Biomedical subjects
J M Kelly
Publications and source records attributed to J M Kelly.
Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I-bound determinant.
We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of V beta 5.2+TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA257-264 determinant, suggesting that the TCR alpha-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.
Fungal catabolic gene regulation: molecular genetic analysis of the amdS gene of Aspergillus nidulans.
Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses of trans-acting and cis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of the amdS gene is regulated by the facB and amdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of the amdR gene mediates omega amino acid induction of amdS. The binding sites for each of these proteins have been localised through amdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, including amdS. Nitrogen control is exerted through the positively acting areA gene product and carbon control is dependent on the creA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in the amdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterized proteins, including a CCAAT-binding complex, which interact with the 5' region of the amdS gene.
Photochemical interactions of methylene blue and analogues with DNA and other biological substrates.
The light-induced reactions of methylene blue and related phenothiazinium dyes with biological substrates are described. The properties of the excited states of the dyes, their reactions with nucleic acids and their photosensitised chemical modifications of nucleic acid bases are examined. Reports on phenothiazinium dye-induced damage to proteins, lipids, biological membranes, organelles, viruses, bacteria, mammalian cells and carcinomas are reviewed.
The effects of growth hormone on protein metabolism in adult growth hormone deficient patients.
OBJECTIVE: Growth hormone treatment given to adult growth hormone deficient patients leads to an increase in lean body mass by an unknown mechanism. The aim of this study was to investigate the actions of growth hormone treatment on protein metabolism in adult growth hormone deficient patients. DESIGN: Double-blind, placebo controlled trial of recombinant human growth hormone (0.018 U/kg/day for 1 month followed by 0.036 U/kg/day for 1 month) with isotopic whole body protein turnover studies at 0 and 2 months. PATIENTS: Eighteen adult growth hormone deficient patients (nine male, nine female of mean age 46.6 (range 30-56). MEASUREMENTS: Whole body isotopic leucine turnover using L-1-13C-leucine measuring leucine Ra (a measure of protein degradation), non-oxidative leucine Rd (a measure of protein synthesis) and leucine oxidation rate. RESULTS: Lean body mass (P < 0.02), circulating insulin-like growth factor I (P < 0.01) and insulin (P < 0.02) were significantly increased at 2 months in the treatment group but there was no change in the placebo group. When expressed in relation to body weight, leucine Ra and non-oxidative leucine Rd increased (P < 0.01) and leucine oxidation decreased (P < 0.02) after 2 months growth hormone treatment. When expressed in relation to lean body mass non-oxidative leucine Rd increased (P < 0.02) and leucine oxidation decreased (P < 0.02) but there was no significant change in leucine Ra after 2 months growth hormone treatment. In the placebo group there were no significant changes in leucine metabolism expressed as lean body mass or body weight after 2 months. changes in leucine metabolism expressed as lean body mass or body weight after 2 months. CONCLUSION: These results indicate that the increase in lean body mass resulting from growth hormone treatment in adult growth hormone deficient patients is due to an increase in protein synthesis.
Isolation of DNA and RNA from Leishmania.
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In vitro ouabain-sensitive respiration and protein synthesis in ruminal epithelial papillae of Hereford steers fed either alfalfa or bromegrass hay once daily.
Seven Hereford steers (457 +/- 8.5 kg BW) with ruminal cannulas were fed either long or short chopped bromegrass (B) or alfalfa (A) hay at 90% of a 3-h ad libitum intake once daily between 0900 and 1200 in a 4 x 4 complete and 3 x 4 incomplete Latin square design. After evacuation of ruminal contents, papillae from the ventral sac of the rumen were excised at 0900, 1200, and 2100 and placed in oxygenated media. Oxygen consumption was determined polarographically, after which either 10(-4) M ouabain or 10(-4) M cycloheximide was added for determination of ouabain-sensitive (OSO2) or cycloheximide-sensitive (CSO2) O2 consumption, respectively. Additionally, protein synthesis was measured by uptake of [3H]phenylalanine. Twenty-four-hour patterns of ruminal fluid pH, osmolality, ammonia N, and VFA were also determined. Steers fed A exhibited a rapid rise in total O2, OSO2, and CSO2 immediately after consumption of a meal; the respiration patterns of ruminal epithelial papillae from animals fed B lagged behind those observed for animals fed A. Patterns of O2 consumption for both diets paralleled those observed for ruminal concentration of products of fermentation; those fed A had a larger magnitude of change in both O2 consumption and fermentation products. Ruminal epithelial O2 consumption seems to be determined by substrate availability and products of fermentation, and Na+,K+ATPase and protein synthetic activity each account for one-fifth of ruminal papillae O2 consumption. Fractional rates of protein synthesis were unaffected by type of forage consumed.
Protein metabolism in growth hormone deficiency, and effects of growth hormone replacement therapy.
The effects of growth hormone (GH) treatment on protein metabolism were studied in adults with GH deficiency (GHD). A double-blind, placebo-controlled trial of recombinant human GH, 0.018 IU/kg/day for 1 month followed by 0.036 IU/kg/day for 1 month, was performed with isotopic whole-body protein turnover studies at 0 and 2 months. In all, 18 adults with GHD (9 men, 9 women; mean age, 46.6 years; range, 30-56 years) were studied. Whole-body isotopic leucine turnover using L-[1-13C]leucine was assessed by measuring leucine Ra (a measure of protein degradation), non-oxidative leucine Rd (a measure of protein synthesis) and leucine oxidation rate. Lean body mass and circulating insulin-like growth factor I were significantly (p < 0.02) increased at 2 months in the GH-treated group, but not in the placebo group. There was no change in leucine Ra in either the placebo or GH-treated groups at 2 months. Leucine oxidation decreased (p < 0.01) and non-oxidative leucine Rd increased (p < 0.02) in the GH-treated group at 2 months. There was no significant change in either leucine oxidation or non-oxidative leucine Rd in the placebo group at 2 months. These results indicate that the increase in lean body mass resulting from GH treatment in adults with GHD is due to an increase in protein synthesis.
A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.
A Trypanosoma cruzi expression vector has been constructed using sequences derived from the flanking regions of the glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. The neomycin phosphotransferase (neor) gene was incorporated as a selectable marker. Using electroporation we have introduced this vector into both T. cruzi and Leishmania cells and conferred G418 resistance. Transformation is mediated by large extrachromosomal circular elements composed of head-to-tail tandem repeats of the vector. The transformed phenotype is stable for at least 6 months in the absence of G418 and can be maintained during passage through the T. cruzi life-cycle. Foreign genes inserted into an expression site within the vector (pTEX) can be expressed at high levels in transformed cells. To our knowledge this paper describes the first trypanosome shuttle vector and the first vector which functions in both trypanosomes and Leishmania.
Cis-acting control elements 5' to aldA, the aldehyde dehydrogenase-encoding gene of Aspergillus niger.
The aldA gene of Aspergillus niger encodes an aldehyde dehydrogenase. Chromosomal deletion of this gene using gene replacement techniques had defined several growth conditions, thought to involve aldehyde and semialdehyde intermediate, that require this enzyme. Reintroduction of aldA clones carrying in vitro-generated deletions in the promoter region into the aldA deletion strain has been used to identify three elements controlling aldA expression: an enhancer required for high-level expression, an ethanol induction-responsive element, and a fructose induction-responsive element.
AIDS, prisoners, and the law.
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Ectomycorrhizal colonization of loblolly pine seedlings during three growing seasons in response to ozone, acidic precipitation, and soil Mg status.
A three-year study was initiated in 1987 to evaluate the impact of O3, acidic precipitation, and soil Mg on ectomycorrhizal colonization of loblolly pine (Pinus taeda L.) seedlings. Thirty-six open-top chambers equipped with a rainfall exclusion/addition system were utilized to administer three levels of O3 (subambient, ambient, or twice ambient) and two precipitation acidity levels (pH 3.8 or 5.2) to seedlings growing in 24-liter plastic pots containing soil having either 35 or 15 mg kg(-1) of exchangeable Mg. Seedlings exposed to the twice ambient O3 treatment exhibited smaller percentages of total ectomycorrhizal short roots at the end of each year of the study, but trends were statistically significant in 1989 only. Changes in number of specific ectomycorrhizal morphotypes in response to O3 were not consistent from year to year. Acidic precipitation treatments had no effect on number or percent of mycorrhizal short roots, and responses of two morphotypes to soil Mg treatments were probably due to differences in the soil environment rather than a result of changes in aboveground processes. Temporal shifts in morphotype frequencies were observed for seedlings in all treatments and indicate that mycorrhizal succession occurred during the study period.
Phenylalanine flooding dose procedure is effective in measuring intestinal and liver protein synthesis in sheep.
We conducted two experiments to evaluate the flooding dose method for measuring intestinal and liver protein synthesis in sheep. Experiment 1 showed that large doses of phenylalanine did not cause marked metabolic disturbances. Experiment 2 examined the effectiveness of flooding with phenylalanine and the time dependency of the protein synthesis calculation. Rams were injected with 1.2 MBq L-[ring 2,6-3H]phenylalanine/kg body wt and slaughtered 20, 40 or 60 min later. Plasma specific radioactivity reached a plateau within 2.5 min and did not change significantly (P > 0.05) throughout the experiment. Tissue intracellular free pool specific radioactivity also remained constant from 20 to 60 min postinfusion. Flooding conditions were achieved in the intracellular free pool of intestinal tissues (specific radioactivity 70-96% of plasma specific radioactivity), although liver flooding was less successful (57-67%). Protein synthesis rates measured after 20 min were significantly (P < 0.05) higher in the liver, jejunum and ileum than those measured at 60 min. Protein synthesis rates also tended to decline with time in the duodenum and colon (P > 0.05). There were no significant differences (P > 0.05) between protein synthesis rates calculated using the intracellular specific radioactivity vs. plasma specific radioactivity in the duodenum, ileum or colon. Therefore, this method represents an improvement over continuous infusion methods for measurements of protein synthesis in visceral tissues.
Incomplete suppression of hepatic glucose production in non-insulin dependent diabetes mellitus measured with [6,6-2H2]glucose enriched glucose infusion during hyperinsulinaemic euglycaemic clamps.
We have minimized methodological errors in the isotope dilution technique by using stable isotope, [6,6-2H2]glucose, thus avoiding the problem of contamination of tritiated glucose tracers and, by maintaining a constant plasma tracer enrichment have reduced error due to mixing transients. Using these modifications we have calculated hepatic glucose production in 20 patients with non-insulin-dependent diabetes mellitus during low (1 mU kg-1 min-1) and high (8 mU kg-1 min-1) dose insulin infusions. Mean fasting hepatic glucose production was 14.2 +/- 0.8 mumol kg-1 min-1. This suppressed by only 68% to 4.6 +/- 0.8 mumol kg-1 min-1 during the low-dose insulin infusion (plasma insulin 0.85 +/- 0.05 nmol l-1) and did not suppress further during the high-dose insulin infusion (plasma insulin 14.55 +/- 0.83 nmol l-1). Hepatic glucose production was significantly higher than zero throughout the study. Thus, we have found that minimization of known errors in the isotope dilution technique results in physiologically plausible and significantly positive values for hepatic glucose production indicating that the liver is resistant to insulin in patients with non-insulin-dependent diabetes mellitus.
Isolation and characterization of the rat liver AVP receptor using [125I][d(CH2)5'sarcosine7]AVP.
1. A vasopressin (AVP) binding protein was purified from rat liver membranes by an improved method using [125I][d(CH2)5'Sarcosine7]AVP, a selective V1 AVP radioligand and a combination of CHAPS solubilization, gel filtration, lectin affinity and FPLC ion exchange chromatography. 2. The purified protein exhibited a maximum binding activity of 2480 pmol/mg protein with a KD of 4.5 nmol/L, which corresponds to a purification of approximately 26,700-fold. The molecular weight of this protein was 70,000 Da. 3. The binding of [125I][d(CH2)5'Sarcosine7]AVP to the solubilized membranes was dependent on the protein concentration, and was inhibited by the unlabelled peptides [d(CH2)5'Sarcosine7]AVP, AVP, and to a lesser degree by peptides with high V2 receptor affinity, such as 1-desamino-D-AVP and [d(CH2)5'D-Ileu2-Ileu4]AVP. 4. In addition, an AVP anti-idiotypic monoclonal antibody bound to both the partially purified and purified lectin affinity AVP binding protein in a concentration-dependent manner. These results indicate that the purified protein displays similar characteristics to the liver membrane-bound AVP V1 receptor.
Activity-based pacing: comparison of a device using an accelerometer versus a piezoelectric crystal.
The EXCEL VR, an accelerometer-based pacemaker (AC), and the Legend, a pacemaker utilizing a piezoelectric crystal (PZ), were compared under ergometric conditions and during stair climbing to assess the appropriateness of their rate responses. The pacemakers, programmed to the manufacturers' nominal settings in order to compare different technologically based sensors under identical conditions, were strapped over subjects' left mid-pectoral region. Placement of the devices was randomized to control for positional effects. Ten healthy subjects (55-72 years) completed a graded exercise treadmill test to 80% of maximum predicted heart rate (HR). An additional group of ten subjects (50-66 years) completed exercise protocols involving bicycle ergometry and stair climbing. Throughout all tests, pacemaker pulse rates and subjects' intrinsic HR were monitored continuously. For the treadmill exercise, the average correlations between the AC and PZ pacemakers' pulse rate and HR for the group as a whole were r = 0.92 and r = 0.82, respectively. Individual subject comparisons were also made between each pacemaker rate and intrinsic HR. The mean difference from intrinsic rate was 11 ppm for the AC pacemaker and 24 ppm for the PZ pacemaker. In addition, the PZ pacemaker's maximal pulse rate was significantly lower (105 +/- 9.6 ppm) than the other two rates (AC 137 +/- 6 ppm; intrinsic HR 129 +/- 2 beats/min). Throughout the bicycle ergometry testing, the intrinsic HR was higher than the AC and PZ pacing rates. However, the AC's rate was significantly higher than the PZ's rate. When subjects ascended stairs, the intrinsic HR and AC rate were closely correlated, but the PZ rate was significantly lower.(ABSTRACT TRUNCATED AT 250 WORDS)
Photo-induced electron transfer from nucleotides to ruthenium-tris-1,4,5,8-tetraazaphenanthrene: model for photosensitized DNA oxidation.
The luminescence quenching of ruthenium-tris-1,4,5,8-tetraazaphenanthrene [Ru(tap)3(2+)] by nucleotides approaches the diffusion rate only with guanosine-5'-monophosphate (GMP), the most reducing nucleotide, and leads to an electron transfer with the production of the monoreduced complex and the oxidized base. The resulting deprotonated GMP(-H).radical recombines with the monoreduced complex according to a bimolecular equimolar process. The pH dependence of the decay of the transient reduced complex, in the presence of an oxidant (oxygen or benzoquinone) indicates the formation of Ru(tap)2(tapH)2+, i.e. the reduced protonated species, subsequent to the electron transfer, with a pKa of 7.6 as confirmed from pulse radiolysis experiments. As the non-protonated reduced complex, Ru(tap)2(tap-.)+, has a higher reducing power than the protonated one, oxygen is able to reoxidize only the non-protonated species, whereas benzoquinone reoxidizes both species but with different rate constants. The flash photolysis of Ru(tap)3(2+) in the presence of DNA and the effect of Mg2+ ions and GMP as supplementary additives also show the existence of a photo-induced electron transfer with the nucleic acid, which can be correlated to the photosensitized cleavage of DNA by this complex.
Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.
We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.