Biomedical subjects
Publications and source records attributed to G Martin.
We have tested conditions for the labeling and tailing the 3'-end of RNAs with yeast poly(A) polymerase. Conditions were optimized for addition of NTP, dNTP, or ddNTP nucleotides to RNA. ATP, GTP, and UTP were useful for adding homopolymer tracts of various lengths. The nonradioactive nucleotides biotin-N6-ATP and digoxigenin-11-UTP also were used efficiently.
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Enhanced 5 year survival rates with adjuvant chemotherapy for colon and rectal cancers are 5% and 9% respectively, according to recent meta-analysis. Despite the NIH consensus statement endorsing adjuvant chemotherapy, many clinicians regard such a seemingly small benefit not justworthy of the expense, inconvenience, discomfort and risk of treatment for their individual patient with colorectal carcinoma. The aim of this study is to evaluate these quality of life issues. The seven criteria considered most important were determined by interviews of treated patients, who emphasized the following quality of life parameters: nausea and vomiting, diarrhea, perineal dermatitis, asthenia, impairment of daily activity, family support, and difficulties of daily transportation to hospital. A numeric scale (1-5) was used to measure their answers (0 = hospitalization, 5 = no modification), and the nonparametric rank coefficient of Kendall was used to compare them. Twenty patients with colon cancer treated with Moertel's protocol and 5 patients with rectal cancer treated with Krook's protocol were evaluated. The study revealed a diminished quality of life for both patients with colon cancer (7 on a scale of 10) and those with rectal cancer (6 on the same scale). By using the same questionnaire at one week interval, the responses remained unchanged (p < 0.001). The effect of radiotherapy seems to be responsible for this difference. This study is one of the first to approach the quality of life from the real interested party's point of view: the patient.
Intracellular fatty acid (FA) concentrations are in part determined by a regulated import/export system that is controlled by two key proteins, i.e. fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which respectively facilitate the transport of FAs across the cell membrane and their esterification to prevent their efflux. The aim of this investigation was to analyze the expression pattern of FATP and ACS and to determine whether their expression was altered by agents that affect FA metabolism through the activation of peroxisome proliferator-activated receptors (PPAR) such as the fibrates and thiazolidinediones. FATP mRNA was ubiquitously expressed, with highest levels being detected in adipose tissue, heart, brain, and testis. Fibrate treatment, which is known to preferentially activate PPARalpha, induced FATP mRNA levels in rat liver and intestine and induced ACS mRNA levels in liver and kidney. The antidiabetic thiazolidinedione BRL 49653, which is a high-affinity ligand for the adipocyte-specific PPARgamma form, caused a small induction of muscle but a robust induction of adipose tissue FATP mRNA levels. BRL 49653 did not affect liver FATP and had a tendency to decrease heart FATP mRNA levels. ACS mRNA levels in general showed a similar pattern after BRL 49653 as FATP except for the muscle where ACS mRNA was induced. This regulation of FATP and ACS expression by PPAR activators was shown to be at the transcriptional level and could also be reproduced in vitro in cell culture systems. In the hepatocyte cell lines AML-12 or Fa 32, fenofibric acid, but not BRL 49653, induced FATP and ACS mRNA levels, whereas in the 3T3-L1 preadipocyte cell line, the PPARgamma ligand induced FATP and ACS mRNA levels quicker than fenofibric acid. Inducibility of ACS and FATP mRNA by PPARalpha or gamma activators correlated with the tissue-specific distribution of the respective PPARs and was furthermore associated with a concomitant increase in FA uptake. Most interestingly, thiazolidinedione antidiabetic agents seem to favor adipocyte-specific FA uptake relative to muscle, perhaps underlying in part the beneficial effects of these agents on insulin-mediated glucose disposal.
There is a need to prevent intestinal colonisation by Salmonella enteritidis and S. typhimurium in newly hatched chicks. Treatment with an undefined bacterial flora is not acceptable to regulatory agencies in some countries because of the potential risk of transmitting pathogens. A defined culture with a potency and stability equivalent to those of an undefined culture has not yet been developed. Since attenuated Salmonella vaccine strains could possess the colonisation characteristics but not the virulence of Salmonella wild-type strains, they could inhibit colonisation of the challenge organism. S. typhimurium live vaccines registered in Germany (Zoosaloral H, Salmonella vac T), S. enteritidis aroA and S. typhimurium aroA strains, S. enteritidis, S. typhimurium and S. infantis wild-type strains or a competitive exclusion product (Broilact) were used as pretreatment cultures and evaluated for their inhibitory effects against S. enteritidis and S. typhimurium colonisation in newly hatched SPF chickens. Day-old chicks were administered a pretreatment culture and infected orally with variants of S. enteritidis or S. typhimurium wild type-strains resistant to nalidixic acid or rifampicin 1 day after pretreatment. On days 2 and 6 after infection, viable numbers of the challenge strain in liver and caeca were determined. The results for birds pretreated with Broilact showed a distinct protective effect against both S. enteritidis and S. typhimurium at a challenge dose of 10(4) cfu/bird. After pretreatment of chicks with S. enteritidis and S. typhimurium wild-type strains, the greatest degree of inhibition of caecal colonisation was produced using isogenic strains. Colonisation after infection with non-isogenic strains could not be prevented but only reduced for a brief period. These effects were also observed after administration of aroA strains of S. enteritidis and S. typhimurium but the protective effect was considerably lower than after pretreatment with wild-type Salmonella strains. Inoculation with attenuated S. typhimurium vaccines resulted in a weak but significantly reduced colonisation by S. typhimurium. Colonisation by S. enteritidis could not be diminished by either of the S. typhimurium vaccine strains. The results indicate in principle the potency of Salmonella vaccine strains to inhibit Salmonella wild-type colonisation in newly hatched chicks. Potential vaccine candidates should be tested for their capacity to prevent intestinal colonisation in newly hatched chicks.
The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate --> alpha-ketoglutarate --> glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through alpha-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.
Based on the same principles as those utilized in a recent study for modeling glucose metabolism (Martin, G., Chauvin, M. F., Dugelay, S., and Baverel, G. (1994) J. Biol. Chem. 269, 26034-26039), a method is presented for determining metabolic fluxes involved in glutamate metabolism in mammalian cells. This model consists of five different cycles that operate simultaneously. It includes not only the tricarboxylic acid cycle, the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> oxaloacetate" cycle and the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA --> citrate --> oxaloacetate" cycle but also the "glutamate --> alpha-ketoglutarate --> glutamate" and the "glutamate --> glutamine --> glutamate" cycles. The fates of each carbon of glutamate, expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites, are described by a set of equations. Since the data introduced in the model are micrograms of atom of traced carbon incorporated into each carbon of end products, the calculation strategy was determined on the basis of the most reliable parameters determined experimentally. This model, whose calculation routes offer a large degree of flexibility, is applicable to data obtained by 13C NMR spectroscopy, gas chromatography - mass spectrometry, or 14C counting in a great variety of mammalian cells.
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The nucleus accumbens (NAcc) may play a major role in opiate dependence, and central NMDA receptors are reported to influence opiate tolerance and dependence. Therefore, we investigated the effects of the selective mu-opioid receptor agonist [D-Ala2-N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) on membrane properties of rat NAcc neurons and on events mediated by NMDA and non-NMDA glutamate receptors, using intracellular recording in a brain slice preparation. Most NAcc neurons showed a marked inward rectification (correlated with Cs+- and Ba2+-sensitive inward relaxations) when hyperpolarized, as well as a slowly depolarizing ramp with positive current pulses. Superfusion of DAMGO did not alter membrane potential, input resistance, or the inward relaxations. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) used to block non-NMDA glutamate receptors and bicuculline to block GABAA receptors, EPSPs evoked by local stimulation displayed characteristics of an NMDA component: (1) long duration, (2) voltage sensitivity, and (3) blockade by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (D-APV). DAMGO (0.1-1 muM) significantly decreased both NMDA- and non-NMDA-EPSP amplitudes with reversal of this effect by naloxone and the mu-selective antagonist [Cys2-Tyr3-Orn5-Pen7]-somatostatinamide (CTOP). To assess a postsynaptic action of DAMGO, we superfused slices with tetrodotoxin and evoked inward currents by local application of glutamate agonists. Surprisingly, 0.1-1 microM DAMGO markedly enhanced the NMDA currents (with reversal by CTOP) but reduced the non-NMDA currents. At higher concentrations (5 microM), DAMGO reduced NMDA currents, but this effect was enhanced, not blocked, by CTOP. These results indicate a complex DAMGO modulation of the NMDA component of glutamatergic synaptic transmission in NAcc: mu receptor activation decreases NMDA-EPSP amplitudes presynaptically yet increases NMDA currents postsynaptically. These new data may provide a cellular mechanism for the previously reported role of NMDA receptors in opiate tolerance and dependence.
Quantitative PCR-based assays of leukaemic cells in remission marrow hold promise for therapeutic guidance, but are not yet sufficiently reliable for clinical application. For B-lineage ALL, these assays usually involve PCR of clonal somatic gene rearrangements of the immunoglobulin heavy chain gene. The most accurate quantification can be achieved by competitive PCR. Here we present a novel approach for the production of reference standards for use in nested competitive PCR of these gene rearrangements, which might enable more reliable assessment of MRD for prognosis and selection of patients for individualised therapy.
OBJECTIVES: To compare lidocaine hydrocarbonate and lidocaine hydrochloride, with and without adrenaline, in the axillary block obtained with a neurostimulator. STUDY DESIGN: Prospective, randomized, double blind study. PATIENTS: Sixty-six patients undergoing surgery of the upper limb under axillary block, allocated into four groups. METHODS: The criteria for evaluation were: onset time, duration and quality of sensory and motor blockades, and blood concentrations of lidocaine in 39 patients. In all patients musculocutaneous, radial, median and ulnar nerves were stimulated and the volume of local anaesthetic administered was 25 mL per square meter of body surface. Group 1 received lidocaine hydrocarbonate 1% (n = 17), group 2, lidocaine hydrocarbonate 1% with adrenaline 1/200,000 (n = 17), group 3, lidocaine hydrochloride 1% (n = 16) and group 4, lidocaine hydrochloride 1% with adrenaline 1/200,000 (n = 16). RESULTS: No significant inter-group differences were found concerning sensory and motor blockades and onset time. The duration of analgesia was longer in groups CO2 + A and HCL + A. The lidocaine blood concentrations were globally lower in group HCL + A. CONCLUSIONS: Considering the cost/benefit ratio and the absence of clinical benefits of lidocaine hydrocarbonate, lidocaine hydrochloride should be preferred.
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The efficacy of three different root canal instruments in maintaining the original shape of curved canals was evaluated. Acrylic blocks were prepared to simulate canals with 40 degrees and 60 degrees curvature. K-Flexofiles and Macfiles were used with a step-back technique utilizing an 'in/out' filing motion. The balanced-force technique was used with Canal Master 'U' instruments. Ten 40 degrees and 10 60 degrees canals were prepared with each instrument. The results were evaluated using superimposed photographic slides at x20 magnification to show the canals before and after the instrumentation. The width of the prepared canals was measured at their convex/concave point to determine the divergence of the instrumented canal shape from the original canal shape. The measurements were taken at the level of the apical foramen and 3 and 6 mm coronal to it. In this way it was possible to recognize all the defects created during the preparation of a canal. Among the tested instruments, the Macfile provoked minimal canal deviation (P = 0.05). The Canal Master 'U' was similarly effective (P = 0.05), but had the highest incidence of instrument fracture. The K-Flexofile demonstrated the most defects in canal preparation and canal deviation, particularly at the level of the apical foramen (P < 0.01).
The three peroxisome proliferator-activated receptors (PPARs), PPAR alpha, delta and gamma, form a subfamily of the nuclear hormone receptor gene family. PPAR alpha has been shown to bind and be activated by leukotriene B4 and fibrates, whereas prostaglandin J2 derivatives and the antidiabetic thiazolidinediones, respectively, are natural and synthetic ligands for PPAR gamma. The availability of ligands and activators for PPAR alpha and PPAR gamma allowed an initial assessment of their respective functions. PPAR alpha and PPAR gamma are shown to function as important regulators in lipid and glucose metabolism, adipocyte differentiation, inflammatory response and energy homeostasis. PPAR alpha seems to mediate its pleiotropic effects mainly through the stimulation of oxidation of lipids, whereas PPAR gamma is a key mediator of lipid storage. The next few years will be very exciting as additional studies will refine our current knowledge about PPAR alpha and PPAR gamma and may reveal a ligand and role for the lonesome orphan among the PPARs, PPAR delta.
Genetic studies and quantitative determination of levels of 3-hydroxykynurenine and kynurenine were performed in an albino strain of a terrestrial isopod Armadillidium vulgare. From the results of matings between the albino and the albino, the red, the dark red, or the wild type individuals, the albino A. vulgare seems to be regulated by an autosomal gene(s) recessive to its wild allele. Litter mating of F1 progenies obtained by crossing the albino and the red mutant or the albino and the dark red mutant yielded progenies at a ratio of 3:6:3:4 for the red, the dark red, the wild, and the albino phenotypes, respectively. The albino gene(s) seems not to be allelic but to be epistatic to the red gene(s) with respect to ommochrome biosynthesis. Quantitative determination of 3-hydroxykynurenine carried out by high-performance liquid chromatography with electrochemical detection revealed that the 3-hydroxykynurenine content in the albino was significantly lower than that in the wild or the red type. The whole content of 3-hydroxykynurenine after enzymatic conversion of kynurenine to 3-hydroxykynurenine was still considerably lower than that found in the wild type, even though it increased after the conversion. The albino gene(s) seems to be associated with a blockage at distinct level(s) of ommochrome biosynthesis.
Twenty-four-hour-old, aerobically grown, Luria-Bertani broth cultures of Salmonella typhimurium F98 suppressed the growth of a spectinomycin-resistant (Spcr) derivative of the same strain inoculated at 10(3) CFU ml(-1). This growth suppression is genus specific and RpoS independent, and it is not solely a result of nutrient depletion (P. A. Barrow, M. A. Lovell, and L. Zhang-Barber, J. Bacteriol. 178:3072-3076, 1996). Mutations in three genes are shown here to significantly reduce growth suppression under these conditions. The mutations were located in the nuo, cyd, and unc operons, which code for the NADH dehydrogenase I, cytochrome d oxidase, and F0F1 proton-translocating ATPase complexes, respectively. When cultures were grown under strictly anaerobic conditions, only the unc mutant did not suppress growth. Prior colonization of the alimentary tract of newly hatched chickens with the S. typhimurium F98 wild type or nuo or cyd mutants suppressed colonization by an S. typhimurium F98 Spcr derivative inoculated 24 h later. In contrast, the S. typhimurium unc mutant did not suppress colonization. The nuo and unc mutants showed poorer growth on certain carbon sources. The data support the hypothesis that growth suppression operates because of the absence of a utilizable carbon source or electron acceptor.
In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.