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J I Harris

Publications and source records attributed to J I Harris.

At least 19 recordsLinked to original sources
PubMed2002

Molecular mediators of Porphyromonas gingivalis-induced T-cell apoptosis.

Porphyromonas gingivalis produces virulence factors which can modify the molecular and cellular components of the host immune response. In the present work we investigated the role of specific virulence factors from P. gingivalis in the induction of apoptosis in Jurkat T cells. P. gingivalis culture supernatants mimicked the effect of butyric acid on T-cell apoptosis and this effect was associated with an increase in histone H4 acetylation. A role for proteases was excluded in experiments which demonstrated that neither protease inhibitors nor use of P. gingivalis mutants defective in protease synthesis had any effect on the stimulation of T-cell apoptosis in this system.

Acetylation↗
PubMed1999

In vitro studies of lymphocyte apoptosis induced by the periodontal pathogen Porphyromonas gingivalis.

Apoptosis has a physiological role in lymphocyte development and function serving to remove self-reactive T-cells in the thymus as well as activated peripheral T-cells when they are no longer required in the immune response. Evidence from the study of several pathogenic bacteria indicate that induction of premature cell death by apoptosis may be an important pathogenic mechanism promoting infection, inflammation and concomitant disease. In this paper we demonstrate that cultures of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) promote lymphocyte apoptosis in peripheral blood mononuclear cells (PBMC). We have used assays designed to investigate the different molecular and cellular changes associated with apoptosis. Thus flow cytometry revealed that whole cultures of P. gingivalis promoted cell shrinkage in the lymphocyte fraction of PBMC and analysis of hypodiploidy confirmed that the cellular changes were associated with nuclear changes characteristic of apoptosis. We also found that apoptosis was promoted in PBMC exposed to both whole P. gingivalis cultures and culture supernatant but not washed bacterial cells; this indicates that molecule(s) secreted into the medium were responsible for this activity and not a factor intrinsic to the bacterial cell. Furthermore heat treatment has no effect on the ability of P. gingivalis cultures to induce lymphocyte apoptosis. In summary, a soluble heat stable component of the supernatant from P. gingivalis cultures promotes lymphocyte apoptosis. These data establish the principle that bacteria-induced apoptosis may be an important feature of the pathogenesis of periodontal disease.

Apoptosis↗
PubMed1980

D-glyceraldehyde-3-phosphate dehydrogenase. The purification and characterisation of the enzyme from the thermophiles Bacillus stearothermophilus and Thermus aquaticus.

1. D-Glyceraldehyde-3-phosphate dehydrogenase from two thermophilic bacteria has been purified by procedures including affinity chromatography on NAD+-Sepharose. 2. Methods for making NAD+-free enzyme are also described. 3. Both the holo and apo forms of the enzyme from Bacillus stearothermophilus have been crystallised. 4. The enzymes are tetrameric and composed of four chemically identical polypeptide chains of molecular weight 36,000. 5. The enzymes are much more stable to heat than their counterparts from mesophiles.

Chromatography, Affinity↗
PubMed1980

D-glyceraldehyde-3-phosphate dehydrogenase. Complete amino-acid sequence of the enzyme from Bacillus stearothermophilus.

1. The complete amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus has been determined. 2. This has been achieved largely by the automated sequence analysis of large fragements derived by chemical cleavage with cyanogen bromide, BNPS-skatole [the product of reaction between N-bromosuccinimide and 2-(nitrophenyl-sulphenyl)-3-methylindole] and hydroxylamine and enzymic hydrolysis with trypsin at arginine residues. 3. The sequence is as follows: (See Text). It has been numbered to maximise homology with the four complete sequences of this enzyme from other sources. Hence the N-terminal residue is numberd 0 and two deletions and two insertions have been introduced. 4. The inability of the B. stearothermophilus apo-enzyme to transfer an acyl moiety from Cys-149 to Lys-183 oberved with muscle enzymes is explained by the replacement of lysine by arginine in the enzyme from the thermophilic organism. 5. The sequences of the S-loop regions, which form the core of the tetrameric enzyme, are similar to each other in B. stearothermophilus and Thermus aquaticus and differ from the highly conserved S-loops of three enzymes from mesophiles.

Amino Acid Sequence↗
PubMed1980

D-glyceraldehyde-3-phosphate dehydrogenase. Amino-acid sequence of the enzyme from the extreme thermophile Thermus aquaticus.

1. The amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus has been elucidated. 2. The polypeptide contains 332 amino acids and its sequence is 70% identical with that of the enzyme from the moderate thermophile Bacillus stearothermophilus. 3. In contrast to less thermostable forms of the enzymes from B. stearothermophilus, pig, lobster and yeast, the T. aquaticus enzyme has only one cysteine residue, namely cysteine-149 which is required for catalysis.

Amino Acid Sequence↗
PubMed1980

Heat stability of a tetrameric enzyme, D-glyceraldehyde-3-phosphate dehydrogenase.

The tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus is more stable to thermal denaturation than its counterpart from lobster muscle [Harris et al. (1980) Eur. J. Biochem. 108, 535-547]. Extra buried ionic bonds between subunits of the thermophilic enzyme make an important contribution to thermal stabilisation. Further stabilisatio of the tetrameric enzyme is derived from additional hydrophobic interactions between the S-loops at the core of the tetramer. In the enzyme from the extreme thermophile Thermus aquaticus, which is even more thermostable, intersubunit ion pairs must also play a role but changes in interactions at the surface appear to be equally important. Thus additional hydrophobic interactions at the edge of subunit interfaces would prevent access of water to the interior of the molecule. Furthermore, the arrangement of charged residues on the surface of the T. aquaticus enzyme would allow maximal surface ion pair formation. The presence of surface ion pairs in other proteins correlates well with thermal stability [Perutz, M. F. and Raidt, H. (1975) Nature (Lond.) 255, 256-258] and would provide a general stabilising influence on the subunit in this case.

Amino Acid Sequence↗
PubMed1980

Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus.

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).

Amino Acid Sequence↗
PubMed1980

Primary structure of triosephosphate isomerase from Bacillus stearothermophilus.

1. Triosephosphate isomerase from Bacillus stearothermophilus is a dimeric enzyme comprising two chemically identical polypeptide chains. 2. The nearly complete amino acid sequence of the subunit polypeptide chain has been established from sequences of tryptic, chymotryptic and lysine-blocked tyrptic fragments of S-[2-14C]carboxymethylated enzyme. Overlaps not established by experimental data have been provisionally established from considerations of sequence homology with previously established sequences for the rabbit, chicken and coelacanth enzymes. The nearly complete sequence of the 249 residues is as follows. (See Text). 3. Comparison of the thermophile and chicken muscle enzymes shows that 40% of the residues are in identical sequence. 4. Correlation of the sequence of the thermophile enzyme with the three-dimensional structure of the muscle enzyme shows that residues in the catalytic site and in the subunit interface are strongly conserved. Possible correlations between sequence changes and thermal stabilisation of the dimeric structure are also noted.

Amino Acid Sequence↗
PubMed1980

Structural comparisons of superoxide dismutases.

The amino-terminal sequences of superoxide dismutase isolated from seven microorganisms have been determined. These include the first sequences of enzyme from anaerobic phototrophes. Five enzymes contain iron and two manganese. The enzymes are all related to each other but not to the Cu/Zn family of superoxide dismutases. These sequences, taken with six others from the same family, show that there is no clear distinction in sequence between Fe and Mn types. Moreover it demonstrates a wide variation between enzymes from different bacteria. Also enzymes from anaerobes do not seem to be a particularly closely related group and are not more closely related to each other than to enzymes from aerobes. Two histidine residues are conserved in all proteins and secondary structure predictions suggest they are in close proximity in the same alpha-helix. These residues may provide ligands for the bound metal.

Amino Acid Sequence↗
PubMed1979

Minigastrin; corrected structure and synthesis.

Evidence is presented that minigastrin is the C-terminal tetradecapeptide amide of gastrin and not the tridecapeptide amide as previously reported. Synthesis of the tetradecapeptide amide sequence, Trp-Leu-[Glu]5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Nh2, was achieved by a series of fragment couplings which were mediated by the dicyclohexylcarbodiimide procedure in presence of either N-hydroxysuccinimide or 1-hydroxybenzotriazole. Purification of all intermediate fragments, and of the final protected tetradecapeptide amide, was by Sephadex LH-20 chromatography. Removal of the protecting groups was effected by treatment with 90% trifluoroacetic acid in the presence of a large excess of scavengers. Purification by ion-exchange chromatography afforded the pure tetradecapeptide amide. This material had full physiological activity.

Amino Acid Sequence↗
PubMed1977

Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The glyceraldehyde 3-phosphate dehydogenase holoenzyme of Bacillus stearothermophilus possesses precise 222 symmetry: in this respect it differs from the reported structure of the lobster muscle enzyme. Pairs of active sites are linked through a flexible polypeptide loop which probably mediates the structural changes giving rise to cooperative effects. Three additional salt bridges made by each subunit to others would make a major contribution to thermostability of the tetramer.

Allosteric Regulation↗
PubMed1977

Superoxide dismutase from Thermus aquaticus. Isolation and characterisation of manganese and apo enzymes.

Superoxide dismutase has been isolated and characterised from the extreme thermophile Thermus aquaticus. The pure enzyme is a reddish-purple manganese-containing protein with a molecular weight of approximately 80000 +/- 5000. Combination of gel electrophoresis in dodecylsulphate and amino acid analysis shows that it is composed of four identical subunit polypeptide chains consisting of approximately 186 amino acids. The tetrameric protein contains two atoms of manganese. A stable manganese-free apoprotein has been prepared by treatment with EDTA in 8 M urea at acidic pH. The apoprotein regains the tetrameric structure in the absence of manganese but is inactive. Reconstitution of active Mn-enzyme was achieved byaddition of Mm2+ apoprotein in 8 M urea at acid pH. Reconstitution was monitored by absorption spectroscopy, manganese analysis and regain of activity and by these criteria the reconstituted enyzme with two atoms Mn per mole is indistinguishable from the native enzyme. The enhanced stability of the thermophile apoenzyme and Mn-enzyme is of advantage for studies of the structure and mechanism of action of superoxide dismutase. The N-terminal amino acid sequence to the 40th residue of the submit was determined by automated Edman degradation. The sequence has a close resemblance to that of the dimeric Mn-enzyme from another thermophile, Bacillus stearothermophilus.

Amino Acid Sequence↗
PubMed1977

A thermostable sequence-specific endonuclease from Thermus aquaticus.

A sequence-specific endonuclease, Taq I, of novel specificity has been partially purified from an extreme thermophile, Thermus aquaticus. The enzyme cleaves bacteriophage lambda DNA at many (greater than 30) sites and bacteriophage psiX174 RF DNA at 10 sites. The enzyme is active at temperatures up to 70 degrees. The cleavage sites on psiX174 RF DNA have been mapped. The sequence recognized and cleaved by Taq I has been shown to be the symmetrical tetranucleotide: (formula: see text).

Base Sequence↗