Biomedical subjects
B E Clarke
Publications and source records attributed to B E Clarke.
Survival of patients with biopsy-proven usual interstitial pneumonia and nonspecific interstitial pneumonia.
This is the first Australian study to examine survival and clinical characteristics in biopsy-proven idiopathic interstitial pneumonia. A cohort of 70 patients from a single institution between January 1990 and December 1999 was reviewed. All patients were Caucasian, 23 (33%) female. Mean age+/-SD at diagnosis was 60+/-12 yrs for males and 54+/-14 yrs for females. A total 24% of patients had never smoked. The histopathological diagnoses were usual interstitial pneumonia (UIP) (n=59), nonspecific interstitial pneumonia (NSIP) (n=7), desquamative interstitial pneumonia (n=3) and acute interstitial pneumonia (n=11). Clinical and functional characteristics of the two main histological subgroups of UIP and NSIP showed significantly older patients in the UIP group and a significantly lower mean forced expiratory volume in one second (FEV1) in the NSIP group. Median survival for UIP was 78 months compared with 178 months for NSIP. No survival difference between treated and untreated patients with UIP was found. Multivariate analysis revealed smoking alone to be predictive of poorer survival. This study demonstrates the best median survival for usual interstitial pneumonia of available series and confirms a survival difference between usual interstitial pneumonia and nonspecific interstitial pneumonia. Furthermore, the reported results may have implications for treatment timing using conventional protocols currently recommended.
Transesophageal echocardiographic abnormalities in a case of cardiac sarcoidosis.
Sarcoidosis is a granulomatous disease that may involve multiple organ systems, including the heart. Manifestations include atrial and ventricular arrhythmias, conduction abnormalities, congestive cardiac failure, pericarditis, and sudden death. Whereas cardiac involvement is a relatively common finding at autopsy, antemortem diagnosis is often missed because the clinical manifestations are nonspecific, and the sensitivity and specificity of investigations are low. We report a case of a 62-year-old woman who had clinically significant cardiac sarcoidosis associated with echocardiographic abnormalities that had not been reported previously in association with this condition.
Life-threatening haemoptysis presenting as a late complication of an ovarian tumour.
Massive haemoptysis may arise as a result of lung malignancy. This case represents the first report of an ovarian granulosa cell tumour metastasising many years after initial tumour resection to the lung causing life-threatening haemoptysis. The management and subsequent clinical course of the patient are discussed as well as the natural history of granulosa-theca cell tumours.
Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity.
The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required.
Localization of the mosaic transmembrane serine protease corin to heart myocytes.
Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125-135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.
New drugs for hepatitis C virus (HCV).
Lack of efficacy and significant side effects have severely limited the use of interferon-alpha (IFN-alpha) as the standard therapy for non-A non-B hepatitis (NANBH) caused by hepatitis C virus (HCV) and alternative, improved therapies are urgently sought. Attempts have been made to improve the potency and tolerability of IFN-alpha by adjusting dosing regimens, methods of delivery and length of treatment. Furthermore, a number of different agents have been used in combination wit IFN-alpha and, from these studies, therapeutic options have been galvanized by the synergistic effects of IFN-alpha and ribavirin. Nevertheless, the majority of patients with HCV still do not sustain lasting therapeutic benefit from this combination and continuing research is required to identify new therapeutic candidates that will have more potent antiviral activity and less severe side effects. This review focuses on the progress that has been made in this area and the prospects for new effective therapies in the near future.
Receptors for hepatitis C virus.
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Microvascular complications in cystic fibrosis-related diabetes mellitus: a case report.
CONTEXT, The prevalence of cystic fibrosis-related diabetes mellitus is increasing and is associated with increased survival from cystic fibrosis. CASE REPORT, This study describes a case of the premature onset of disabling and widespread microvascular complications resulting from cystic fibrosis-related diabetes mellitus. Previously asymptomatic retinopathy was diagnosed on recognition of diabetic nephropathy. CONCLUSIONS, The treatment of pulmonary exacerbations has become more complex due to the nephrotoxic potential of intravenous aminoglycoside drugs which are frequently used to control chronic Pseudomonas infection in cystic fibrosis.
A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain.
The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 microM at pH 7.0 and 25 degrees C. The enzyme had maximal affinity for NA with 12 or more nucleotides. A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector. The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s-1 and 2.7 s-1, respectively. The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 microM-1 s-1 (3.9 s-1) and 290 microM-1 s-1 (2.27 s-1), respectively. The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 microM-1 s-1 (<0.5 s-1) and 0.9 microM-1 s-1 (<10(-1) s-1), respectively. These data were consistent with a random kinetic mechanism.
Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.
Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.
Unique features of internal initiation of hepatitis C virus RNA translation.
The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.
Induction of T-helper cell response to hepatitis B core antigen in chronic hepatitis B: a major factor in activation of the host immune response to the hepatitis B virus.
The T helper (Th) cell response to hepatitis B core antigen (HBcAg) was analyzed in 76 chronic hepatitis B virus (HBV) carriers with varying degrees of hepatic inflammation and HBV replication. Fifty-five patients had active viral replication, 28 with minimal histological changes and normal alanine transaminase (ALT) and 27 with active hepatic inflammation and elevated ALT. The remaining 21 chronic hepatitis B surface antigen (HBsAg) carriers had undetectable HBV replication, minimal histological activity, and normal ALT. In addition, 34 chronic HBV carriers were studied prospectively during treatment with alpha-interferon. The HBcAg-specific Th cell response was evaluated by a proliferative assay using 3H-thymidine uptake and gamma-interferon production by peripheral blood mononuclear cells. The proliferative response and gamma-interferon production of patients with active hepatic inflammation were significantly higher than in patients with minimal histological changes and in controls. In the longitudinal analysis during alpha-interferon treatment, 22 of 34 patients sustained an ALT flare accompanied by a parallel, significant Th cell response, which preceded or coincided with the ALT flare. The elevation in the Th cell response and the ALT flare were followed by a significant rise in the serum immunoglobulin (Ig) M anti-HBc index. Ten of twenty-two patients with an enhanced Th cell response and an ALT flare seroconverted after alpha-interferon treatment. The Th cell activity in the 10 responders rapidly subsided after hepatitis B e antigen (HBeAg) to anti-HBe seroconversion, whereas in the 12 nonresponders it remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)
In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease.
The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.
Approaches to the development of novel inhibitors of hepatitis C virus replication.
Chronic infection with the hepatitis C virus is a major health problem. Conventional therapy, with interferon-alpha is effective in only a minority of patients. The failure of current treatments has led to a major initiative to identify new antiviral agents. In the absence of a tissue culture model for hepatitis C infection the pharmaceutical industry has been obliged to investigate the basic biology of hepatitis C viral replication. Such studies have identified novel translational regulatory elements and new proteolytic enzymes which may serve as targets for new antiviral drugs.
Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro.
The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.
Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.
The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.
The nature and spatial distribution of amino acid substitutions conferring resistance to neutralizing monoclonal antibodies in human rhinovirus type 2.
A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1.7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.