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Biomedical subjects

J F Martin

Publications and source records attributed to J F Martin.

At least 55 records · Page 3Linked to original sources
PubMed1998

Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein.

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [Alpha-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

Adenosine Diphosphate↗
PubMed1998

S-nitrosoglutathione reduces the rate of embolization in humans.

BACKGROUND: Antiplatelet agents presently used in the secondary prevention of cardiovascular disease fail to prevent the majority of cases of recurrent stroke and systemic embolization. An evaluation of the efficacy of new agents is hampered by a lack of in vivo models in humans. Asymptomatic cerebral embolic signals (ES) may be detected with the use of transcranial Doppler ultrasonography. These signals are particularly common after carotid endarterectomy, and this provides a situation in which new antiplatelet agents can be evaluated. With this model, we determined the effectiveness of S-nitrosoglutathione (GSNO), a nitric oxide donor with relative platelet specificity, in reducing cerebral embolization. METHODS AND RESULTS: Transcranial Doppler ultrasound recordings from the ipsilateral middle cerebral artery were made after carotid endarterectomy in 12 control patients and 12 patients receiving intravenous GSNO from the induction of anesthesia until 2 hours after skin closure. Recording times were 0.5 to 3.5, 6 to 7, and 24 to 25 hours after skin closure. The Doppler signal was recorded onto tape, and analysis for ES was performed, with the investigators blinded to treatment group. All patients received aspirin 300 mg/d before surgery and 5000 IU of heparin during surgery. The median (range) number of ES detected during the initial 3-hour postoperative recording was markedly reduced in the GSNO group compared with the control group: 7.5 (0 to 61) versus 38.5 (1 to 219) (P=0.018). This difference persisted until 6 hours after surgery. CONCLUSIONS: Despite the administration of aspirin and heparin, frequent embolization occurred and was markedly reduced after the administration of GSNO. This demonstrates the potential use of platelet-specific nitric oxide donors in the treatment of thromboembolic disease. This model of cerebral embolism may allow determination of the effectiveness of new antiplatelet agents in humans.

Aged↗
PubMed1998

Thrombopoietin stimulates VEGF release from c-Mpl-expressing cell lines and haematopoietic progenitors.

Vascular endothelial growth factor (VEGF) production was analysed in megakaryocytic cell lines and CD34+ haematopoietic progenitors following treatment with thrombopoietin (TPO). In CMK cells TPO caused a time- and dose-dependent increase in the levels of VEGF released into the medium. A similar effect was observed in UT-7/mpl cells transfected with the TPO receptor c-Mpl, but not in parental UT-7 cells. In CD34+ haematopoietic progenitor cell cultures TPO stimulated VEGF mRNA expression and VEGF protein release. Production of VEGF in CD34+ cultures increased with TPO-induced megakaryocytic differentiation, but not with erythroid or myelomonocytic differentiation induced respectively by erythropoietin and granulocyte-macrophage colony-stimulating factor. These results demonstrate that TPO stimulates VEGF release in c-Mpl-expressing cells and suggest that this process is an integral feature of the megakaryocytic differentiation programme.

Animals↗
PubMed1998

Changes in platelet size and count in unstable angina compared to stable angina or non-cardiac chest pain.

AIMS: An increase in platelet aggregability is associated with unstable angina and myocardial infarction. Platelet size and activity correlate and mean platelet volume was found to be increased before acute myocardial infarction. We measured the mean platelet volume and platelet count in patients with stable angina, unstable angina and non-cardiac chest pain. METHODS AND RESULTS: We studied 981 patients (734 men; 247 women) defined clinically as stable angina (n = 688), unstable angina (n = 108) and unstable angina requiring immediate angioplasty (n = 52). After coronary angiography the patients were subdivided into single (n = 269), double (n = 304) and triple-vessel disease (n = 311) and the control group of non-cardiac chest pain (n = 97). There was no significant difference in platelet count between the control group and patients with 1, 2, or 3-vessel disease. However, the platelet size in patients with coronary artery disease was significantly larger (single: 8.7 +/- 1.19 fl; double: 8.7 +/- 1.12 fl; triple-vessel disease: 8.8 +/- 1.18 fl) than the control group (8.2 +/- 0.95 fl) (P < 0.01). Patients with stable angina similarly had no, significant difference in platelet count compared to the control group but did have a significantly increased mean platelet volume (8.7 +/- 1.13; P < 0.01). In contrast, patients with unstable angina had a decreased platelet count (245 +/- 56 x 10/l) compared to either stable angina (262 +/- 62 x 10/l; P < 0.05) or the control group (261 +/- 58 x 10/l; P < 0.05); furthermore, the mean platelet volume (9.4 +/- 1.23 fl) was significantly greater than for stable angina (P < 0.01). Patients with unstable angina requiring immediate PTCA had an even lower platelet count (231 +/- 55 x 10/l) and higher mean platelet volume (10.4 +/- 1.03 fl) (P < 0.01) than the rest of the population with unstable angina. CONCLUSIONS: In stable angina the platelet count is unchanged compared to patients with normal coronary arteries but the platelet size is increased. However, in unstable angina there is a decrease in platelet count and an even larger increase in platelet size. We interpret this as meaning that unstable angina might be associated or preceded by an increase in platelet destruction rate that is not completely compensated for by an increase in platelet production rate. The large, more reactive platelets might be causally related to an ongoing coronary artery obstruction in unstable angina.

Adult↗
PubMed1998

The nine genes of the Nocardia lactamdurans cephamycin cluster are transcribed into large mRNAs from three promoters, two of them located in a bidirectional promoter region.

The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Base Sequence↗
PubMed1998

The folate branch of the methionine biosynthesis pathway in Streptomyces lividans: disruption of the 5,10-methylenetetrahydrofolate reductase gene leads to methionine auxotrophy.

In enterobacteria, the methyl group of methionine is donated by 5-methyltetrahydrofolate that is synthesized from N5,10-methylenetetrahydrofolate by the 5,10-methylenetetrahydrofolate reductase. The Streptomyces lividans metF gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids with a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of the transcription initiation site showed that the metF gene is expressed, forming a leaderless mRNA. A 13-bp tandem repeat located immediately upstream of the promoter region shows homology with the consensus MetR-binding sequence of Salmonella typhimurium. Expression of metF in multicopy plasmids in S. lividans resulted in accumulation of a 32-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Disruption of the metF gene led to methionine auxotrophy. Integration of the disrupting plasmid at the metF locus was confirmed by Southern hybridization in three randomly isolated transformants. The methionine auxotrophy was complemented by transformation of the auxotrophs with an undisrupted metF gene. These results indicate that the folate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.

Amino Acid Sequence↗
PubMed1998

S-nitrosoglutathione inhibits platelet activation and deposition in coronary artery saphenous vein grafts in vitro and in vivo.

OBJECTIVE: To investigate platelet activation and deposition in human saphenous vein and internal mammary artery grafts following coronary artery bypass in vitro and in vivo, as well as inhibition of activation by the platelet selective nitric oxide donor S-nitrosoglutathione (GSNO). DESIGN: Controlled in vitro and in vivo studies. SETTING: Tertiary cardiac centre. PATIENTS: 24 patients undergoing coronary artery bypass surgery requiring vein and artery grafts. INTERVENTIONS: In vitro: human platelet rich plasma was perfused through segments of vein and artery, with or without GSNO 10(-6) M, and the platelet count was measured in the effluent. In vivo: indium-111 labelled antibody against the platelet alpha granule protein GMP-140 was injected at the end of coronary bypass grafting and gamma counts were compared between vein and artery grafts with or without systemic infusion of GSNO (40 nmol/min). RESULTS: In vitro: platelet count in perfused vein (< 70% of baseline) decreased more than in artery segments (89-94% of baseline) (p < 0.001). The platelet count was unchanged with GSNO in vein and artery segments. In vivo: gamma counts were greater at all time points over vein than artery grafts (p < 0.05), and were reduced by infusion of GSNO (p < 0.05). CONCLUSIONS: Platelet activation is greater in vein than in artery grafts in vitro and in vivo. Activation, which contributes to early vein graft failure, was inhibited by GSNO.

Aged↗
PubMed1998

Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.

Prx1 and Prx2 are closely related paired-class homeobox genes that are expressed in very similar patterns predominantly in mesenchyme. Prx1 loss-of-function mutants show skeletal defects in skull, limbs and vertebral column (Martin, J. F., Bradley, A. and Olson, E. N. (1995) Genes Dev. 9, 1237-1249). We report here that mice in which Prx2 is inactivated by a lacZ insertion had no skeletal defects, whereas Prx1/Prx2 double mutants showed many novel abnormalities in addition to an aggravation of the Prx1 single mutant phenotype. We found defects in external, middle and inner ear, reduction or loss of skull bones, a reduced and sometimes cleft mandible, and limb abnormalities including postaxial polydactyly and bent zeugopods. A single, or no incisor was present in the lower jaw, and ectopic expression of Fgf8 and Pax9 was found medially in the mandibular arch. A novel method to detect &bgr ;-galactosidase activity in hydroxyethylmethacrylate sections allowed detailed analysis of Prx2 expression in affected structures. Our results suggest a role for Prx genes in mediating epitheliomesenchymal interactions in inner ear and lower jaw. In addition, Prx1 and Prx2 are involved in interactions between perichondrium and chondrocytes that regulate their proliferation or differentiation in the bones of the zeugopods.

Amino Acid Sequence↗
PubMed1997

VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries.

Thickening of the arterial intima and smooth muscle cell (SMC) proliferation remain major problems after vascular surgery and other types of vascular manipulations. We studied the effect of endothelial cell (EC)-specific vascular endothelial growth factor (VEGF) gene transfer on the thickening of the intima using a silicone collar inserted around carotid arteries that acted both as the agent that caused intimal SMC growth and as a reservoir for the transfected gene. The model preserved EC integrity and permitted direct extravascular gene transfer without any intravascular manipulation. Compared to beta-galactosidase (lacZ)-transfected control arteries, plasmid/liposome-mediated VEGF gene transfer significantly reduced intimal thickening 1 week after the gene transfer. Administration to the experimental animals of the nitric oxide (NO) synthase inhibitor L-NAME abolished the difference in intimal thickening between VEGF and lacZ-transfected arteries. Furthermore, VEGF caused NO release from cultured human umbilical vein EC. It is concluded that extravascular VEGF gene transfer attenuates intimal growth and could be useful for the prevention of intimal thickening during vascular surgery. Our results further suggest that VEGF may reduce SMC proliferation via a mechanism that involves VEGF-induced NO production from the endothelium.

Animals↗
PubMed1997

Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses.

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.

Adenoviridae↗
PubMed1997

Inducible nitric oxide synthase gene transcription and protein activity in the rat heart during endotoxaemia.

Septicaemia leads to an impairment of myocardial contractility in animals and humans. Cytokines released during endotoxaemia are capable of increasing inducible nitric oxide synthase (iNOS) expression in vitro in myocytes, endothelial cells and macrophages. The aim of this study was to assess whether iNOS gene transcription occurs in the myocyte in vivo. Rats were injected with intraperitoneal endotoxin. Myocardial sections obtained 4, 6 and 8 hours after infection were hybridised with oligonucleotides complementary to iNOS cDNA. Myocardial homogenates were used to measure NOS enzyme activity and to detect iNOS mRNA. Uninfected control animals did not demonstrate myocardial iNOS expression. Myocardium from endotoxaemic animals contained iNOS mRNA and high calcium-independent NOS enzyme activity. In situ hybridisation did not localise iNOS to myocytes but to cells located between myocytes. Endotoxaemia leads to iNOS gene transcription and calcium-independent NOS enzyme activity in the rat myocardium. In situ hybridisation demonstrates that iNOS is not transcribed by the myocyte in vivo.

Animals↗
PubMed1997

Food acquisition habits, nutrient intakes, and anthropometric data of Havasupai adults.

OBJECTIVE: To describe the dietary patterns, anthropometric data, and food sources of Havasupai adults (> or = 18 years old) and determine the effect of age and gender. DESIGN: Dietary intakes (one 24-hour recall), anthropometric measures (body mass index [BMI], waist-to-hip ratio [WHR]) and demographic data, including sites of food purchases, were obtained. Food sources of selected nutrients were calculated from diet recalls. SETTING/SUBJECTS: 92 adults (60 women, 32 men) from the Havasupai Reservation, Supai, Ariz. STATISTICAL ANALYSES: Descriptive statistics were generated for demographic data. Nutrient intakes, BMI, and WHR were compared across gender and age groups ("Younger" [18 to 59 years old] vs "older" [> or = 60 years old]) by one-way analysis of variance. Two-tailed t tests identified significant differences in selected food practices by age group. RESULTS: Diets were moderately high in fat (35% of energy), saturated fat (12%), and sugar (14%); intakes of zinc, calcium, vitamin A, vitamin B-6, and folate were frequently inadequate (less than two thirds of the Recommended Dietary Allowance). Of the 92 subjects, 76 (83%) were obese (BMI > or = 27). Fifty-four of the 60 women (90%) and 24 of the 32 men (75%) exhibited abdominal obesity (no age effect). Thirty-nine of the subjects (42%) consumed at least one food item purchased off the reservation on the day of the recall; the remaining 53 subjects (58%) consumed only food purchased or acquired on the reservation. Older Havasupai were significantly more dependent on the tribal store and other village food sources than were younger adults. Food sources of key nutrients did not differ by age or gender. APPLICATIONS: The dietary patterns of isolated populations may be shaped by the unique limitations of their food sources as well as by factors such as age and gender. Individual and community-wide efforts to improve nutrient intakes and food patterns must recognize these geographic limitations. For populations such as the Havasupai, cooperative marketing and health promotion efforts between tribal officials, health care providers, and managers of the cafe and tribal store could improve the availability and consumption of a wider range of health-promoting foods.

Adult↗
PubMed1997

Megakaryocytes and platelets in vascular disease.

Platelets are anucleate cells with no DNA. They are derived from their precursor, the megakaryocyte (MK), whose differentiation is characterized by nuclear polyploidization through a process called endomitosis. Changes in the MK-platelet-haemostasis axis may precede acute thrombotic events. Changes in MK ploidy distribution may be associated with the production of large platelets. Mean platelet volume (MPV) is an important biological variable as it is a determinant of platelet reactivity. Large platelets are denser and more active haemostatically. MPV is increased in patients after myocardial infarction (MI) and is a predictor of a further ischaemic event and death when measured after MI. It has been suggested that changes not only in platelets but also in the parental MK are associated with chronic and acute vascular events. The regulation of megakaryocytopoiesis depends on several haematopoietic factors such as thrombopoietin. An understanding of the signalling system that controls platelet number and size might give insight into a role of platelet production in thrombogenesis and atherogenesis.

Arteriosclerosis↗
PubMed1997

A cross-sectional study of platelet volume in healthy normotensive women with bilateral uterine artery notches.

High uterine artery resistance characterized by bilateral notches seen on Doppler ultrasound in the second half of pregnancy is associated with an increased risk of adverse outcome related to pre-eclampsia and intrauterine growth retardation. We sought to establish whether there was a difference in platelet volume in healthy, normotensive women with high-resistance uterine artery Doppler findings compared to those with normal uterine artery Doppler findings. Forty-seven women were allocated prospectively into 'bilateral notch' and 'no notch' groups at color Doppler imaging of the uterine arteries carried out at a mean of 26 weeks' gestation. The difference in platelet volume between the two groups and the relationship of adverse outcome with raised platelet volume and high-resistance uterine artery Doppler findings was investigated. Twenty-three women had no evidence of uterine artery notches and 24 had bilateral uterine artery notches. Platelet volume in the women with bilateral notches was greater than in those with no notches (8.28 fl vs. 7.46 fl; p = 0.01). However, unlike high-resistance uterine artery Doppler findings, increased platelet volume was not associated with adverse outcome. Uterine artery Doppler flow abnormalities have not previously been studied in combination with hematological or biochemical markers. Our findings show, for the first time, that women with bilateral uterine artery notches have an increased platelet volume compared to those with normal uterine artery Doppler findings many weeks before clinical signs of pre-eclampsia or fetal growth retardation are evident. Women with abnormal uterine artery flow at this gestation may have other cardiovascular and hematological differences compared to those with normal flow.

Arteries↗
PubMed1997

Length of the follicular phase, time of insemination, coital rate and the sex of offspring.

The penetrability of cervical mucus improves over the follicular phase. When the length of the follicular phase varies due to variation in the timing of the luteinizing hormone surge, mucus penetrability will also improve as the phase lengthens. As selection for Y spermatozoa decreases with improvements in mucus penetrability, sex ratios at conception should decline in longer follicular phases. Sex ratios should also decline as the time of insemination approaches ovulation unless hormonally-induced improvements in penetrability are reduced by the debris left by earlier inseminations.

Cervix Mucus↗
PubMed1997

Compartmental shifts of calcium and magnesium as a result of swimming and swimming training in rats.

To describe accurately the mineral changes (Ca and Mg) provoked by swimming, the aims of this study were to analyze those tissues that, with regard to their mineral content, can better classify individuals performing both swimming until exhaustion and swimming as training and to know the shifts of these minerals between different tissues after a single session of swimming until exhaustion and after training. Wistar rats were distributed into 12 groups, six male and six female (N = 10): 1) control rest group (CR); 2) trained rest group (TR); 3) control exercise group (CE); 4) trained exercise group (TE); 5) control recovery group (CER) and 6) trained recovery group (TER). The most informative tissues of Ca and Mg compartmental shifts during exercise have been determined. Discriminant analysis selected heart Ca, muscle Ca and bone Ca, bone Mg, erythrocyte Mg, and serum Mg as the most significant variables. The animals were classified by means of two canonical axes: the first one relates to training situation and sex, and the second one shows the special characteristics of trained male rats. Another independent discriminant analysis applied to male and female groups separately showed that the first canonical axis (control/trained) is basically defined by heart Ca, bone Ca, and erythrocyte Mg (male), and by heart Ca, bone Ca, and bone Mg (female), while the second axis, related to the exercise situations, is defined by the serum Mg levels in both sexes. We think that discriminant analysis is a statistical method capable of explaining physiological processes and classifying individuals performing exercises of different length. It suggests that the homeostasis of Ca and Mg is somewhat different for males and females. Serum magnesium must be considered to distinguish exercise situations. The analysis of these tissues could inform us about the mineral status of the rats and then we could correct possible deficiencies in our research. In this work we have only found different mineral redistributions among tissues. The trained animals have a better mineral recovery capacity than the untrained ones. Training has a different physiological repercussion in male and female rats on the basis of their respective maximal swimming times after training and their mineral behavior.

Animals↗
PubMed1997

A model for the design and evaluation of algorithms for closed-loop cardiovascular therapy.

Developing a clinically useful closed-loop drug delivery system can be extremely time consuming and costly. One approach to reducing the time and cost associated with developing closed-loop systems is to reduce the number of animal experiments and perform an extensive set of simulation studies. Through simulations, a closed-loop controller's performance can be evaluated over a complete spectrum of the patient population, including boundary conditions. Simulation studies are repeatable, offering significant advantages in comparing modifications in control algorithms. Finally, simulation studies can be performed in a fraction of the time required for animal studies, at a fraction of the cost. We have developed a simulator, that included a nonlinear pulsatile-flow cardiovascular model, a physiological regulatory mechanism, and the pharmacology of four frequently titrated cardiovascular drugs. This simulator has already been used in the design and evaluation of two closed-loop algorithms-a self-tuning regulator (STR) and a multiple model adaptive controller (MMAC)-for blood pressure control during and after cardiac surgery.

Algorithms↗