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Biomedical subjects

J M Kelly

Publications and source records attributed to J M Kelly.

At least 19 recordsLinked to original sources
PubMed1998

Hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 studied with a stable isotope technique in men with visceral obesity.

OBJECTIVE: To test the hypothesis that the hepatic secretion of very-low-density lipoprotein (VLDL) apolipoprotein B-100 (apoB) is increased in men with visceral obesity and to examine whether the oversecretion of this apolipoprotein is related to insulin resistance and increased hepatic availability of lipid substrates. SUBJECTS: 16 obese men (body mass index (BMI) > 30 kg/m2, waist circumference > 100 cm) and 16 non-obese, age matched men, were studied. MEASUREMENTS: The hepatic secretion of VLDL apoB was measured using a primed (1 mg/kg), constant (1 mg/kg/h), intravenous infusion of 1-[13C]-leucine. Isotopic enrichment of VLDL apoB was determined using gas-chromatography mass spectrometry and a multi-compartmental model (SAAM-II) was used to estimate the fractional turnover rate of VLDL apoB. RESULTS: Plasma concentrations of total cholesterol, triglyceride, glucose, insulin, mevalonic acid and lathosterol, as well as dietary fat intake, were significantly higher (P < 0.05) in obese than control subjects. The obese subjects had significantly lower high-density-lipoprotein cholesterol (P < 0.01). VLDL apoB pool size and hepatic secretion rate (mg/kg fat free mass/d) were significantly higher in the obese than non-obese subjects (P < 0.02). The fractional catabolic rate of VLDL apoB was lower in the obese subjects compared with controls, but the difference did not attain conventional significance (P=0.053). In pooled analysis, there was a significant positive association (P < 0.05) between VLDL apoB secretion rate (mg/kg fat free mass/d) and waist-to-hip ratio (WHR), waist circumference, and fasting plasma triglyceride, insulin and glucose concentrations. In multiple linear regression analysis, the association between VLDL apoB secretion and fasting insulin concentration was independent of age, apolipoprotein E (apoE) genotype, mevalonic acid concentration, free fatty acid concentration and fat intake. CONCLUSION: Our findings are consistent with the hypothesis that in visceral obesity, insulin resistance and the associated increased lipid substrate supply to the liver contribute to hepatic oversecretion of apoB; expansion in the VLDL apoB pool size may also be due to a catabolic defect related to insulin resistance.

Adult↗
PubMed1997

Pyruvate decarboxylase and anaerobic survival in Aspergillus nidulans.

The presence of pyruvate decarboxylase activity has been demonstrated in Aspergillus nidulans, and a gene encoding a pyruvate decarboxylase has been isolated from this organism and physically characterized. The isolation of the pdcA gene in A. nidulans confirms the existence of the alcoholic fermentation pathway in this fungus, despite it being an obligate aerobic organism. Southern analysis showed that it is most probably a single copy gene. Several potential binding sites for a GATAR-binding protein were identified in the sequence just prior to the start point of transcription, and mutant alleles of the GATAR-binding protein-encoding gene, areA, affected pdcA mRNA levels in a manner that suggested that it influences pdcA expression in nitrogen repressing conditions. Other previously reported cases of AREA action are in nitrogen-limiting conditions. Interestingly, the production of ethanol was affected in a similar way by the same areA alleles, suggesting that changes in pdcA mRNA level are reflected in the changes in the level of ethanol production. The experiments presented here confirm that PDC levels are a major determinant of ethanol production under these conditions.

Alleles↗
PubMed1997

Overexpression of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, is associated with enhanced metacyclogenesis.

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected T. cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandemly repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteine proteinase inhibitor Cbz-Phe-Phe-CHN2 (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

Animals↗
PubMed1997

Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans.

CreA is the major regulatory protein involved in carbon catabolite repression in Aspergillus nidulans. Previously we have reported the molecular characterization of a number of in vivo selected mutant alleles and showed that they were unlikely to represent total loss of function alleles (Shroff et al., 1996) and that a deletion of the creA gene and surrounding DNA has an extremely severe effect on morphology under both carbon catabolite repressing and carbon catabolite nonrepressing conditions (Dowzer and Kelly, 1991). Here we present an analysis of in vivo selected creA mutations with an extreme morphological phenotype and show that some of these alleles would be predicted to result in no functional CreA. The most extreme of these alleles resulted in a truncation of the protein within the first zinc finger. Precise gene disruptions, leaving the flanking sequences intact, show essentially the same phenotype as this truncated allele. Thus, a strain containing a null allele is viable, and the leaky-lethal phenotype of previous deletion alleles (Dowzer and Kelly, 1991) must be due to the deletion of additional 3' genomic sequence. A strain containing an allele that results in a deletion of the final 80 amino acids shows reduced sensitivity to carbon catabolite repression for a number of systems, thus localizing a region of the protein involved in repression. Surprisingly, the phenotypically most extreme allele studied is not a null allele, but results in an amino acid substitution that would disrupt the zinc finger region and abolish binding to DNA. This is the only allele that produces a full-length protein, predicted to be nuclear localized, but which completely abolishes DNA binding. The phenotype may be more extreme than the null alleles due to the nuclear located CreA protein titrating interacting proteins.

Alleles↗
PubMed1997

Molecular characterization of mutants of the acetate regulatory gene facB of Aspergillus nidulans.

The facB gene of Aspergillus nidulans encodes a DNA binding transcriptional activator required for growth on acetate as a sole carbon source. FacB contains N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA binding and leucine zipper-like heptad repeat motifs and central and C-terminal acidic alpha-helical regions. facB recessive loss of function mutants are deficient in acetate induction of acetyl-CoA synthase, isocitrate lyase, malate synthase, acetamidase, and NADP-isocitrate dehydrogenase. Characterization of lesions in facB mutant alleles has localized important functional regions of the FacB protein. Two extreme mutants are shown to lack the C-terminal region of the protein. Two temperature sensitive mutants contain amino acid substitutions in the DNA binding domain and are shown to affect acetate induction of amdS-lacZ expression and confer temperature sensitive in vitro DNA binding. Two temperature sensitive facB mutations result in thermolability of acetyl-CoA synthase, isocitrate lyase, and malate synthase but not acetamidase or NADP-isocitrate dehydrogenase in crude extracts. This suggests that FacB may have a structural role in acetate metabolism in addition to its regulatory function.

Acetates↗
PubMed1997

Direct association between the hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 and plasma mevalonic acid and lathosterol concentrations in man.

Apolipoprotein B-100 (apo B) is the principal structural and functional protein of the pro-atherogenic lipoproteins, but its homeostasis in man has not been clearly established. The hepatic availability of cholesterol substrate may be a determining factor. We examined whether there was a direct correlation between plasma concentrations of mevalonic acid (MVA) and lathosterol (indices of in vivo cholesterol synthesis) and hepatic secretion of very-low-density lipoprotein (VLDL) apo B in 13 normolipidaemic, healthy male subjects. The secretion of VLDL apo B was measured using a primed constant intravenous infusion of 1-[13C]-leucine (1 mg/kg per h) over 8 h. Gas-chromatography mass spectrometry (GCMS) was used to derive isotopic enrichment of apo B and fractional turnover rate was calculated using a monoexponential function. There was a highly significant positive correlation between the absolute secretion rate (ASR) of VLDL apo B and the plasma concentrations of mevalonic acid (r = 0.72, P = 0.005) and lathosterol (r = 0.81, P = 0.001) and the lathosterol:cholesterol ratio (r = 0.79, P = 0.001). In multiple regression analysis, these correlations remained significant after adjusting for waist circumference, age, apolipoprotein E genotype and dietary fat intake. The data further support the notion that the availability of cholesterol substrate regulates the hepatic secretion rate of apo B.

Adult↗
PubMed1997

Photoreactions of ruthenium(II) and osmium(II) complexes with deoxyribonucleic acid (DNA).

The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.

DNA↗
PubMed1997

Determinants of the kinetics of very low-density lipoprotein apolipoprotein B-100 in non-obese men.

1. Apolipoprotein B-100 (ApoB) is the principal structural and functional protein of the pro-atherogenic lipoproteins. Elevated plasma apoB is an independent risk factor for coronary artery disease. In the present study we aimed to assess the factors that determine the kinetics of apoB in the very low-density lipoprotein (VLDL) in healthy men. 2. We studied 17 non-obese men who were consuming an ad libitum diet and had the following characteristics: mean (+/-SD) age 45.5 +/- 9.7 years, body mass index (BMI) 25.1 +/- 1.4 kg/m2, waist:hip ratio 0.91 +/- 0.04, serum cholesterol 5.2 +/- 0.6 mmol/L, triglycerides 1.08 +/- 0.53 mmol/L and high-density lipoprotein-cholesterol 1.24 +/- 0.31 mmol/L. Daily dietary intake was as follows: total fat 76 +/- 26 g, carbohydrate 238 +/- 67 g, protein 103 +/- 33 g and alcohol 20 +/- 16 g. 3. The kinetics of VLDL ApoB were studied using a primed, constant infusion (1 mg/kg per h) of 1-[13C]-leucine over 8 h with measurement of isotopic enrichment of ApoB using gas chromatography/mass spectrometry. The fractional turnover rate of VLDL ApoB was estimated using a monoexponential function. The mean (+/-SD) absolute hepatic secretion rate (ASR) of ApoB was 8.5 +/- 4.6 mg/kg per day and the fractional catabolic rate (FCR) was 7.9 +/- 5.6 pools/day. The ASR was significantly correlated with the waist:hip ratio (r = 0.60; P = 0.04), but not with age, BMI, weight or nutrient intake. The FCR was significantly and inversely correlated with plasma triglycerides (r = -0.53; P = 0.03) and alcohol intake (r = -0.48; P = 0.05). 4. In conclusion, the hepatic secretion of VLDL ApoB in nonobese, healthy men is primarily determined by the waist:hip ratio, a measure of visceral fat. This is consistent with the hypothesis that the rate of lipid substrate supply in the liver regulates the output of ApoB. The fractional catabolism of VLDL ApoB may, however, be inversely related to alcohol intake and appears to determine the plasma concentration of triglycerides.

Adult↗
PubMed1997

Inhibition of cholesterogenesis decreases hepatic secretion of apoB-100 in normolipidemic subjects.

We examined the effect of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the kinetics of very low-density lipoprotein apolipoprotein B-100 (VLDL apoB) in 13 normolipidemic men in a placebo-controlled crossover study. Simvastatin significantly decreased the plasma concentrations of low-density lipoprotein (LDL) cholesterol by 36%, triglycerides by 26%, mevalonic acid by 34%, and lathosterol by 32%. Hepatic secretion of VLDL apoB was measured using a primed constant intravenous infusion of [1-13C]leucine with monitoring of isotopic enrichment of apoB by gas chromatography-mass spectrometry; fractional turnover rate was derived using a monoexponential function. Simvastatin decreased VLDL apoB pool size by 53% and the hepatic secretion rate of VLDL apoB by 46% but did not significantly alter its fractional catabolism. The change in hepatic VLDL apoB secretion was significantly and independently correlated with changes in plasma mevalonic acid and lathosterol concentrations and the lathosterol-to-cholesterol ratio. The data support the hypothesis that the rate of de novo cholesterol synthesis directly regulates the hepatic secretion of VLDL apoB in normal subjects.

Adult↗
PubMed1996

Cloning and expression of a second human natural killer cell granule tryptase, HNK-Tryp-2/granzyme 3.

Cytotoxic lymphocytes possess a number of serine proteases (granzymes) usually localized in cytoplasmic granules. To date, the DNA sequences of four human granzymes have been reported. A fifth human granzyme (granzyme 3) has been biochemically purified and its N-terminal amino acid sequence has been reported. This enzyme was described as possessing tryptase activity, cleaving synthetic substrates after arginine or lysine. We recently cloned a rat granzyme tryptase (RNK-Tryp-2), and used this cDNA to screen human cDNA libraries. Isolation of cDNA fragments of a human gene could be overlapped to provide a complete cDNA sequence, which we designated HNK-Tryp-2. The N-terminal amino acid sequence deduced from HNK-Tryp-2 was identical to that reported for granzyme 3. This gene appears to be a single copy gene that is expressed in isolated natural killer cells and T cells as well as in tissues containing these cells.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Amino Acid Sequence↗
PubMed1996

Mucin-like glycoprotein genes are closely linked to members of the trans-sialidase super-family at multiple sites in the Trypanosoma cruzi genome.

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10-12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.

Amino Acid Sequence↗
PubMed1996

Analysis of mutations in the creA gene involved in carbon catabolite repression in Aspergillus nidulans.

The molecular nature of a number of creA mutant alleles has been determined. Three alleles analysed are missense mutations in the DNA binding domain and predicted to reduce but not abolish binding. Of the other four alleles, two result from frameshifts: one has a nonsense mutilation and the other has an inversion. All four alleles result in truncations of the protein after the zinc finger domain, such that the protein no longer contains at least the carboxy terminal 145 amino acids, so identifying a region required for repression. Transcriptional analysis of creA indicates that the transcript is autoregulated and analysis using 5' rapid amplification of cDNA ends indicates that transcriptional start points exist in clusters over a region of 200 bp located up to 595 bp 5' of the translational start point. The two major clusters have potential CREA-binding sites (SYGGRG) at appropriate positions to allow autoregulation. Autoregulation leads to the creA transcript being most abundant in carbon catabolite nonrepressing conditions, and this, together with the phenotypes of the mutant alleles, has led to the suggestion that CREA has effects under conditions generally not considered as carbon catabolite repressing, as well as in carbon catabolite repressing conditions.

1-Propanol↗
PubMed1995

Expression of recombinant human Met-ase-1: a NK cell-specific granzyme.

Human Met-ase-1 is a member of a family of cytotoxic lymphocyte serine proteases (granzymes), but is expressed specifically in CD3- large granular lymphocytes with natural killer cell activity. We have devised a polymerase chain reaction strategy to delete the predicted hexapropeptide of human Met-ase-1 (Ser-6 to Gln-1), to enable its expression and activation in mammalian COS cells. In addition, using peptide immunization we have derived a unique and specific monoclonal antibody detecting human Met-ase-1. Western blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the human Met-ase-1 gene encodes a serine proteinase that specifically hydrolyzes substrates containing a methionine (Met-) side chain at P1. The expression of active human Met-ase-1 and the generation of a specific anti-human Met-ase-1 monoclonal antibody will now enable a detailed structure/function analysis of key amino acids that confer this unusual serine protease specificity.

Animals↗