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Biomedical subjects

M Kehoe

Publications and source records attributed to M Kehoe.

At least 37 records · Page 2Linked to original sources
PubMed1993

Distinct IgG1 and IgG3 subclass responses to two streptococcal protein antigens in man: analysis of antibodies to streptolysin O and M protein using standardized subclass-specific enzyme-linked immunosorbent assays.

The IgG subclass composition of antibodies to two streptococcal protein antigens in sera following infection was analysed by enzyme-linked immunosorbent assays (ELISA). The assays were standardized using 5-iodo-4-hydroxy-nitrophenacetyl (NIP)-specific chimeric antibodies, to permit quantitative comparisons between subclasses. Antibodies to streptolysin O (SLO) were predominantly IgG1, with only minor contributions from the other subclasses. In contrast, antibodies to M protein were distributed between the IgG1 and IgG3 subclasses, and in approximately half the sera IgG3 predominated. The ratio of IgG1:IgG3 was greater for SLO than for M protein in 22/23 sera. Little or no IgG4 antibody was detected to either antigen. Functional affinities of the IgG1 and IgG3 antibodies, determined by inhibition ELISA, were comparable for the two antigens. The demonstration that two protein antigens encountered during streptococcal infection elicit antibody responses with markedly different subclass profiles has implications for IgG subclass regulation and vaccine development.

Antibodies, Bacterial↗
PubMed1992

Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells.

The Listeria monocytogenes hemolysin listeriolysin O (LLO) plays a major role in mediating the escape of L. monocytogenes from a vacuolar compartment. In a previous report, it was shown that Bacillus subtilis expressing LLO could escape from a host vacuolar compartment and grow in the cytoplasm (J. Bielecki, P. Youngman, P. Connelly, and D. A. Portnoy, Nature [London] 345:175-176, 1990). In the present study, two related thiol-activated hemolysins, streptolysin O (SLO) and perfringolysin O (PFO), were expressed in B. subtilis and their ability to mediate intracellular growth was monitored by visual inspection and by assaying for CFU. Like LLO, PFO was active within the vacuolar environment, whereas SLO showed negligible activity. However, expression of PFO seemed to damage the host cells. The pH of the vacuole probably had little to do with these results, since all three hemolysins showed full or enhanced activity at pH 5.5, although LLO showed greatly reduced activity at pH 7. In addition, neutralization of the pH within host vacuoles by using weak bases had little effect on the lysis of the vacuole. The lack of SLO activity is probably caused by its lower specific activity; the purified protein had 10-fold less activity on a molar basis. These results suggest that LLO is not unique in its capacity to mediate intracellular growth of B. subtilis.

Animals↗
PubMed1990

Cloning and expression of the carbon monoxide dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2.

Carbon monoxide dehydrogenase (CODH) from Pseudomonas thermocarboxydovorans strain C2 is composed of three non-identical subunits. A gene library of C2 DNA in lambda vector L47.1 was generated and screened using anti-CODH serum. Western blotting experiments revealed a protein which co-migrated with and had the same immunological reaction as the large subunit of CODH in some of the clones isolated from the library. The coding region was pinpointed to a 4 kb fragment which was subcloned into plasmid. Western blotting experiments showed that all three subunits of CODH were coded for by the subclone. However, no CODH activity was detected.

Aldehyde Oxidoreductases↗
PubMed1989

Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Bacillus cereus phospholipase C.

The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43,000), suggesting post-translational processing. The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

Amino Acid Sequence↗
PubMed1989

The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.

Site-directed mutagenesis of the TGC codon in a cloned streptolysin O (SLO) gene exchanged the single Cys residue in SLO for either Ala or Ser. The parent wild-type SLO (SLO.Cys-530) and the SLO.Ala-530 and SLO.Ser-530 mutant toxins, expressed in Escherichia coli, were purified and analyzed. Wild-type SLO.Cys-530 and the SLO.Ala-530 mutant showed no significant differences in their specific hemolytic activities, while the SLO.Ser-530 mutant had a reduced (ca. 25%), but still considerable, specific hemolytic activity as compared with that of wild-type SLO. The parent and mutant toxins extracted from lysed erythrocyte membranes had similar sedimentation profiles on sucrose density gradients, suggesting that the mutations did not affect the ability of SLO to form oligomers in membranes. These results show that the widely held assumption that the in vitro cytolytic activity of SLO requires an essential Cys residue is not true.

Amino Acid Sequence↗
PubMed1988

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins.

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.

Amino Acid Sequence↗
PubMed1988

Lesbians over 60 speak for themselves.

With all its faults, this study, begun in 1983, is the first attempt to reach a nationwide representation of lesbian elders in order to gather information about their background, their relationship with their families of origin, and, for those married to men, with their husbands, children, and grandchildren. It explores their sexual behavior, both their physical and psychological health, and how aging has affected them. With the relatively meagre data available, it tries to make some comparison between lesbians and gay men over 60. All of this is just a beginning, an effort, it is hoped, that will encourage researchers to investigate further this hidden population that is another fascinating component of our diverse society. Through our research, we established that gay women of advanced age, as we have long surmised, are everywhere. Like their younger cohorts, they live in all regions of America, rural as well as urban. They come in all colors and are from all occupations: professional, business, clerical, trades, domestic work, and land management, to name a few. Many of them are now retired. Their formal education ranges from high school diplomas to doctorate degrees. A few are financially well off, while another few live below the poverty line. The rest enjoy reasonable comfort in their own homes, on modest incomes. In both political and religious affiliation, they tend to be liberal. Their sexual history runs the gamut from exclusively homosexual, through equally homosexual and heterosexual, to asexual. As they age and sex becomes a less significant part of their relationships, companionship grows more important. They prefer to associate with other lesbians within 10 years of their own age, whom they meet in community social groups, through friends, or at work--but not in bars. They do not relish the thought of ending their lives in any institution for the aged, but would consider a gay/lesbian intergenerational retirement community acceptable. Their most serious problems, even with the advantaged group surveyed, are those that affect many women of advanced age in our society: loneliness and economic worries. Although the feminization of poverty has been a topic under public scrutiny for some time, because it involves women of all ages, less attention has been paid to it in relation to the elderly. The equally disabling condition brought on by their isolation and loneliness has also been disregarded.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Psychological↗
PubMed1986

Lesbians over 65: a triply invisible minority.

Questionnaire responses from 50 lesbians, 65 to 85 years of age, were used to describe their present status, their educational background, their economic and occupational condition, their personal and psycho/social concerns, as well as their perception of their own physical and mental health. The data suggests that the 65+ lesbian is a survivor, a balanced personality, coping with aging in a satisfactory manner.

Aged↗
PubMed1984

Cloning and expression in Escherichia coli of the streptolysin O determinant from Streptococcus pyogenes: characterization of the cloned streptolysin O determinant and demonstration of the absence of substantial homology with determinants of other thiol-activated toxins.

A gene bank of Streptococcus pyogenes Richards was constructed in Escherichia coli by using the bacteriophage replacement vector lambda L47.1, and hybrid phage expressing streptolysin O (SLO) were identified among the recombinants. DNA sequences encoding SLO were subcloned from an slo+ hybrid phage into a low-copy-number vector plasmid to yield an slo+ hybrid plasmid, pMK157. This plasmid contains 5.6 kilobase pairs of cloned streptococcal DNA sequences, is stable, and expresses SLO at easily detectable levels in E. coli. Transposon gamma delta insertion mutants and in vitro-generated deletion mutants of pMK157 were isolated and analyzed. This analysis showed that a single gene is sufficient for production of SLO in E. coli and allowed this slo gene to be mapped to within +/- 100 base pairs. Two forms of the slo gene product, with molecular weights of 68,000 and 61,000, were detected in E. coli minicells harboring slo+ plasmids and by immunoblotting of E. coli whole cells harboring slo+ plasmids. Southern blotting hybridization experiments with the cloned SLO DNA sequences as probes failed to demonstrate homology between the cloned SLO determinant and DNA isolated from bacteria expressing thiol-activated cytolysins related to SLO.

Bacterial Proteins↗
PubMed1984

Primary sequence of the alpha-toxin gene from Staphylococcus aureus wood 46.

The complete DNA sequence of a cloned alpha-toxin gene from Staphylococcus aureus was determined. The amino acid sequence of the alpha-toxin protein, predicted from the DNA sequence, was described and compared with published data. The primary product of the cloned alpha-toxin gene contained a 26-amino-acid leader sequence which possessed characteristic features of a signal sequence involved in secretion. The mature alpha-toxin protein had a molecular size of 33,000 and contained only three short regions of high hydrophobicity in addition to a number of short, weakly hydrophobic regions.

Amino Acid Sequence↗
PubMed1984

Norms for hand grip strength.

Norms for hand grip strength of healthy children are presented. Sex and age specific centiles for age 5 to 18 years have been determined using a portable strain gauge dynamometer with an accuracy of 0.5 N. The test group comprised 1417 healthy, urban school children from a middle class suburb of Brisbane. Mean maximum grip strength (of four tests, two with each hand) and mean peak grip strength (best of four tests) were recorded. Mean values of peak grip strength were 10 to 15% higher than the average maximum grip in all age groups. At all ages girls had a reduced grip strength compared with boys and although boys manifested a continual, approximately linear increase in grip strength through all age groups, girls manifested an approximately linear increase up to 13 years after which mean hand grip usually remained constant. By the age of 18 years boys had a mean grip strength some 60% higher than girls. Correlations with height and weight are also presented. "Handedness' influenced grip strength and was most noticeable in children aged over 10 years. The clinical use of hand grip strength centiles for the early indication of neurological and muscular disorders and for following the natural history of neuromuscular disease is discussed.

Adolescent↗
PubMed1983

Cloning, expression, and mapping of the Staphylococcus aureus alpha-hemolysin determinant in Escherichia coli K-12.

A fragment of Staphylococcus aureus DNA encoding the alpha-hemolysin determinant was cloned from strain Wood 46 by inserting Sau3A-generated genomic DNA fragments between the BamHI sites of the lambda replacement vector L47.1. Phages expressing alpha-hemolysin were detected by overlaying plaques formed from several thousand independent recombinant phage with erythrocytes and looking for zones of hemolysis. One phage expressing alpha-hemolysin was purified and named lambda w alpha 3. This was subsequently shown to contain a 10.2-kilobase pair insert of S. aureus DNA. A 7.6-kilobase pair HindIII fragment encoding the alpha-hemolysin was subcloned from lambda w alpha 3 into the plasmid vector pACYC184 to form the hybrid plasmid pDU1148. Escherichia coli K-12 cells harboring pDU1148 synthesized a low level of alpha-hemolysin which remained associated with the cells and was not secreted into culture supernatants. When the same strain was stabbed onto blood agar plates, no zones of hemolysis were detected after overnight growth at 37 degrees C but hemolysis developed if the plates were left at room temperature for 48 h. By introducing specific deletions or Tn5 insertions into plasmid pDU1148, the alpha-hemolysin gene was mapped to a region within a 3.3-kilobase pair EcoRI-HindIII fragment which was subcloned onto the vector plasmid pBR322. A specific enzyme-linked immunosorbent assay with peroxidase-labeled rabbit anti-alpha-hemolysin antibodies was used to measure the levels of alpha-hemolysin antigen expressed in E. coli K-12 cells harboring pDU1148 or a variety of pDU1148::Tn5 and pDU1148 deletion mutants.

Chromosome Mapping↗
PubMed1983

Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212.

Bacillus subtilis↗