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Biomedical subjects

E Hunter

Publications and source records attributed to E Hunter.

At least 91 records · Page 5Linked to original sources
PubMed1994

Epidemiology of Pseudomonas cepacia colonization among patients with cystic fibrosis.

Colonization with Pseudmonas cepacia in patients with cystic fibrosis (CF) has been associated with increased morbidity and early death, compared with colonization by P. aeruginosa. The mode of acquisition of P. cepacia is not fully understood, although person-to-person spread appears likely. Recent epidemiologic studies support the importance of social contact in the spread of P. cepacia among patients with CF. This study was undertaken to investigate the epidemiology of P. cepacia colonization among patients with CF attending the CF clinic at our center. Isolates of P. cepacia were collected from patients at two CF treatment centers, including ours. Additional isolates were collected from patients without CF in the hospital ICU, from other teaching hospitals, and from the environment. Profiles of enzymes were obtained by ultrathin polyacrylamide gel electrophoresis of P. cepacia extracts. A predominant electromorphic type (ET) was found among P. cepacia isolates from patients at both centers, suggesting a common source or person-to-person transmission. The majority of hospital isolates fell into a single, different ET. Surveillance swabs of respiratory equipment at our CF clinic did not grow P. cepacia. Attendance of patients at CF summer camp correlated strongly with P. cepacia colonization (P < 0.0001).

Adolescent↗
PubMed1994

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.

Amidohydrolases↗
PubMed1994

Expression of the TM protein of Rous sarcoma virus in the absence of SU shows that this domain is capable of oligomerization and intracellular transport.

The SU and TM subunits of the Rous sarcoma virus glycoprotein, which are derived from a single polypeptide precursor, have been expressed independently with a simian virus 40 vector. The TM protein retains the ability to form an oligomer which resembles the TM oligomer derived from the wild-type glycoprotein complex present in virions. Oligomerization of the recombinant TM protein is more rapid than that observed for the intact glycoprotein expressed from the simian virus 40 vector and is required for its transport out of the endoplasmic reticulum. Oligomeric TM is terminally glycosylated in the Golgi complex but is less stable than the intact wild-type protein and does not accumulate at the cell surface. The SU protein, in contrast, does not form detectable oligomers but is efficiently secreted into the culture medium. These observations suggest that the oligomerization domain of the Rous sarcoma virus glycoprotein lies in the TM protein and that it can function independently of SU.

Amino Acid Sequence↗
PubMed1994

Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity.

Viral protease-mediated cleavage within the cytoplasmic domain of the transmembrane (TM) glycoprotein of the type D retrovirus, Mason-Pfizer monkey virus, removes approximately 16 amino acids from the carboxy terminus of the protein. To determine the functional significance of this cleavage in the virus life cycle, we introduced premature stop codons into the TM coding domain, resulting in the production of truncated glycoproteins. Progressive truncated of the cytoplasmic domain identified the carboxy-terminal third as being required for efficient incorporation of the glycoprotein complex into budding virions and profoundly increased the fusogenic capability of the TM glycoprotein. These results, together with the ability of matrix protein mutations to suppress TM cleavage, imply that this portion of the glycoprotein interacts specifically with the capsid proteins during budding, suppressing glycoprotein fusion function until virus maturation has occurred.

Amino Acid Sequence↗
PubMed1994

Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli.

A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli. An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique. In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA. Selection for the antibiotic thus increases the frequency of mutants. High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector. The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed. A retroviral proteinase precursor, toxic for E. coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained.

Amino Acid Sequence↗
PubMed1993

One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.

We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells. Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose. After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site. We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR. The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells. The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed.

Animals↗
PubMed1993

Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant baculovirus.

Mason-Pfizer monkey virus (M-PMV) is a primate retrovirus that shows type D morphogenesis in mammalian cells. Immature intracytoplasmic A type particles (ICAPs) preassemble in the infected cell cytoplasm migrate to the plasma membrane and are released by budding. This is in contrast to retroviruses that show type C morphogenesis, where assembly and budding occur concurrently at the plasma membrane. We expressed the M-PMV structural genes (gag-pro-pol) in insect cells using a recombinant baculovirus. The polyprotein precursors assembled predominantly intracellularly, although a small proportion also assembled at the membrane. The protease enzyme was active since mature particles were identified in the culture supernatant. We also expressed the M-PMV mutants, D26N and gag-STOP, which carry a nonfunctional protease or fail to express the protease gene, respectively. These baculovirus recombinants generated a homogeneous population of immature M-PMV capsids having exclusively type D morphogenesis. Sufficient quantities of polyprotein precursors were synthesized to be visualized directly on a Coomassie-stained protein gel, and the capsids were subject to purification. These results provide the first expression of type D retrovirus particles using the baculovirus expression system.

Animals↗
PubMed1993

Analysis of retroviral assembly using a vaccinia/T7-polymerase complementation system.

The nature of protein-protein interactions during retroviral assembly is not well understood, and mutational analyses of the potential signals involved in the viral assembly process has been difficult, particularly with the avian retroviruses due to the level of viral proteins expressed in the clonal cell lines containing defective viral genomes. We describe here a complementation system in which the retroviral gag/pol and env gene products were expressed independently from different plasmids under the control of the bacteriophage T7 promoter, in avian cells. Coexpression of the T7 polymerase from a vaccinia virus vector resulted in a high level of biosynthesis of retroviral structural proteins and efficient assembly of virus particles. Electron microscopy and protein composition analyses demonstrated that these virions were indistinguishable from those produced from RSV-infected cells. Through the use of mutant glycoprotein genes it was possible to demonstrate the specificity of the assembly process and the applicability of this system to other retroviral systems is described.

Animals↗
PubMed1993

Alcohol-related problems among Aboriginal drinkers in the Kimberley region of Western Australia.

The paper reports on the prevalence of alcohol-related problems among drinkers in a stratified random sample of the adult Aboriginal population of the Kimberley region of Western Australia. Subjects were 265 current drinkers who were identified in the total sample of 516 Kimberley Aboriginal men and women over the age of 15 years. Participants' reports were obtained on their frequency and quantity of alcohol consumption, and their lifetime experience of 16 alcohol-related problems. The majority of Aboriginal drinkers in the Kimberley consumed harmful amounts of alcohol, and there was a high prevalence of the 16 alcohol-related problems which showed a high degree of internal coherence, with the first principal component accounting for 45% of the total variance. The number of alcohol-related problems which respondents reported was strongly related to the quantity and frequency of self-reported alcohol consumption.

Adult↗
PubMed1993

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.

Animals↗
PubMed1993

Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.

Amino Acid Sequence↗
PubMed1993

The snake on the caduceus: dimensions of medical and psychiatric responsibility in the Third Reich.

The literature on the activities of medical professionals during the tumultuous years of the Third Reich has, over the last decade, increased substantially. However, many questions remain unanswered and the subject is likely to receive further attention following recent access to previously restricted archival material in Eastern Europe. In this paper, based on the English language literature, the author explores the role of medicine, and in particular psychiatry, in defining the bio-medical vision that was central to Nazi ideology.

Complicity↗
PubMed1993

Rapid purification of recombinant baculovirus using fluorescence-activated cell sorting.

Expression of foreign proteins in the baculovirus-insect cell expression system has been limited by difficulties in rapid identification and purification of recombinant virus. Although the process of identifying recombinant virus has been greatly facilitated by the introduction of vectors that lead to insect cell co-expression of beta-galactosidase with foreign genes of interest, isolation of pure recombinant virus using plaque purification may still take several weeks to months to accomplish. Using a fluorescent beta-galactosidase substrate, we have established that insect cells harboring recombinant virus can be rapidly isolated using fluorescence-activated cell sorting. Pure recombinant virus can then be readily obtained using this cellular fraction, with a pure viral culture generally obtained within 2-3 weeks of insect cell transfection.

Animals↗
PubMed1992

A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein.

The envelope glycoprotein precursor of retroviruses undergoes proteolytic cleavage in the Golgi complex to yield the mature surface and transmembrane (TM) glycoproteins of the virus. We report here that the TM glycoprotein of Mason-Pfizer monkey virus undergoes a second proteolytic processing event during a late maturation step that can follow virus release and Gag polyprotein cleavage. Cleavage results in the conversion of the cell-associated TM glycoprotein (gp22) to a virus-associated gp20. Processing continues after virus release and yields virions that contain predominantly gp20. A mutation within the active site of the Mason-Pfizer monkey virus aspartyl protease was shown to block both TM glycoprotein cleavage and the processing of the Gag polyprotein precursor. The role of the viral protease in cleavage of the TM glycoprotein localizes the cleavage site to the cytoplasmic domain of this protein. Surprisingly, point mutations within the matrix (MA) coding region of the gag gene can affect the extent to which gp22 is processed to gp20 and in one case [p10(MA)-A79V] results in greater than 90% inhibition of gp22 cleavage. The data provide genetic evidence of a specific interaction between the capsid proteins and the cytoplasmic domain of the TM glycoprotein of a retrovirus. This interaction is required for cytoplasmic domain cleavage to occur and may play a critical role in virus assembly and viral infectivity.

Amino Acid Sequence↗
PubMed1992

Evidence for heat-stable liver cytosol substance(s) capable of causing oxidative activation of fructose 1,6-bisphosphatase.

The endogenous fructose 1,6-bisphosphatase (FBPase) in chicken liver extract undergoes a drastic increase in activity if the pH of the extract is in the alkaline range. Greater and more consistent activation occurs when purified FBPase, placed inside dialysis sack, is incubated in liver extract. Maximal activation (over 16-fold) is accompanied by the disappearance of 4 highly reactive sulfhydryl groups (SH) per molecule of enzyme. The activating effect of the extract remains essentially unchanged after heating to 100 degrees C. Activation can be reversed by dithiothreitol. These data show the existence in liver cytosol of heat-stable substance(s) capable of activating FBPase presumably by forming disulfide bonds with the enzyme's highly reactive SH groups.

Animals↗
PubMed1992

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.

Amino Acid Sequence↗
PubMed1992

Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector.

In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.

Betaretrovirus↗