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K Gould

Publications and source records attributed to K Gould.

50 records · Page 3Linked to original sources
PubMed1989

Recognition of influenza virus proteins by cytotoxic T lymphocytes.

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.

Animals↗
PubMed1988

Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen.

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.

Animals↗
PubMed1986

The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

Animals↗
PubMed1984

The gene structure of human anti-haemophilic factor IX.

The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5' non-coding and a 1390 residue long 3' non-coding region, but excluding the poly(A) tail. The factor IX gene is approximately 34 kb long and we define, by the sequencing of 5280 residues, the presumed promoter region, all eight exons, and some intron and flanking sequence. Introns account for 92% of the gene length and the longest is estimated to be 10 100 residues. Exons conform roughly to previously designated protein regions, but the catalytic region of the protein is coded by two separate exons. This differs from the arrangement in the other characterized serine protease genes which are further subdivided in this region.

Amino Acid Sequence↗
PubMed1976

Oxolinic acid in the treatment of typhoid fever due to chloramphenicol-resistant strains of Salmonella typhi.

Of 28 strains of Salmonella typhosa collected in late 1972 in Vietnam, 4 had minimum inhibitory concentrations to chloramphenicol of >100 mug/ml. Median minimum inhibitory concentrations of all strains to oxolinic acid were 0.39 mug/ml; ampicillin, 6.25 mug/ml; amoxicillin, 0.39 mug/ml. Widespread typhoid fever appeared in mid-1973 with more than three-fourths of strains found to be resistant to chloramphenicol. Peak serum concentrations of oxolinic acid average 3.0 mug/ml after the oral ingestion of 1.0 g. In July 1974, a pilot study was begun to evaluate the efficacy of oxolinic acid in vivo, recognizing the discrepancy between in vitro and in vivo results with many agents evaluated in the treatment of typhoid fever. Seven patients with typhoid fever, six with positive blood cultures, were treated with oxolinic acid (1.5 g twice daily by mouth, a daily dose that averaged 75 mg/kg per day) for 5 to 12 days. In four of six patients, blood cultures became negative at 2 to 3 days, with another being negative at 6 days. Despite negative blood cultures, all but one patient remained clinically ill with temperatures of >39.5 C at 4 to 9 days. All strains were susceptible to 0.19 mug of oxolinic acid per ml, and resistant strains did not occur. One patient died after being changed to ampicillin, one left against advice, three responded to amoxicillin, and one died with pseudomonas bacteremia. Toxicity to oxolinic acid did not occur.

Adolescent↗
PubMed1975

Adherence of bacteria to heart valves in vitro.

The abilities of 14 strains of aerobic gram-positive cocci and gram-negative bacilli to adhere in vitro to human or canine aortic valve leaflets were compared. 2-mm sections of excised valve leaflets were obtained by punch biopsy and were incubated under standardized conditions in suspensions of bacteria. Valve sections were subsequently washed and homogenized, and quantitative techniques were used to determine the proportions of bacteria from the initial suspensions that had adhered to the valve sections. Comparable results were obtained when these adherence ratios were determined by two independent methods based either on measurements of bacterial viability or of radioactivity in 51Cr-labeled bacteria. For each bacterial strain, the adherence ratio was constant over a wide range of concentrations of bacteria in the incubation medium. Strains of enterococci, viridans streptococci, coagulase-positive and coagulase-negative staphylococci and Pseudomonas aeruginosa (adherence ratios 0.003-0.017) were found to adhere more readily to valve sections than strains of Escherichia coli and Klebsiella pneumoniae (adherence ratios 0.00002-0.00004). The organisms that most frequently cause bacterial endocarditis were found to adhere best to heart valves in vitro, suggesting that the ability to adhere to valvular endothelium may be an important or essential charcteristic of bacteria that cause endocarditis in man.

Animals↗
PubMedDate not supplied

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence.

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.

Animals↗
PubMedDate not supplied

Pulmonary "cyclosporinoma" mimicking infection after heart transplantation.

Tissue from a transthoracic needle biopsy, which was performed as part of the investigation of an undiagnosed left upper lobe opacity in a patient after orthotopic heart transplantation, revealed numerous macrophages loaded with oil globules. No causative organisms were present. High-pressure liquid chromatography showed a similar profile to the oil in which cyclosporine is suspended. The lesion appears to have been caused by aspiration of cyclosporine.

Adult↗