integrin alphaVbeta3/antagonists & inhibitors ; Integrin beta3/*metabolism ; Mice ; Receptors, Virus/*metabolism ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the alpha v beta 3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of alpha v mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the alpha v beta 3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the alpha v beta 3 protein. No staining was evident in control aortas for the alpha v beta 3 antigen, tenascin or alpha v mRNA. The upregulation of av mRNA and the alpha v beta 3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.
Adult ; Aorta, Abdominal/immunology/pathology ; Aortic Aneurysm, Abdominal/*pathology ; Biological Markers/analysis/metabolism ; Female ; Humans ; Integrin alphaVbeta3/analysis/genetics/metabolism ; Male ; Neovascularization, Pathologic/genetics/*metabolism ; Nitric-Oxide Synthase/analysis/metabolism ; RNA, Messenger/analysis/metabolism ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, P.H.S. ; Tenascin/analysis/metabolism

Effects of diets on hepatic aflatoxin B1 (AFB1)- DNA binding and AFB1-induced glutathione S- transferase placental (GST-P) form positive hepatic foci have been examined in young male Fischer rats. Animals were fed either AIN-76A or Purina Chow (PC) diet for 1 wk before AFB1- DNA binding studies in vivo and in vitro. Animals were injected i.p. with AFB1 (1 mg/kg body wt) and 3 days later were given either AIN-76A or PC diet with or without 0.1% phenobarbital (PB) in their drinking water. All animals were sacrificed 10 wks after AFB1 dosing for analysis of AFB1-induced GST-P positive hepatic foci by immunochemistry. Two h after i.p. injection of AFB1, hepatic AFB1-DNA binding in AIN-76A fed rats was twice as much as those in PC fed animals without affecting GSH levels. There was no significant effect of diet on either cytochrome P-450 content, GSH levels or microsomal cytochrome P-450 mediated AFB1-DNA binding to exogenous DNA. There was a 40% increase in cytosolic GSH S-transferase activity with 1-chloro-2,4-dinitrobenzene as a substrate in PC fed animals compared to those given AIN- 76A diet. The number and area of AFB1-induced GST-P positive hepatic foci were twice and fivefold as much in AIN-76A fed compared to those in PC fed rats. The number of AFB1-induced GST-P positive foci was increased 5-10 fold in the presence of PB in both groups. In summary, the present data indicate that feeding of PC diet compared to AIN-76A diet inhibits the initiation phase whereas AIN-76A stimulates the promotion phase of AFB1 hepatocarcinogenesis in rats by inhibiting AFB1-DNA binding and increasing AFB1-induced hepatic foci respectively.
Aflatoxin B1/metabolism/*pharmacology ; Animals ; Cell Transformation, Neoplastic ; Cytochrome P-450 Enzyme System/metabolism ; DNA/*metabolism ; *Diet ; Glutathione Transferase/analysis/*metabolism ; Hepatocytes/drug effects/*enzymology ; Liver Neoplasms/*etiology ; Microsomes, Liver/enzymology ; Rats ; Research Support, U.S. Gov't, P.H.S.

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonulease activity was found to be at 37degrees C to 42degrees C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.
Amino Acid Substitution ; Animals ; Anti-Bacterial Agents/pharmacology ; Baculoviridae ; Capsid/*enzymology ; Cell Line ; Cysteine/genetics ; Endoribonucleases/antagonists & inhibitors/chemistry/genetics/isolation & purification/*metabolism ; Enzyme Activation/drug effects ; Heat ; Insects ; Leishmania guyanensis/*virology ; RNA/chemistry ; RNA Viruses/*enzymology/genetics ; Recombinant Proteins/antagonists & inhibitors/genetics/isolation & purification/metabolism ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S. ; Substrate Specificity/genetics ; Transduction, Genetic

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease whose etiopathogenesis is not well understood. Although soluble (s) forms of 4-1BB (s4-1BB) and 4-1BB legand (s4-1BBL) have been detected in the sera of RA patients, their significance is not known. We compared the serum levels of s4-1BB and s4-1BBL in RA patients with those in systemic lupus erythematosus (SLE) and Behcet's disease (BD) patients. Serum levels of s4-1BB and s4-1BBL were significantly higher in RA patients compared with healthy controls, SLE or BD patients, and the abundance was correlated with disease severity in patients with RA. The serum levels of s4-1BB in RA patients were inversely corroborated with 4-1BB expression levels on activated T lymphocytes. In addition, there was a correlation between serum levels of s4-1BB and s4-1BBL. The augmented secretion of s4-1BB and s4-1BBL levels into the serum may reflect the clinical symptoms of RA and levels of s4-1BB and s4-1BBL in sera at the time of diagnosis may be indicative of the severity and outcome of RA.
Adult ; Aged ; Antigens, CD/metabolism ; Arthritis, Rheumatoid/*blood/drug therapy/immunology/*pathology ; Behcet Syndrome/blood/immunology ; Comparative Study ; Female ; Humans ; Immunosuppressive Agents/metabolism/therapeutic use ; Leukocytes, Mononuclear/metabolism ; Lupus Erythematosus, Systemic/blood/immunology ; Male ; Middle Aged ; Random Allocation ; Receptors, Nerve Growth Factor/*blood ; Receptors, Tumor Necrosis Factor/*blood ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S. ; Severity of Illness Index ; Statistics ; Tumor Necrosis Factor-alpha/*metabolism

No abstract available.
Animal ; Cattle ; Chick Embryo ; Comparative Study ; Human ; Methylation ; Protein Methyltransferases/metabolism* ; Proteins/metabolism* ; Rats ; Species Specificity ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S.

No abstract available.
Animal ; Arteries/pathology ; Arteriosclerosis/pathology* ; Diet, Atherogenic ; Disease Models, Animal ; Support, U.S. Gov't, P.H.S. ; Swine

We studied the effects of organic and inorganic mercury (Hg) on the uptake of L-[3H] glutamate (L-GLU) in cultured mouse astrocytes. Following exposure to mercuric chloride (MC) [0.2 approximately 5.0 microM], selective and dose-dependent inhibition of L-GLU uptake to 50% of control levels was observed, whereas 2-deoxyglucose (2-DG) uptake was not significantly affected. Methylmercuric chloride (MMC) also inhibited L-GLU uptake but 50% reduction was reached only at a concentration of 10 microM. Inhibition of L-GLU uptake by MMC appears to be closely linked to voltage-sensitive calcium channels as evidenced by the lack of L-GLU uptake inhibition by MMC in calcium-free medium or in the presence of the channel blocker verapamil. Exposure to a variety of divalent metallic ions, including CuCl2, FeCl2 and ZnCl2, did not affect L-GLU uptake in astrocytes in vitro. Exposure to PbCl2, however, resulted in a decline in L-GLU uptake, though to a much smaller degree than that observed with Hg compounds. Selective impairment of astroglial L-GLU transport may represent a critical early pathogenetic feature of Hg-induced neurotoxicity.
Animal ; Astrocytes/*drug effects/metabolism ; Cells, Cultured ; Glutamic Acid/*metabolism ; Mercury/*toxicity ; Mice ; Mice, Inbred C57BL ; Support, U.S. Gov't, P.H.S.

The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.
Animals ; Basement Membrane/drug effects/metabolism ; Cattle ; Cells, Cultured ; Diabetic Nephropathies/metabolism ; Endothelial Cells/cytology/*drug effects/*metabolism ; Gene Expression/drug effects ; Glucose/*pharmacology ; Heparan Sulfate Proteoglycan/genetics/*metabolism ; Kidney Glomerulus/*cytology ; Sulfur Radioisotopes/diagnostic use ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S.

A chemopreventive approach to cancers of the upper aerodigestive tract (including those of head and neck and lung) to reduce the incidence and mortality rates for these cancers has become an important strategy because therapies such as surgery, radiation, and chemotherapy have only marginally improved the five-year survival rate over the last two decades. However, chemopreventive trials have been hampered by serious feasibility problems, including high cost, the requirement of large numbers of patients, and long-term follow-up necessary to determine cancer incidence, which served as the study end point. Thus, the use of biomarkers, the identification of which would serve as an intermediate end point of the study has recently emerged as a subject of great interest. To try to understand the process of tumorigenesis from normal tissues through the premalignant tissue stage to malignant lesions, there has recently been a search for genetic and/or phenotypic changes that qualify as candidates for biomarkers. These candidates include genomic markers, certain specific genetic markers (such as oncogenes, growth factors and their receptors, and tumor suppressor genes), cell proliferation markers, and cell differentiation markers. This review covers genomic markers (including micronuclei and specific chromosomal alterations) and specific genetic markers (such as the ras gene family, the myc family, erb B1, int-2/hst-1, and the p53 tumor suppressor gene). As a consequence of genetic alteration, we also reviewed cell proliferation markers such as proliferating cell nuclei antigen (PCNA) and the squamous cell differentiations markers, including keratins, involucrin, and transglutaminase 1. These biomarker candidates are important adjuncts to the development of the new chemopreventive agents and to the rational design of future intervention trials. However, it should be emphasized that these biomarkers must first be validated in clinical trials; only then can they replace cancer incidence as the sole end point in chemoprevention trials.
Chromosome Aberrations ; Digestive System Neoplasms/*diagnosis/prevention & control ; Genetic Markers ; Human ; Respiratory Tract Neoplasms/*diagnosis/prevention & control ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tumor Markers, Biological/*blood