Biomedical subjects
Publications and source records attributed to W Martin.
Hydroxocobalamin inhibited nitrergic nerve-induced relaxations in rat anococcygeus and bovine retractor penis muscles in a concentration-dependent manner. In the rat anococcygeus muscle, the inhibition was greater in light than in dark conditions, whereas in the bovine retractor penis the inhibition was similar under both conditions.
The evolutionary histories of the 12 enzymes that catalyze the reactions of the Calvin cycle in higher-plant chloroplasts are summarized. They are shown to be encoded by a mixture of nuclear genes of cyanobacterial and proteobacterial origin. Moreover, where cytosolic isoforms of these enzymes are found they are almost invariably encoded by genes of clearly endosymbiont origin. We infer that endosymbiosis resulted in functional redundancy that was eliminated through differential gene loss, with intruding eubacterial genes repeatedly replacing pre-existing nuclear counterparts to which they were either functionally or structurally homologous. Our findings fail to support the 'product-specificity corollary', which predicts re-targeting of nuclear-encoded gene products to the organelle from whose genome they originated. Rather it would appear that the enzymes of central carbohydrate metabolism have evolved novel targeting possibilities regardless of their origins. Our findings suggest a new hypothesis to explain organelle genome persistence, based on the testable idea that some organelle-encoded gene products might be toxic when present in the cytosol or other inappropriate cellular compartments.
Thirty percent of patients undergoing percutaneous transluminal coronary angioplasty develop recurrent disease within a year. This is usually due to the rapid accumulation of intimal smooth muscle cells and extracellular matrix, which causes luminal narrowing, and is probably orchestrated by several mitogenic and chemotactic factors, of which platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) appear to be particularly important. We have investigated the effects of administering a combination of neutralizing antibodies directed against PDGF-BB and bFGF on neo-intima development following balloon catheter injury in the rat carotid artery. Purified sheep anti-PDGF-BB and anti-bFGF immunoglobulins (IgGs) were administered singly and in combination prior to mechanical injury and daily until sacrifice, 8 days later. Plasma titres of exogenous anti-PDGF-BB and anti-bFGF were maintained at levels 10-20-fold higher than those required to neutralise the mitogenic and chemotactic effects of 20 ng/ml of PDGF-BB, or 10 ng/ml bFGF in vitro. Used singly, anti-PDGF IgG treatment was associated with a 47% reduction in intimal thickness and a 59% reduction in intimal:medial area ratio; anti-bFGF IgG administration caused a 53% reduction in intimal thickness, and a 50% reduction in intimal:medial area ratio. Treatment with a combination of these antibodies resulted in a 83.8% reduction in intimal thickness (P < 0.05), and a 91% reduction in intimal:medial area ratio (P < 0.01). The latter treatment was also associated with a significantly higher intimal cell density (14.2 +/- 1.6 x 10(3) nuclei/mm2) compared to animals receiving non-immune IgG (7.8 +/- 0.8 x 10(3) nuclei/mm2; P < 0.025), although intimal and medial cell proliferation indices were not significantly different between the groups (P > 0.05). Our results suggest that in this particular model, PDGF-BB and bFGF are the major factors controlling neointimal hyperplasia, and that these growth factors are operating principally via an effect on smooth muscle cell migration and extracellular matrix protein accumulation.
OBJECTIVES: Despite laboratory evidence of leucocyte involvement in reperfusion injury, cardiac surgical clinical trials do not support the therapeutic effectiveness of leucocyte filtration. Furthermore, the direct effects of crystalloid cardioplegia and reperfusion on the capillaries of the heart have yet to be elucidated. We tested the effects of cardioplegic arrest and reperfusion both with and without leucocyte depletion, in a model of cardiopulmonary bypass that mimics clinical cardiac surgical conditions. METHODS: Four groups of Landrace pigs were studied. Group A (n = 6) underwent 30 min of hypothermic (28 degrees C) cardiopulmonary bypass. Groups B (n = 6), C (n = 6) and D (n = 6) also had 90 min of cardioplegic arrest. Group C was then reperfused with whole blood, while Group D was reperfused with leucocyte-depleted blood. Microvascular methylmethacrylate corrosion casts were made at the end of the experimental period. Myocardial vascular anatomy was defined by electron microscopy and capillary abundance derived from this and from the weight of casts from representative areas. Leucocyte deposition was assessed using radioisotope-labelled leucocytes. Ischaemic damage to tissues was graded according to light and electron microscopic findings. RESULTS: In Group A the mean (+/- S.D.) vascular cast weight/volume of myocardium (density) was 125 +/- 9 mg/mm3. After cardioplegic arrest (Group B), it fell to 74 +/- 7 mg/mm3 (P < 0.0001) due to absence of capillaries, although arterioles, venules and non-nutritive bypass vessels remained patent. Following reperfusion with whole blood (Group C), capillary numbers partially recovered but luminal diameters were reduced with a cast density of 94 +/- 5 mg/mm3 (P < 0.0001 versus Group A and B). Leucocyte-depleted (87-92%) reperfusion in Group D did not affect cast density (90 +/- 3 mg/mm3; P = 0.17). Coronary vascular resistances in Groups C and D rose slightly, but not significantly, during reperfusion. CONCLUSIONS: Following cardioplegic arrest, microvascular changes are marked. These changes are partially reversed by 30 min reperfusion. Leucocyte depletion does not ameliorate these effects in this model.
1. In this study the impairment induced by hydrogen peroxide of vascular reactivity and the role of endogenous catalase in protection against this impairment was assessed in isolated rings of rat aorta. 2. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout depressed, in a time-dependent manner, the subsequent ability of endothelium-containing and endothelium-denuded rings to contract to phenylephrine. 3. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) to inhibit endogenous catalase had no effect by itself on subsequent phenylephrine-induced contraction. However, pretreatment with 3-amino-1,2,4-triazole did lead to a profound enhancement of the ability of hydrogen peroxide (1 mM, present for the final 30 min of the 90 min incubation, followed by washout) to depress phenylephrine-induced contraction in both endothelium-containing and endothelium-denuded rings. 4. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout inhibited, in a time-dependent manner, the subsequent ability of acetylcholine (10 nM-3 microM) to induce endothelium-dependent relaxation. Furthermore, incubation with hydrogen peroxide 1 mM (30 min, followed by washout) also inhibited relaxation induced by glyceryl trinitrate (1-100 nM) or isoprenaline (10 nM-3 microM) in endothelium-denuded rings. 5. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) had no effect by itself on relaxation induced by acetylcholine, glyceryl trinitrate or isoprenaline. In contrast, pretreatment with 3-amino-1,2,4-triazole led to profound enhancement of the ability of hydrogen peroxide (1 mM, present for final 30 min of the 90 min incubation) to block relaxation to acetylcholine, glyceryl trinitrate or isoprenaline. 6. On the basis of the actions of 3-amino-1,2,4-triazole, it is likely that endogenous catalase plays an important role in the protection of vascular reactivity of rat aorta against oxidant damage by high (1 mM) but not lower (0.1 mM) concentrations of hydrogen peroxide. The data are consistent with the promotion of non-selective damage to the vascular smooth muscle cells by hydrogen peroxide, but endothelial damage may also be sustained.
Several studies have indicated that growth factors, such as platelet derived growth factor (PDGF), may be important in atherogenesis. These factors are released from platelets, or expressed by cells of the arterial wall. In order to study their role in atherogenesis more directly, rabbits were immunized with PDGF-BB, platelet cytosolic protein, or human serum albumin (HSA), until high titres of antibody were attained. Atherosclerotic lesions were subsequently induced by feeding the animals with a 2% cholesterol enriched diet. At the end of approximately 3 months, the extent of aortic lesion development was assessed by image analysis of en face preparations of aortae stained with Oil Red-O, and histological segments of aortae taken at the level of the first intercostal artery branch point. The endogenous antibodies were characterized with respect to their cross-reactivity, and ability to neutralize PDGF and platelet cytosol-induced cell proliferation and migration in vitro. The endogenous, anti-PDGF-BB antibody was isoform specific, and neutralized the mitogenic and chemotactic properties of PDGF-BB and rabbit platelet cytosolic protein in vitro. The anti-platelet cytosol antibody partially inhibited the chemotactic and mitogenic properties of rabbit platelet cytosolic protein. Compared to non-immune rabbits (n = 5), animals immunized with HSA (n = 4) had a significantly larger area of aortic lesion involvement (P < 0.01), whereas aortic lesions in rabbits immunized with PDGF-BB (n = 5), or platelet cytosolic protein (n = 7) were significantly smaller than either non-immune animals, or animals immunized with HSA (P < 0.05). The same pattern was observed for other measures of aortic lesion involvement including aortic intima:media ratio at the level of the first intercostal artery. These data suggest that PDGF-BB, and possibly other platelet-associated growth factors, are involved in cholesterol-induced atherosclerosis.
Characterization of flow properties of an artificial ventricle may aid development of designs to minimize thrombosis. Techniques for determining two such flow properties, viz. ventricular clearance rate and ejection fraction, are compared and validated here for polyurethane and silicone rubber ventricles operated in a mock circulatory loop at various stroke volumes and pulse rates. Ventricular clearance rats were measured both by clinical radionuclide tracer techniques and by an optical tracer method. Ejection fractions were measured by radionuclide imaging and validated by direct measurements of flow rate and ventricular volume. Results from the two methods for ventricular clearance are in close agreement. The optical tracer method is superior in spatial resolution, convenience and economy, but the radionuclide tracer method for ejection fraction gives excellent agreement with the absolute measurements.
Explore the source record for details and available documents.
We studied the new catechol-O-methyltransferase inhibitor tolcapone, 100 and 200 mg, three times daily (tid) in a randomized, double-blind, parallel-group trial involving 202 parkinsonian patients who were experiencing the "wearing-off" phenomenon on levodopa therapy. After 3 months, patients receiving tolcapone had a significant decrease in mean daily levodopa dose requirement compared with placebo-treated patients (p < 0.01). In patients treated with tolcapone 200 mg tid, daily "off" time, measured using patient diaries, was reduced from baseline by 3.25 hours; this reduction was significantly different from that seen in the placebo group (p < 0.01). Moreover, the number of daily levodopa intakes was reduced significantly in each tolcapone group compared with placebo (p < 0.01). We found significant improvements in motor function and overall efficacy in the tolcapone groups (p < 0.01). The most frequent adverse events were associated with levodopa treatment. Dyskinesia developed or worsened in 18% of patients receiving placebo, in 51% receiving tolcapone 100 mg tid, and in 64% receiving 200 mg tid, with most cases occurring within the first 30 days of the study. Diarrhea was the most frequent nondopaminergic event, occurring in 14% on placebo, 13% on tolcapone 100 mg tid, and 19% on 200 mg tid. Overall 18% of patients withdrew because of adverse events: 15% on placebo, 17% on tolcapone 100 mg tid, and 22% on 200 mg tid. We conclude that tolcapone as an adjunct offers promise for the relief of the "wearing-off" phenomenon in levodopa-treated parkinsonian patients.
The purpose of this study was to determine the relaxant effects in vitro of two nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, which are currently available for use in vivo, on contractions of non-labouring myometrium from pregnant women. Since nitric oxide also mediates relaxation by increasing the concentration of cGMP, sensitivity to 8-bromo-cGMP (a cGMP analogue) was also determined. The effects of the K(+)-channel opener lemakalim and of the Ca(2+)-channel blocker nifedipine were studied for comparison. After the addition of glyceryl trinitrate (0.1-100 mumol l-1), sodium nitroprusside (0.1-100 mumol l-1) or 8-bromo-cGMP (0.001-3 mmol l-1), the spontaneous rhythmic contractility of myometrial strips was inhibited in a concentration-dependent manner: the maximum inhibition produced by the highest tested concentration of each drug was 40 +/- 7%, 53 +/- 8% and 39 +/- 8% of the original degree of contraction, respectively. Myometrial contractions were completely abolished by lemakalim and by nifedipine and verapamil at concentrations of > or = 10(-5) mol l-1. The nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, attenuate myometrial contractions and are therefore useful as tocolytic agents. However, at equimolar concentrations in vitro, the ability of glyceryl trinitrate and sodium nitroprusside to attenuate myometrial contractions is less than that of lemakalin, nifedipine and verapamil. Controlled trials are required to determine the side-effects and clinical efficacy of each of these agents in vivo.
Homospermidine synthase (HSS) catalyzes the synthesis of the polyamine homospermidine from 2 mol putrescine in an NAD(+)-dependent reaction. In this study, the enzyme was purified from anaerobically grown cultures of the photosynthetic bacterium Rhodopseudomonas viridis to electrophoretic homogeneity using a three-step procedure. The enzyme was shown to be a homodimer of 52-kDa subunits. Six endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers designed against these peptides, specific PCR products from R. viridis DNA were obtained that were used as hybridization probes to isolate the hss gene from a library constructed in lambda EMBL4. The hss gene and flanking regions were sequenced and were shown to exist as a single copy in the R. viridis genome. HSS is translated from a monocistronic mRNA and possesses no detectable similarity to previously sequenced gene products. Escherichia coli, which lacks HSS activity, was transformed with an expression plasmid containing the hss coding region under the control of a bacteriophage T7 promoter. Upon induction, transformed F. coli cells accumulate enzymatically active and highly stable R. viridis HSS at levels corresponding to 40-50% of the soluble protein in crude extracts.
To validate their correlation with tissue iron concentration, proton transverse relaxation measurements have been made at 2.35 T (100 MHz) in 25 samples of excised, frozen, but unfixed human gray matter tissue obtained from the globus pallidus, putamen, caudate, thalamus, and cortex of five postmortem brains free of neurological disease. The iron concentration was independently measured, using atomic absorption spectroscopy. The proton transverse relaxation measurements exploited the interecho time dependence of the apparent transverse relaxation rate, R2app, obtained from a Carr-Purcell-Meiboom-Gill (CPMG) sequence. An empirical semilogarithmic relationship between R2app and the interecho time provided a measure of the relaxation enhancement due to iron, namely, a slope p, which demonstrated a significant correlation (r = 0.78, P < 0.001) with tissue iron concentration. Moreover, a simple rate difference, delta R2app, determined between interecho time values of 6 and 60 ms, was also found to correlate significantly with iron concentration (r = 0.81, P < 0.001). Both of the foregoing correlations were better than that of R2app itself. When the tissue samples were subdivided into brain structure groups, the intergroup differences in rho reflected their known differences in iron accumulation and correlated with those of the mean group iron content, determined by atomic absorption spectrometry.
Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme. We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms. A very high calculated net negative charge of 63 for PGK from H. vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol. We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes. The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria. Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol. Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e. contain more genes of eubacterial origin, than is generally assumed.
Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.
A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.
Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.
Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15-35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.
Explore the source record for details and available documents.