To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35 S]methionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10 5 -fold increase) after trypsin but not chymotrypsin treatment. This trypsinenhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.Astroviruses were first identified in the stools of children with gastroenteritis and characterized by electron microscopy (EM) (18). The particles were approximately 28 nm in diameter, and some had a star-like morphology. Since the initial observation in humans, similar viruses have been identified in a variety of other mammalian species. Lee and Kurtz first demonstrated growth of the virus in tissue culture and showed that trypsin is necessary for ongoing astrovirus replication (16). The development of enzyme immunoassays (EIAs) (11,12) and reverse transcriptase PCR assays has allowed larger numbers of samples to be screened with more-sensitive tests (13,14,21). It has become apparent that astroviruses are an important cause of gastroenteritis in a variety of settings, including nosocomial infections, daycare outbreaks, and diarrhea in outpatients (5-10, 19, 20, 24, 26).The astrovirus genome consists of a single-stranded positivesense RNA of approximately 7,200 nucleotides with three open reading frames (ORFs). The first, ORF1a, encodes a serine 3C type of viral protease (27). The second, ORF1b, is separated from ORF1a by a frame shift and encodes a viral RNA polymerase (17). The third, ORF2, encodes an 87-kDa polypeptide which functions as the capsid precursor. During infection, a subgenomic RNA that encodes the capsid precursor is found in abundance in the cell cytoplas...

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP) (7), and placed on ice. At the end of experiments, the abdominal cavity was exposed via a midline incision and a blood sample was drawn from the inferior vena cava. The portal vein was then cannulated with a 21-gauge needle, which was tied in place. The vena cava above the liver was then incised and the liver was flushed with 3 ml of cold 0.15 M NaCl under a pressure of 50 cm of water.The liver was then removed and weighed. A mixed endosome fraction was prepared by minor modifications of methods developed for rat liver (18). Two livers were pooled and homogenized in 0.5 M sucrose, and an endosome-enriched subcellular fraction was obtained after several steps by centrifugation in a Percoll gradient. This material was diluted with 2 vol of 0.15 M NaCI, and the endosomes were obtained by centrifugation onto a sucrose cushion. The fluffy white material just above the sucrose cushion was harvested with a Pasteur pipette, resuspended in 0.15 M NaCl, and recentrifuged as above. The purified endosomes were again harvested from just above the sucrose cushion for analysis. Their appearance by electron microscopy closely resembled that shown previously for hepatocytic endosomes from rats (18 RESULTSLDLR (-/-) mice had increased levels of cholesterol in LDL, as reported previously (7), and also in IDL, but only a slight increase in VLDL-cholesterol (Table 1). The concentrations of apoB-100 and apoB-48 in VLDL of normal and LDLR (-/-) mice were similar. The total concentration of apoB-100 in plasma, however, was increased almost 4-fold, mainly in LDL, and that of apoB-48 was increased only about 1.4-fold, also mainly in LDL, with a lesser increase in IDL. The concentration of apoE was increased 2.5-fold, predominantly in the LDL and IDL fractions, with a minor increase in VLDL.To determine the effectiveness of GST-RAP in blocking removal of activated a2-macroglobulin by the liver, the hepatic content of 125I was determined at intervals after intravenous

Background. Antiplatelet therapy with aspirin and antithrombotic therapy with heparin both prevent the complications of unstable angina; however, no definitive data exist on the relative clinical efficacy of the two drugs.Methods and Results. Aspirin (325 mg bid) or heparin (5000-U intravenous bolus followed by a perfusion titrated to the APTT) were compared in a double-blind randomized trial of 484 patients in two cohorts enrolled sequentially. The study was initiated at admission to hospital at a mean of 8.3±7.8 hours after the last episode of pain. End points were assessed 5.7±3.3 days later, when the decision for long-term management was made. Myocardial infarction occurred in 2 (0.8%) of the 240 patients randomized to heparin and in 9 (3.7%) of the 244 randomized to aspirin (P=.035), an odds ratio of 0.22 and a risk difference of 2.9%o (95% confidence limits, 0.3% to 5.6%) with heparin. The only death resulted from a

The 10-kb deletion ("French Canadian mutation") of the low-density lipoprotein (LDL) receptor gene is the most common mutation causing familial hypercholesterolemia among subjects of French Canadian descent. In affected subjects, it results in a null allele of the LDL receptor gene and provides a unique opportunity to examine single-allele regulation of this gene in humans. We sought to ascertain the response of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in subjects with the French Canadian mutation of the LDL receptor gene and to correlate this response with biochemical variables and the haplotype of the nondeletion LDL receptor allele. The prevalence of nonresponders to high doses of HMG CoA reductase inhibitors (denned as <15% decrease in LDL cholesterol [LDL-C] from baseline values after dietary intervention) was ascertained in 105 patients heterozygous for the 10-kb deletion after excluding first-degree relatives and those on combined lipid-lowering therapy or other lipid-lowering agents. Lipoprotein cholesterol levels were examined after a diet period (30% calories as fat) and after receiving HMG CoA reductase inhibitors as monotherapy for a minimum of 3 months. The mean reduction in total cholesterol was 45±23%, in LDL-C 33±15%, and in F amilial hypercholesterolemia (FH) is a genetic disorder characterized by elevated plasma levels of total cholesterol due to elevated low-density lipoprotein (LDL) cholesterol (LDL-C) and cutaneous lipid deposits (xanthelasmas, xanthomas, and corneal arcus) as well as the presence of premature coronary artery disease in probands and family members.1 ' 2 Mutations within the gene coding for the LDL receptor have been identified as the cause of FH.3 Multiple mutations have been described, 4 and five classes of phenotypes at the cellular level have been established. Class 1 defects do not produce LDL receptor protein (null allele); class 2 defects encode proteins that are blocked between the endoplasmic reticulum and the Golgi complex; class 3 defects encode proteins that do triglycerides 32±49% (all P<.005). There was a slight increase in high-density lipoprotein cholesterol of 8.5±18% (P>.05). Overall, 68.4% of patients had more than a 30% decrease in LDL-C levels and 23% had a decrease ranging from 15% to 30%; 8.6% of patients failed to respond, and some even showed an increase in their LDL-C levels (mean change, 1.2±11.9%). This response was independent of age, gender, triglycerides, or high-density lipoprotein cholesterol levels. There was no significant overall effect of the apolipoprotein E phenotype in predicting the degree of total cholesterol or LDL-C reduction. Haplotypes of the LDL receptor gene were determined with the use of five polymorphic markers. The 10-kb deletion is known to be present on a single haplotype, thus allowing the haplotype determination of the nondeletion allele. Analysis of the nondeletion allele of the LDL receptor did not reveal a difference between responders and nonresponders, suggesting that a defec...

Plasma levels of endothelin-1 are elevated in acute myocardial infarction with higher levels in complicated infarctions. Measurements of levels in unstable angina could help clarify whether the elevation is the consequence of cell necrosis or is in some way related to the pathophysiology of acute coronary syndromes. Plasma endothelin-1 levels were determined by radioimmunoassay in 29 patients with unstable angina and 6 with a myocardial infarction. Blood samples were obtained at admission before drug administration and 6 and 72 h later. Levels were also determined in 27 control subjects and in 29 patients with stable angina. Admission levels were similar in unstable angina, 0.635 ± 0.052 pg/ml [log(l +x)], and myocardial infarction, 0.746 ± 0.122, and significantly higher than in controls, 0.428 ± 0.047, and stable angina patients, 0.449 ± 0.052 (p < 0.01). In unstable angina, levels decreased progressively to normal at 6 h, 0.557 ± 0.049 pg/ml, and 72 h 0.474 ± 0.054, as opposed to an increase to 0.868 ± 0.109 (p < 0.05) after 6 h in myocardial infarction followed by a decrease to 0.597 ± 0.122 at 72 h. The elevation in unstable angina did not correlate with other clinical or laboratory characteristics. Unstable angina is associated with an increase in endothelin-1 plasma levels during the acute phase, suggesting a role of this endothelium-derived vasoactive peptide in the pathophysiology of acute coronary artery syndromes.