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Biomedical subjects

R A Hall

Publications and source records attributed to R A Hall.

At least 55 records · Page 3Linked to original sources
PubMed1999

Identification and analysis of truncated and elongated species of the flavivirus NS1 protein.

The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the viral polyprotein. In this study, we determined the basis of structural heterogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by glycosylation analysis, pulse-chase experiments and terminal amino acid sequencing. Inhibition of N-linked glycosylation by tunicamycin revealed that NS1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1(o)) and 47 kDa (NS1'). Pulse-chase experiments established that no precursor-product relationship existed between NS1(o) and NS1' and that both were stable end products. Terminal sequencing revealed that the N- and C-termini of NS1(o) were located at amino acid positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. Expression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1' was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (deltaNS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing revealed a 120-residue truncation at the N-terminus of this protein. This corresponds closely with the in-frame 121-codon deletion at the 5' end of the NS1 gene of defective MVE viral RNA (described by Lancaster et al. in 1998), suggesting that deltaNS1 may be a translation product of defective viral RNA.

Aedes↗
PubMed1999

Complications of surgical feeding jejunostomy in trauma patients.

OBJECTIVE: To determine the complication rate of feeding jejunostomy (FJ) performed as an adjunct to trauma celiotomy. METHODS: Retrospective analysis of 222 patients from January of 1988 to May of 1998. RESULTS: Thirty-seven total FJ-related complications occurred in 22 patients (10%). Major FJ-related complications occurred in nine patients (4%): two small bowel perforations, two small bowel volvuli with infarction, two intraperitoneal leaks, and three small bowel necroses. Patients suffering major FJ-related complications were similar to those without complications, except for the FJ type. Patients with major FJ-related complications were more likely to have had a Witzel tube jejunostomy than a needle catheter jejunostomy (p = 0.03). Three deaths were related to major FJ complications, for a FJ-related mortality rate of 1.4%. CONCLUSIONS: FJ has a major complication rate of 4% in severely injured patients. Major complications occur more frequently with larger, Witzel-type tubes. Needle catheter jejunostomy appears to be a safer method of surgical jejunal access in trauma patients.

Abbreviated Injury Scale↗
PubMed1999

Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase.

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.

Animals↗
PubMed1999

The use of micro-dose aprotinin with continuous infusion in coronary artery bypass surgery.

BACKGROUND: To evaluate the efficacy of aprotinin at a dose far less than standard. METHODS EXPERIMENTAL DESIGN: Retrospective, case-control study. SETTING: community-based, teaching hospital PATIENTS: one hundred one patients undergoing primary, non-emergent, coronary artery bypass during two, six-month periods were studied. INTERVENTIONS: during the first period aprotinin was not administered, and these patients served as controls (n = 52). During the second period all patients received aprotinin via a micro-dose regimen (n = 49). MEASURES: postoperative bleeding and blood product usage served as determinants of efficacy. RESULTS: A significant difference existed in postoperative bleeding with the mean thoracic drain outputs being reduced in the aprotinin group both at 6 hours (p = 0.0003) and in total (p = 0.0004). This was further supported by significantly higher hematocrits (p = 0.03) on the first postoperative day in patients receiving aprotinin. Likewise, there was a significant reduction in total blood product exposures (p = 0.04) and platelet usage (p = 0.02) in the aprotinin group with a tendency towards decreased red cell usage. Further, when all patients with a hematocrit < or =30% prior to bypass were excluded, the significant reduction in total blood product exposures persisted (p = 0.04), and there was a significant reduction in red cell usage (p = 0.04) with a trend towards decreased platelet usage (p = 0.06) in the aprotinin group. CONCLUSIONS: Micro-dose aprotinin significantly reduces postoperative bleeding and blood product usage in primary, non-emergent, CABG patients.

Aprotinin↗
PubMed1998

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

Amino Acid Sequence↗
PubMed1998

GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Arboviruses in the Australian region, 1990 to 1998.

Arboviruses continue to be major human pathogens in the Australian region. This report provides a summary of the activities of these viruses over the past eight years, and comments on new findings relevant to their respective ecologies. Of particular interest and concern is the propensity of these viruses to spread. The examples discussed include the initiation of dengue epidemics in north Queensland by virus imported in viraemic travellers; the spread of Japanese encephalitis virus to the Australasian region and its probable enzootic establishment in the south-west of Papua New Guinea; the potential spread of Ross River virus to other countries, as demonstrated by the 1979-80 outbreak in the South Pacific, and the recent occurrence in military personnel from the United States of America after an exercise; and the recent spread of Barmah Forest virus into Western Australia.

Animals↗
PubMed1998

The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin.

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Animals↗
PubMed1998

Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF).

Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.

Adenosine Triphosphate↗
PubMed1998

The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange.

Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

Adrenergic beta-2 Receptor Agonists↗
PubMed1998

Identification of Australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA.

An ELISA using a panel of specific monoclonal antibodies was developed to identify all alpha and flaviviruses isolated from mosquitoes caught throughout Australia. This technique is sensitive and rapid and is more specific than the traditional methods used to identify flaviviruses. The ability to identify unknown virus isolates from field-caught mosquitoes quickly and accurately improves the efficiency of arbovirus surveillance programs and allows health authorities to give an early warning of an increased health risk from a mosquito-borne virus in a particular region.

Aedes↗
PubMed1998

An audit of prostate-specific antigen and clinical symptoms in general practice.

The objective was to devise local guidelines for the referral of patients with suspected prostatic carcinoma following evaluation by a retrospective audit of the value of the prostate-specific antigen concentration, together with age, urological symptoms, and digital rectal examination in the diagnosis of carcinoma of the prostate. Relevant details were collected from the notes of 582 patients from general practice and hospital. The significant diagnostic factors were ascertained by stepwise logistic regression. Prostate-specific antigen concentration, digital rectal examination and significant terminal dribbling were the most powerful factors in the diagnosis of carcinoma of the prostate. When prostate-specific antigen concentration was considered in isolation, a value of 6.5 ng/ml appeared appropriate for referral. Age was not significant, perhaps due to the narrow patient age range. The significant diagnostic factors were built into an algorithm calculating the probability of carcinoma of the prostate. This algorithm, together with prostate-specific antigen concentration results and digital rectal examination findings, forms the basis of the referral guidelines and a subsequent prospective study.

Adult↗
PubMed1998

Development of a thyroid function strategy for general practice.

A study was carried out to investigate a thyroid stimulating hormone (TSH) frontline strategy that could potentially result in a more straightforward interpretation of thyroid function tests, a reduction in the number of inappropriate referrals to medical outpatients, an improvement in the 'turnaround time' of results, and a reduction in the number of unnecessary tests carried out, thereby reducing costs.

Decision Trees↗
PubMed1997

Differential surface expression and phosphorylation of the N-methyl-D-aspartate receptor subunits NR1 and NR2 in cultured hippocampal neurons.

The trafficking and phosphorylation of the NR1 and NR2 subunits of the N-methyl-D-aspartate-type glutamate receptor complex were studied in cultured rat hippocampal neurons. Surface expression was examined by modifying surface receptors via treatment of intact neurons with either the protease chymotrypsin or the cross-linking reagent bis(sulfosuccinimidyl)suberate, followed by quantification of anti-NR1 and anti-NR2B Western blot immunostaining. These studies revealed that only 40-50% of total NR1 immunoreactivity is found at the cell surface, as compared to more than 90% of total NR2B immunoreactivity. Metabolic labeling of the cultures with 32P revealed that NR2 subunits are highly phosphorylated under basal conditions, whereas basal phosphorylation of NR1 subunits is barely detectable. Following stimulation of the cultures with glutamate/glycine or phorbol esters, NR1 phosphorylation was found to be enhanced by 3-5-fold, whereas phosphorylation of NR2 subunits was enhanced by less than 2-fold. To determine whether the difference in the basal NR1 versus NR2 phosphorylation could be due to tyrosine phosphorylation of NR2, phosphoamino acid analyses of NR2 were performed. These analyses revealed phosphorylation on serine but not on threonine or tyrosine; immunoprecipitation and deglycosylation experiments using anti-phosphotyrosine antibodies confirmed that NR2 subunits in the primary hippocampal cultures are not detectably phosphorylated on tyrosine residues. These results demonstrate that the NR1 and NR2 subunits, which assemble into heteromeric complexes to form functional N-methyl-D-aspartate receptors, are trafficked in neurons with differential efficiency to the plasma membrane and exhibit different levels of basal versus stimulated serine phosphorylation.

Animals↗
PubMed1997

Quantitation of AMPA receptor surface expression in cultured hippocampal neurons.

Protein and messenger RNA levels of the AMPA-type glutamate receptor subunits 1-3 are high in many brain regions, but it is not known how much of the glutamate receptor protein is expressed on the surface of neurons in the form of functional receptors. To provide insight into this matter, western blot immunoreactivities for glutamate receptors 1 and 2/3, as well as binding of the specific ligand [3H]AMPA, were quantified following three independent treatments modifying surface receptors in intact primary hippocampal cultures: (i) proteolysis of surface receptors by chymotrypsin, (ii) cross-linking of surface receptors with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate, and (iii) biotinylation of surface receptors with the membrane-impermeant reagent sulfosuccinimidyl-2(biotinamido)ethyl-1,3-dithiopropionate. All three of these methods demonstrated that 60-70% of total glutamate receptor subunit 1 protein and 40-50% of total glutamate receptor 2/3 protein are expressed on the surface of hippocampal neurons. Parallel studies revealed that 52% of total [3H]AMPA binding sites could be precipitated with avidin beads following biotinylation of intact cultures, providing an estimate of [3H]AMPA binding site surface expression in accord with the estimates of the surface expression of glutamate receptor subunits 1-3. Experiments examining the surface expression of 32P-labeled glutamate receptor subunit 1 demonstrated that approximately 65% of the phosphorylated form of the subunit is located in the plasma membrane, an estimate similar to the that derived via western blot for the entire glutamate receptor subunit 1 population in the same samples. Moreover, no significant change in the surface expression profile of the glutamate receptor subunits 1-3 was observed following stimulatory treatments known to increase glutamate receptor phosphorylation. These data indicate that slightly more than half of the AMPA receptors in cultured hippocampal neurons are located in the plasma membrane, and that AMPA receptor surface expression is not rapidly altered by glutamate receptor phosphorylation.

Animals↗
PubMed1997

Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells.

The surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross-linking with the membrane-impermeant reagent bis (sulfosuccinimidyl) suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by > 90%, whereas immunoreactivity for the lower band was reduced by only 10-20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.

Animals↗
PubMed1997

Genetic stability among temporally and geographically diverse isolates of Barmah Forest virus.

An increase in the incidence of polyarthritis caused by Barmah Forest (BF) virus, and its recent emergence into Western Australia, prompted a study of the molecular epidemiology of this Australian mosquito-borne alphavirus. The nucleotide sequence of a 500-basepair region of the 3' end of the envelope (E2) gene of the prototype BF virus strain (BH2193) was compared with other members of the alphavirus genus, and to a panel of isolates of BF virus collected more for than 20 years from different geographic regions of Australia. The BF virus was shown to be genetically distinct from other members of the alphavirus genus. A high degree of sequence homology (98-100%) was found between the BF isolates, with no evidence of geographic or temporal divergence. This nucleotide homogeneity was similar to that observed with other Australian mosquito-borne viruses with avian vertebrate hosts, such as Sindbis, Murray Valley, and Kunjin viruses, but it contrasts to the heterogeneity reported for Ross River virus, an alphavirus with mammalian vertebrate hosts.

Alphavirus↗