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Biomedical subjects

J F Martin

Publications and source records attributed to J F Martin.

At least 73 records · Page 4Linked to original sources
PubMed1997

Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.

Amino Acid Isomerases↗
PubMed1997

The adventitia, endothelium and atherosclerosis.

Despite many theories, the initiating circumstances for the development of atherosclerosis remain obscure. The development of animal models of atherosclerosis was based upon the different theories of the origins of atherosclerosis which suggested that it originates at the intimal surface of the vessel. A more recent model of atherosclerosis involves perivascular manipulation of the vessel by positioning of a hollow silastic collar around the artery. In this model, several of the features seen in early human atherosclerosis are generated within a period of 7 days. It is hypothesized that arterial wall hypoxia following occlusion of the vasa vasorum may be the initial lesion of atherosclerosis, and hence that in some cases atherosclerosis is a disease of the outer layers of the arterial wall.

Animals↗
PubMed1997

Induction and role of NO synthase in hypotensive hepatic failure.

Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by NG-monomethyl-L-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.

Acetylcholine↗
PubMed1997

Megakaryocyte ploidy and platelet changes in human diabetes and atherosclerosis.

Altered platelet morphology and function have been reported in patients with diabetes. They are likely to be associated with the pathological processes and increased risk of vascular disease seen in these patients. Mean platelet volume (MPV), platelet count, and megakaryocyte (MK) ploidy (DNA content) were measured in (1) nondiabetics with normal coronary arteries, (2) nondiabetics with coronary artery atherosclerosis, (3) diabetics without evidence of vascular complications, and (4) diabetics with vascular disease. The platelet count (+/- SD) was increased in all groups but only significantly in the diabetics with vascular disease (236 +/- 65 versus 250 +/- 54 versus 257 +/- 64 versus 295 +/- 90 [P < or = .05] x 10(9)/L, for groups, I, II, II, and IV, respectively). The MPV was significantly increased in patients with atherosclerosis (7.0 +/- 0.4 versus 8.0 +/- 1.2 [P < or = .05] versus 7.2 +/- 0.9 versus 8.1 +/- 0.9 [P < or = .05] IL). Geometric mean MK ploidy was significantly increased in all groups compared with controls (16 +/- 1.5 versus 18.7 +/- 1.8 [P < or = .05] versus 19.8 +/- 1.6 [P < or = .05] versus 20.1 +/- 2.7 [P < or = .05]). Furthermore, some patients with vascular disease and/or diabetes had a modal ploidy shift from 16 (the normal mammalian modal ploidy) to 32, with a concomitant reduction of MKs in the 8 and 16 ploidy classes. This shift was seen particularly in the diabetics with vascular disease (P = .007). Interleukin-6 (IL-6) levels were measured and were elevated in patients with atherosclerosis; the highest levels were found in the diabetic patients (0.7 +/- 0.9 versus 5.3 +/- 5.5 [P < or = .05] versus 2.5 +/- 2.8 versus 6.7 +/- 5.5 [P < or = .05] ng/L). In the diabetic patients with atherosclerosis, fibrinogen levels were also increased (2.85 +/- 0.76 versus 3.34 +/- 1.32 versus 2.43 +/- 1.50 versus 5.59 +/- 1.72 [P < or = .05] g/L). Furthermore, IL-6 levels correlated with MK ploidy (r = .45, P = .009) and fibrinogen levels (r = .5, P = .0001). This study demonstrates that patients with vascular disease, particularly diabetics, have an altered MK ploidy distribution, showing a shift toward higher ploidy in association with an increased platelet mass (count x volume). Changes in platelets in diabetes probably reflect MK changes, which themselves are a response to systemic change.

Aged↗
PubMed1997

A study of platelet activation during human cardiopulmonary bypass and the effect of S-nitrosoglutathione.

Cardiac surgery is complicated by the occurrence of post-operative bleeding due to platelet dysfunction. This is largely caused by platelet activation and consumption during cardiopulmonary bypass. Patients undergoing cardiac surgery requiring cardiopulmonary bypass were studied to determine whether early platelet changes due to bypass could be inhibited using the platelet-selective nitric oxide donor S-nitrosoglutathione (GSNO). Flow cytometry was used to measure platelet surface expression of P-selectin (an alpha-granule protein) and glycoproteins (GP) IIb/IIIa and Ib (mediators of aggregation and adhesion) before and 5 and 10 min after commencing cardiopulmonary bypass, in 6 controls and 6 patients receiving GSNO 50 microg/min. Platelet P-selectin expression increased during bypass both in controls and patients receiving GSNO. Glycoproteins IIb/IIIa and Ib fell during bypass in control and GSNO-treated patients. There was no difference between control and GSNO-treated groups. Thus no significant platelet inhibition by S-nitrosoglutathione was demonstrated under these conditions.

Aged↗
PubMed1997

HLA-DQA1*0501 and DQB1*02 homozygosity and disease susceptibility in Spanish coeliac patients.

Coeliac disease (CD) susceptibility is strongly associated with HLA-DQA1*0501 and DQB1*02 alleles. There are discordant reports on whether homozygosity increases the risk. We genotyped HLA-DQA1*0501 and DQB1*02 in 50 CD patients, 100 parents, and 50 controls. Most patients (96%) were positive for DQA1*0501 (RR = 18.07, p < 0.001), 94% for DQB1*02 (RR = 17.55, p < 0.001), and 92% for both alleles together (RR = 31.82, p < 0.001). DQA1*0501 was found in 52% of controls, DQB1*02 in 44%, and only 24% had both alleles. Patients homozygosity or heterozygosity was estimated by assessing-in each case-whether one or both parents were carriers of the allele of risk. The frequencies of parents both positive for DQA1*0501 (58%) and for DQB1*02 (53.1%) were higher than expected by the fact that the proband is a carrier. These findings suggest that the frequency of homozygosity is increased among CD patients, and therefore, homozygosity for either DQA1*0501 or DQB1*02 represents a risk factor added to the fact of being a carrier.

Alleles↗
PubMed1996

New type of hexameric ornithine carbamoyltransferase with arginase activity in the cephamycin producers Streptomyces clavuligerus and Nocardia lactamdurans.

The ornithine carbamoyltransferases (OTCases) from the beta-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans have been purified to near-homogeneity by delta-N-phosphonoacetylornithine-Sepharose 4B affinity chromatography. The S. clavuligerus and N. lactamdurans OTCases monomers had a molecular mass of 37 kDa. The native OTCases of S. clavuligerus, N. lactamdurans and Streptomyces coelicolor had molecular masses of 248, 251 and 247 kDa respectively, which correspond to a hexameric structure. The apparent K(m) values for ornithine and carbamoylphosphate of the S. clavuligerus enzyme were respectively 2.3 and 6.0 mM at pH 8.0. The enzyme showed a reverse activity on citrulline and used lysine and putrescine as substrates. The hexameric complex showed coupled arginase-OTCase activities and was able to convert arginine into citrulline in a carbamoylphosphate-dependent manner. The requirement for carbamoylphosphate might prevent the arginase-OTCase complex from carrying out a futile cycle of arginine biosynthesis and degradation.

Allosteric Regulation↗
PubMed1996

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells.

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.

Aphidicolin↗
PubMed1996

Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum.

Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum. The efficiency of transformation of P. chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. The AMA1 region works as an origin of replication in P. chrysogenum and A. nidulans but not apparently in Acremonium chrysogenum.

Base Sequence↗
PubMed1996

The effects of S-nitrosoglutathione on platelet activation, hypertension, and uterine and fetal Doppler in severe preeclampsia.

OBJECTIVE: To determine the effects of the platelet-specific nitric oxide donor S-nitrosoglutathione on women with severe preeclampsia. METHODS: Ten women with severe preeclampsia or preeclampsia with severe fetal compromise at 21-33 weeks' gestation each received a 60-90-minute intravenous infusion of 50-250 micrograms/minute of S-nitrosoglutathione. Each was hypertensive, despite conventional oral antihypertensive therapy in eight. Maternal blood pressure, heart rate, platelet activation, uterine artery, and fetal Doppler indices were measured during the infusion. RESULTS: A dose-dependent reduction in mean arterial pressure from 125 mmHg (95% confidence interval [CI] 117-133) to 103.5 (95% CI 97-111) (P < .005) and an increase in pulse rate from 73.7 beats per minute (95% CI 64.3-84.5) to 89.1 (95% CI 81.2-97.8) (P < .02) was observed during the infusion. Mean uterine artery resistance index fell from 0.76 (95% CI 0.73-0.81) to 0.70 (95% CI 0.65-0.75) (P < .009). Platelet activation measured by P-selectin expression was reduced from 3.02% (95% CI 2.09-4.36) to 1.22% (95% CI 0.94-1.58) (P < .01). Fetal Doppler indices (umbilical artery, middle cerebral artery, and thoracic aorta) showed no significant changes during the infusion. CONCLUSION: S-nitrosoglutathione infusion reduced material mean arterial pressure, platelet activation, and uterine artery resistance without further compromising fetal Doppler indices. This study suggests that platelet-specific nitric oxide donors may prove beneficial in the management of severe preeclampsia.

Adult↗
PubMed1996

Expression of nitric oxide synthase in ulcerative colitis.

Nitric oxide (NO) is generated from L-arginine by a family of enzymes called the NO synthases. Previous investigators have proposed that the expression of this inducible enzyme (iNOS) may account for the characteristic vasodilatation, oedema and impairment of get motility seen in active ulcerative colitis. Using a specific antibody to iNOS, we have investigated the distribution of this enzyme in colonic tissue from patients with histologically proven ulcerative colitis. Eight patients with ulcerative colitis expressed calcium-independent citrulline activity (9.96 +/- 2.34 pmol citrulline mg-1 protein min-1) and showed immunoreactivity to the iNOS antibody within the inflammatory infiltrate of the lamina propria, and also within the cytoplasm of the epithelial cells lining the colon. Five age-matched controls showed no calcium-independent citrulline activity (0.2 +/- 0.08 pmol citrulline mg-1 protein min-1) and no immunoreaction to the antibody. We conclude that this enzyme is present in colonic tissue including the epithelium from patients with active colitis. Inhibition of this enzyme may provide a novel therapeutic option for patients with active ulcerative colitis.

Adult↗
PubMed1996

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.

Amino Acids↗
PubMed1996

Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.

There are four members of the myocyte enhancer factor 2 (MEF2) family of transcription factors in vertebrates, MEF2A, -B, -C, and -D, which have homology within a MADS box at their amino termini and an adjacent motif known as the MEF2 domain. These factors activate muscle gene expression by binding as homo- and heterodimers to an A/T-rich DNA sequence in the control regions of muscle-specific genes. To understand the mechanisms of muscle gene activation of MEF2 factors, we generated a series of deletion and site-directed mutants of MEF2C. These mutants demonstrated that the MADS and MEF2 domains mediate DNA binding and dimerization, whereas the carboxyl terminus is required for transcriptional activation. Amino acids that are essential for MEF2 site-dependent transcription but which do not affect DNA binding were also identified in the MEF2 domain. This type of positive-control mutant demonstrates that the transcription activation domain of MEF2C, although separate from the MEF2 domain, is dependent on this domain for transcriptional activation through the MEF2 site. MEF2 mutants that are defective for DNA binding act as dominant negative mutants and can inhibit activation of MEF2-dependent genes by wild-type MEF2C.

Amino Acid Sequence↗
PubMed1996

MEF2B is a potent transactivator expressed in early myogenic lineages.

There are four members of the myocyte enhancer binding factor 2 (MEF2) family of transcription factors, MEF2A, -B, -C, and -D, that have homology within an amino-terminal MADS box and an adjacent MEF2 domain that together mediate dimerization and DNA binding. MEF2A, -C, and -D have previously been shown to bind an A/T-rich DNA sequence in the control regions of numerous muscle-specific genes, whereas MEF2B was reported to be unable to bind this sequence unless the carboxyl terminus was deleted. To further define the functions of MEF2B, we analyzed its DNA binding and transcriptional activities. In contrast to previous studies, our results show that MEF2B binds the same DNA sequence as other members of the MEF2 family and acts as a strong transactivator through that sequence. Transcriptional activation by MEF2B is dependent on the carboxyl terminus, which contains two conserved sequence motifs found in all vertebrate MEF2 factors. During mouse embryogenesis, MEF2B transcripts are expressed in the developing cardiac and skeletal muscle lineages in a temporospatial pattern distinct from but overlapping with those of the other Mef2 genes. The mouse Mef2b gene maps to chromosome 8 and is unlinked to other Mef2 genes; its intron-exon organization is similar to that of the other vertebrate Mef2 genes and the single Drosophila Mef2 gene, consistent with the notion that these different Mef2 genes evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1996

Platelet activation in acute myocardial infarction and unstable angina is inhibited by nitric oxide donors.

Platelet activation and thrombus formation within the coronary artery are major factors in acute myocardial infarction (AMI) and unstable angina (UA), and continuing platelet activation is associated with an adverse prognosis. We assessed platelet activation by using flow cytometry to measure platelet surface expression of P-selectin and glycoprotein IIb/IIIa in 20 patients with AMI and 20 with UA, all of whom were treated with aspirin. Platelet studies were repeated after the infusion of a nitric oxide donor (glyceryl trinitrate or S-nitrosoglutathione) that produced a fall in mean arterial pressure of no more than 10 mm Hg. P-selectin was expressed on 2.5% (range, 1.4% to 6.3%) of platelets from AMI and 2.3% (range, 1.6% to 3.3%) from UA subjects compared with 1.0% (range, 0.6% to 1.9%) of platelets from 20 control volunteers without angina (P < .001). Glycoprotein IIb/IIIa expression was 101.6 +/- 2.7 arbitrary units of relative fluorescence in AMI and 100.2 +/- 3.3 in UA compared with 87.8 +/- 2.5 in control subjects (P < .01). In both AMI and UA, S-nitrosoglutathione reduced P-selectin (P < .001) and glycoprotein IIb/IIIa (P < .05) expression, as did glyceryl trinitrate (P < .02 and P < .01, respectively). In 3 of 20 patients receiving glyceryl trinitrate the lowest dose was not tolerated due to headache or hypotension. These findings show that platelet activation persists in AMI and UA despite aspirin treatment and that this can be inhibited by using glyceryl trinitrate or S-nitrosoglutathione. S-nitrosoglutathione is better tolerated at the doses required.

Adult↗