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S Clegg

Publications and source records attributed to S Clegg.

67 records · Page 4Linked to original sources
PubMed1983

Organization of genes responsible for the production of mannose-resistant fimbriae of a uropathogenic Escherichia coli isolate.

A group of insertion mutants was used to define the genes of plasmid pDC5 required for the expression of mannose-resistant fimbriae. Minicell experiments identified four polypeptides (71,000, 45,000, 27,000, and 17,000 daltons) concerned with fimbrial production, the smallest of these being the fimbrial subunit. The approximate location of the structural genes encoding these polypeptides and one possible additional polypeptide not identified in minicell experiments has been established. Complementation experiments in vivo showed that these genes are arranged in more than one operon. The direction of transcription of the fimbrial genes was established by creating beta-galactosidase fusions by using the mini-Mu d1681 kanamycin resistance transposon.

DNA Transposable Elements↗
PubMed1982

Serological heterogeneity among fimbrial antigens causing mannose-resistant hemagglutination by uropathogenic Escherichia coli.

Serological agglutination tests were used to determine cross-reactivity among mannose-resistant fimbrial hemagglutinins of uropathogenic Escherichia coli. Of 47 E. coli strains demonstrating mannose-resistant activity, 12 were agglutinated by one fimbrial antiserum and two reached with a second serum. The mannose-resistant hemagglutinins (fimbriae) of urinary E. coli exhibit antigenic heterogeneity.

Agglutination Tests↗
PubMed1982

Cloning of genes determining the production of mannose-resistant fimbriae in a uropathogenic strain of Escherichia coli belonging to serogroup O6.

Chromosomal DNA from a uropathogenic strain of Escherichia coli was partially digested with the restriction enzyme EcoRI. The partial digests were ligated into a cosmid containing an ampicillin-resistant determinant and packaged into lambda phage particles. An ampicillin-resistant transductant of E. coli HB101 was found to possess mannose-resistant hemagglutinating activity associated with a 50-kilobase-pair plasmid. Subcloning of the mannose-resistant fimbrial genes revealed that the genetic determinants were encoded by a 6.9-kilobase-pair DNA fragment of a recombinant plasmid. Chimeric plasmids smaller in size were unable to transform E. coli to fimbrial production. Physical maps of the recombinant plasmids were prepared showing restriction endonuclease sites within the inserted DNA fragments. The hemagglutinating activities of the wild-type strain and of the recombinant derivative were compared. Both strains agglutinated human erythrocytes in the presence of D-mannose to the same degree and also failed to produce fimbriae after incubation at 18 degrees C. Also, both strains were agglutinated by antifimbrial serum at a high titer, whereas no such activity was observed when a strain of E. coli which did not possess a plasmid was used.

Cloning, Molecular↗
PubMed1982

Antigenic heterogeneity of haemagglutination type VI fimbriae produced by Escherichia coli isolated from patients with bacteremia.

In previous work it was observed that the majority of Escherichia coli strains isolated from the blood of bacteremia patients possess fimbriae which mediate mannose-resistant hemagglutination (HA) of human and monkey erythrocytes; this HA pattern (HA type VI) is different from those of enterotoxigenic E. coli possessing the colonization factor antigen CFA/I or CFA/II, which are also mannose-resistant HA positive. We and other investigators have found that HA type VI fimbriae may function as colonization factor antigens, especially in urinary tract infections. The work reported here was concerned with determining the number of antigenic types of fimbriae responsible for the HA type VI property. Fimbriae were isolated by hydrophobic interaction chromatography from four isolates of E. coli possessing the same HA pattern but belonging to different serotypes; antifimbrial antiserum was prepared by hyperimmunization of rabbits. Homologous and heterologous antifimbrial antibody titers were determined by the indirect enzyme-linked immunosorbent assay, bacterial agglutination by the tube method, and the HA inhibition test. Antiserum against the fimbriae of strain LY-71 (O4:H1) produced a high homologous titer but low or insignificant titers with fimbriae of strains LY-63 (O6:H31), LY-154 (O25:H(-)) and LY-156 (O2:H(-)). Antiserum against the fimbriae of strain LY-63 produced high titers with its own and with LY-71 fimbriae but insignificant titers with the fimbriae of strains LY-154 and LY-156. LY-154 and LY-156 fimbriae were highly cross-identical but antigenically different from those of LY-63 and LY-71. Twenty-five other test strains all identified with at least one of the prototype strains, as determined by HA inhibition tests performed with the four antifimbrial sera, although these belonged to a wide variety of serotypes. These data indicate the existence of at least two major different antigenic types of HA type VI fimbriae and possibly three, depending on the nature of the unidirectional crossidentity observed between the fimbriae of strains LY-63 and LY-71.

Antigens, Bacterial↗
PubMed1981

Identification of a toxic impurity in commercial diphenylamine.

The impurity in commercial diphenylamine which induces polycystic kidney disease in rats has been identified as N,N,N'-triphenyl-p-phenylenediamine. The structure of the compound was confirmed by mass and proton magnetic resonance spectrometry and by comparison with a synthetic standard prepared by the Ullman coupling of iodobenzene and p-phenylenediamine or N,N-diphenyl-p-phenylenediamine. Laboratory studies with highly purified diphenylamine indicated that N,N,N'-triphenyl-p-phenylenediamine can be formed by heating diphenylamine.

Aniline Compounds↗
PubMed1980

Enzyme-linked immunosorbent assay for quantitating the humoral immune response to the colonization factor antigen of enterotoxigenic Escherichia coli.

The enzyme-linked immunosorbent assay technique was used to quantitate, in milligrams per milliliter, anti-colonization factor antigen/I (CFA/I) immunoglobulin G (IgG) in acute- and convalescent-phase sera of individuals who experienced diarrhea associated with CFA/I-positive enterotoxigenic Escherichia coli. Purified CFA/I was used as antigen to coat polystyrene Microtiter plate wells for the determination of anti-CFA/I antibody. A reference anti-CFA/I IgG preparation was obtained by affinity chromatography of a high-titered serum with a CFA/I-Sepharose 4B column; IgG was the only class of immunoglobulin detectable in this serum as anti-CFA/I. Goat anti-human IgG conjugated to alkaline phosphatase was used in the enzyme-linked immunosorbent assay. Quantitation of IgG in the reference anti-CFA/I serum was achieved by comparison with a known sample of pure human IgG. Anti-CFA/I in test sera was quantitated by titration with CFA/I-coated Microtiter plate wells in the enzyme-linked immunosorbent assay, using a standard curve obtained with the reference anti-CFA/I serum. Anti-CFA/I IgG in paired sera was determined as percentage of total IgG by using the radial immunodiffusion technique to quantitate total IgG for each test serum. Diarrhea with isolation of CFA/I-positive enterotoxigenic E. coli was associated with a significant rise in serum anti-CFA/I IgG when these values were expressed as either milligrams of IgG per milliliter or as percentage of total IgG, although the response varied quantitatively and nonresponders were detected. None of the matched controls showed an anti-CFA/I IgG response. Further elucidation of the immune response to enterotoxigenic E. coli can now be accomplished by applying these methods to determine the class and specificity of immunoglobulins in external secretions such as saliva and intestinal contents.

Antibodies, Bacterial↗
PubMed1980

Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli.

Antigens, Bacterial↗
PubMed1979

The fimbrial and non-fimbrial haemagglutinins of Escherichia coli.

Both cultures of Escherichia coli were examined for mannose-sensitive (MS) haemagglutinin in rocked-tile tests with guinea-pig red cells at ambient temperature, and agar plate cultures were examined for mannose-resistant eluting (MRE) haemagglutinins against 14 species of red cells in tests mixed at 3--5 degrees C in the presence of .05% (w/v) D-mannose. Ox, sheep, human, pig, horse, guinea-pig, and fowl red cells were required to detect the various patterns of MRE haemagglutination with the different species of cells. Of 387 strains in 155 O serogroups, 95 formed both MS and MRE haemagglutinins (MS+/MRE+), 198 formed only MS (MS+/MRE-), 21 only MRE (MS+/MRE+), and 73 neither (MS-/MRE-). Strains of more than one of these types, and MRE+ strains with different cell specificities were found in many of the serogroups. Some strains in 144 O serogroups had MS haemagglutinin and some in 50 an MRE haemagglutinin. The presence of MS haemagglutinin in a culture was invariably associated with the presence of type-1 fimbriae on the bacteria. All MS+ strains shared a common antigen in their type-1 fimbriae and three groups of these strains possessed also a group-specific fimbrial antigen. The presence of certain kinds of MRE haemagglutinin in over half the MRE+ strains was associated with that of type-MRE fimbriae, but fimbriae were not detected in the other MRE+ strains. The antigens of the MRE haemagglutinins in different strains were heterogeneous and differed from those of the type-1 fimbriae of MS+ strains. Three series of strains from normal faeces, and from patients with infantile diarrhoea and urinary-tract infections each included a minority possessing neither type of haemagglutinin, but this observation did not preclude a role of the haemagglutinins in colonization or pathogenicity.

Adult↗
PubMed1979

Purification and characterization of the CFA/I antigen of enterotoxigenic Escherichia coli.

The fimbral colonization factor antigen CFA/I of enterotoxigenic Escherichia coli was purified and characterized. The initial purification step was release of these fimbriae from the bacterial cells by homogenization with a Waring blender. Common fimbriae and flagellar antigen were avoided by careful control of growth conditions and the use of a nonmotile (H-) mutant of the prototype strain H-10407 (O78:H11). The essential purification steps were membrane filtration (Millipore Corp.), ammonium sulfate fractionation, and negative diethylaminoethyl-Sephadex column chromatography. Yields were approximately 4.0 mg of CFA/I protein per g (wet weight) of bacteria. Purified CFA/I is a fimbrial molecule 7.0 nm in diameter and has an average molecular weight of 1.6 X 10(6), as determined by sedimentation equilibrium. CFA/I is a polymer of identical subunits of molecular weight 23,800 with an N-terminal valine, 37% hydrophobic amino acid residues, and 11 residues of proline per mol. The purified antigen retains its morphology, antigenicity, and biological activity. Purified antigen retains its morphology, antigenicity, and biological activity. Purified CFA/I exhibits mannose-resistant hemagglutination of human group A, bovine, and chicken erythrocytes, as do CFA/I-positive bacteria. This was demonstrated by sensitizing latex microbeads with the purified antigen since cell-free CFA/I fimbriae do not hemagglutinate erythrocytes. Thus, CFA/I detached from the bacteria are monovalent; however, purified CFA/I antigen retains an affinity for the epithelial cells of rabbit small intestine and blocks adhesion of CFA/I-positive bacteria. These results demonstrate that purified CFA/I is a good candidate for use in an oral vaccine for immunoprotection against diarrhea caused by CFA/I-positive enterotoxigenic E. coli.

Amino Acids↗
PubMed1979

Fimbriae of Escherichia coli K-12 strain AW405 and related bacteria.

Fimbriate strains of Escherichia coli K-12 of the AW405 series agglutinated erythrocytes of several animal species. The hemagglutination was mannose senstivie, and the fimbriae were type 1. When cultured for extended periods in static broths, they did not form fimbrial pellicles but formed thick, nonfimbrial pellicles, the appearance of which was not associated with the selective outgrowth of fimbriate-phase bacteria.

Aerobiosis↗
PubMedDate not supplied

The DNA sequence and comparative analysis of human chromosome 20.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

Animals↗
PubMedDate not supplied

The use of hydralazine to manipulate tumour temperatures during hyperthermia.

Hydralazine is an antihypertensive drug which theoretically could increase tumour temperatures during hyperthermia via reduction in tumour blood flow from a vascular 'steal' phenomenon. Doses that are therapeutically effective in reducing blood pressure in hypertensive patients would probably cause postural hypotension and other side-effects in normotensive patients beyond the hyperthermia treatment session, however. This study was designed to evaluate whether hydralazine, when administered at a safe dose for normotensive patients (0.125 mg/kg, i.v.) would be effective in increasing tumour temperatures during hyperthermia. The working hypothesis was that hydralazine at a dose of 0.125 mg/kg would be effective in raising tumour temperatures during hyperthermia treatment with minimal change in blood pressure. Fourteen human and five canine subjects were given hydralazine (0.125 mg/kg, i.v.) at the midpoint of a hyperthermia session. Temperatures and blood pressures were monitored before and after drug administration. Although hydralazine resulted in slight reduction in blood pressure, it was ineffective in increasing tumour temperatures in human patients (average maximum rise in median temperature was 0.26 +/- 0.32 degrees C). In canine subjects the same dose of hydralazine was effective in reducing blood pressure in four of five subjects studied (mean maximum drop was 22.7 +/- 4.1 mmHg) and the median temperature rose 0.8 +/- 0.7 degrees C. In the canine subjects the greater the decrease in blood pressure, the greater the increase in temperature. These results suggest that a rise in tumour temperature induced by hydralazine is dependent on creating a drop in blood pressure. Future studies in this laboratory will include tumour blood flow manipulation with antihypertensives which have a shorter half-life and a titratable effect. Using this approach, hypotension, which seems to be required to raise tumour temperature, will be more controllable in terms of magnitude and duration.

Animals↗