Focusing of submicron beams for TeV-scale e+e- linear colliders.
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Journal Article↗
Biomedical subjects
G Roy
Publications and source records attributed to G Roy.
Amino acid current through anion channels in cultured human glial cells.
During volume regulation in hypotonic media, glial cells release a large portion of their amino acids. These amino acid losses appear to be mediated by a diffusion type of transport and a swelling-activated chloride channel seems to be involved. The objective of this project was to provide direct evidence that amino acids could diffuse through a Cl- channel. Using a human glial cell line, Cl- currents activated in hypotonic media were measured in whole-cell patch clamp. To measure the currents produced by amino acids, it was necessary to increase the pH of external solutions to basic values reaching 9.6 and 10.0 to raise the concentration of the anionic form of these amino acids. Introducing external hypotonic media containing high concentrations of amino acids, like glycine, taurine, glutamine and glutamate, it was possible to measure their respective current-voltage curves with NMDG-Cl-filled pipettes. From the reversal potentials, their permeability ratios with respect to chloride were determined. It was found that the low molecular weight amino acids, like glycine, were most permeant, while the larger ones, like glutamine, had a lower permeability with respect to chloride. The amino acids with two carboxyl groups, like glutamate, had a much lower permeability ratio. The reversal potentials for some metabolites, like lactate and malate were also measured for comparison. These results demonstrate that amino acids can diffuse through anion channels and that activation of these channels in pathological conditions could be at least partly responsible for the observed increase in external amino acids.
Amino Acids↗
Structural organization of the mouse DNA repair gene, N-methylpurine-DNA glycosylase.
N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.
3T3 Cells↗
Effect of thyroxine on experimental bronchospasm in guinea pigs.
Effect of Thyroxine was studied in histamine induced bronchospasm in guinea pigs. Chronic treatment with the drug significantly protected against experimental bronchospasm. Thyroxine also potentiated salbutamol evoked bronchodilation in this experimental model. Up-regulation of beta-2 adrenoceptors in bronchial smooth muscle may be the probable mechanism of action of thyroxine.
Albuterol↗
Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance.
Oxyanions in the form of pentavalent antimony compounds are currently the drug of choice for treating leishmaniasis. Leishmania mutants resistant to high concentrations of the oxyanion arsenite were obtained in a stepwise selection procedure. Amplification of the H locus P-glycoprotein-related gene ltpgpA, as part of extrachromosomal circles, is a frequent event in arsenite-resistant cells, but was observed only in cells resistant to high concentrations of the metalloid salts. Revertants grown in the absence of the drug lost their ltpgpA-containing amplicon and part of their resistance. The results of previous transfection experiments in Leishmania had suggested that ltpgpA is only involved in low level resistance to arsenite and antimonite. The results of this study using transfection of ltpgpA alleles isolated from arsenite-resistant mutants or of whole circular amplicons containing ltpgpA demonstrate clearly that this P-glycoprotein-related gene is involved in low level resistance to oxyanions, including the pentavalent antimony-containing drug Pentostam. By site-directed mutagenesis, the LtpgpA protein was shown to require an intact nucleotide-binding site to confer arsenite resistance. Under the experimental conditions used, decreased accumulation of the 73AsO2- drug was not observed in the ltpgpA transfectants. One possibility is that LtpgpA-mediated arsenite resistance could result from sequestration of the toxic anion in an intracellular compartment. The results indicate that despite the fact that ltpgpA amplification is a frequent event in oxyanion-resistant mutants, it contributes only slightly to the overall resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene.
The short chain dehydrogenase gene ltdh, which is derived from the H locus of Leishmania, confers high level resistance to the dihydrofolate reductase inhibitor methotrexate via a novel mechanism. Resistance is correlated with LTDH overproduction. High level resistance of ltdh transfectants was observed even in poor media where reduced folates are essential for the synthesis of thymidylate precursors. The ltdh transfectants were also capable of growing for several passages, at slightly reduced rates, in unsupplemented folate-deficient medium (fdDME-L), unlike the wild-type cells that could grow only in fdDME-L if supplemented with various folates or pterins. An homozygous ltdh mutant was obtained by gene targeting. This mutant became hypersensitive to methotrexate, but unlike wild-type cells, methotrexate toxicity could not be circumvented by the addition of thymidine. Although the homozygous mutant was capable of growing in fdDME-L supplemented with folate derivatives, it lost its ability to thrive in fdDME-L supplemented with pterins. Our results support the model in which LTDH is a key enzyme responsible for the conversion of a pterin derivative into an essential cofactor. This essential cofactor can also be converted into reduced folates at sufficient levels for growth when LTDH is overproduced, rendering dihydrofolate reductase dispensable. The novelty and uniqueness of LTDH opens the possibility of developing parasite-specific inhibitors.
Animals↗
Channels for ions and amino acids in kidney cultured cells (MDCK) during volume regulation.
A cell line from canine kidney (MDCK) was studied in hypotonic conditions to determine the transport mechanisms involved during regulatory volume decrease (RVD). It was found that RVD was related to a loss of K+, Cl- and amino acids. Membrane potential measurements during RVD have shown a hyperpolarization followed by a depolarization, implying that ion fluxes were probably occurring through channels. Quinine was found to inhibit RVD, the ion fluxes and the amino acid losses. A long preincubation in Ca(++)-free EGTA medium had no effect on RVD in cells exposed to the hypotonic medium. Single channel experiments with the patch clamp technique have shown that cell swelling rapidly activates K+ channels and, after a 30 sec delay, Cl- channels. The K+ channel was highly selective and had a linear I-V curve with a conductance of 25 pS. The Cl- channel was an outward rectifier with conductances of 63 and 26 pS. It was not very selective; replacing Cl- with NO3- did not change the I-V curve and did not affect RVD. The amino acid losses during RVD were studied with HPLC. It was found that three of them, glutamate, taurine and glycine, had large concentrations and were lost at different rates. Their fluxes during RVD depended linearly on their external concentrations, meaning that the transport process was diffusional. Single channel measurements with glutamate, aspartate or taurine in place of Cl- have shown that these amino acid losses occurred through the Cl- channel.
Amino Acids↗
High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system.
Leishmania tarentolae cells selected for resistance to the oxyanions pentavalent or trivalent antimonials or to trivalent arsenicals exhibited cross-resistance to the other oxyanions. The basis for resistance in these mutants was studied by transport experiments using radioactive arsenite. All mutants exhibiting high level resistance to arsenite showed a marked decrease in the steady-state accumulation of arsenite. Decreased accumulation was also observed in antimonials-resistant mutants cross-resistant to various concentrations of arsenite. Cells depleted of endogenous energy reserves with metabolic inhibitors were loaded with radioactive arsenite; following addition of glucose, rapid efflux of arsenite was observed from arsenite mutant cells. Mutants resistant to high levels of arsenicals exhibited amplification of the P-glycoprotein related gene ltpgpA or of a linear amplicon of unknown function. However, the efflux-mediated arsenite resistance did not correlate with the amplification of the ltpgpA gene or with the presence of the linear amplicon. The calcium channel blocker verapamil and arsenite act in synergy in cells exhibiting the efflux system. Overall the oxyanion efflux system in Leishmania shares several properties with other resistance efflux systems mediated by transporters.
Animals↗
Autonomous replication of bacterial DNA plasmid oligomers in Leishmania.
Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.
Animals↗
Easy adaptation of protein structure to sequence.
An investigation into the conservation of coarse, medium and fine grain structural properties has been performed over a data set of 175 protein tertiary structures in 34 different families, each characterized by a common core fold and a library of conserved sites formed for each family. It is shown that, while the conservation of coarse and medium grain properties correlates to the structural deviation between the proteins, fine grain properties are poorly conserved except in functional sites. This flexibility in fine grain properties suggests that folding can be viewed as an optimization process whereby side chains have freedom to position themselves as best as possible given environmental conformational constraints and that given a basic framework, the local structure is able to adapt easily to sequence variation. The conserved cores of the 34 families are used to estimate a minimal core size of 35% of the fold, consistent with buried residue considerations. Finally, conservation in side chain chi 1 torsion angles is combined with structural deviation, sequence deviation and resolution to suggest a set of example structure pairs suitable for testing automatic homology modelling programs.
Hydrogen Bonding↗
Improving medication administration through an enhanced occurrence reporting system.
Accurate data about medication errors are essential to prevention efforts. A multidisciplinary team at Bon Secours Hospital trialed an innovative method of reporting medication errors. It increased the number of reported medication errors six times compared with the same time period for the previous year on the pilot unit. It also saved time for the person reporting the error. The continuous quality improvement methodology was used to implement the new process.
Attitude of Health Personnel↗
beta -delayed alpha decay of 17N.
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Journal Article↗
Tensor polarization in elastic electron-deuteron scattering in the momentum transfer range 3.8 <= Q <= 4.6 fm-1.
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Journal Article↗
Constraints on the low-energy E1 cross section of 12C( alpha, gamma )16O from the beta -delayed alpha spectrum of 16N.
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Journal Article↗
Nuclear localization of the PEP protein tyrosine phosphatase.
PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.
Amino Acid Sequence↗
Channels for amino acids and metabolites activated by cell volume regulation.
Cell volume regulation after swelling has been widely demonstrated and attributed to a loss of KCl in the external medium. But it was also shown in a few cell types that organic cell osmolytes, particularly amino acids, were contributing to volume regulation. These effluxes occurred through a diffusional type of transport. Electrophysiological experiments with patch-clamp electrodes have demonstrated the activation of selective K+ channels in parallel with chloride channels during volume regulation. These chloride channels appear much less selective for a large variety of anions. Many cell osmolytes, not only negatively charged but also neutral ones, are permeable through these channels. When the cells swell in hypotonic media, they loose amino acids and other neutral and negative osmolytes through these channels. It is possible that these channels in astrocytes play an important role in pathological conditions, e.g. brain ischemia, by releasing glutamate in the external medium and contributing to the damaging effect of glutamate in ischemia.
Amino Acids↗
Research as a tool for the teaching of epidemiology.
At a medical school in India, undergraduates have been given the opportunity to volunteer to conduct research as a means of improving their knowledge and understanding of epidemiology. First-year clinical students have conducted case-control studies with emphasis on methodological detail. Second-year students have been involved in community-based epidemiological studies. At the intern level, projects related to social factors in health and disease and to health administration have been encouraged. This initiative has been largely welcomed by the students and has yielded highly encouraging results.
Education, Medical, Undergraduate↗
Frequent amplification of a short chain dehydrogenase gene as part of circular and linear amplicons in methotrexate resistant Leishmania.
The H locus of Leishmania codes for a short chain dehydrogenase gene (ltdh) that is involved in antifolate resistance. Leishmania tarentolae cells, selected in a step by step fashion for resistance to the antifolate methotrexate (MTX), frequently amplified ltdh in response to drug selection. Both circular and linear extrachromosomal amplicons were generated de novo from the chromosomal H locus and several contained inverted duplications. At least four different rearrangement points were used during the formation of amplicons, with one of them used preferentially. All mutants highly resistant to MTX, whether or not they have the H locus amplified, showed a decreased steady-state accumulation of MTX. Nevertheless, two types of transport mutants were clearly discernable. In the first type, accumulation was reduced four to five-fold, whereas in the second class of mutants, accumulation was reduced more than 50-fold. The ltdh gene was amplified in all the mutants with the transport mutation of the first type, but not in all the mutants with a more pronounced decrease in the steady-state accumulation of MTX. Both types of transport mutation, leading to the reduction in MTX accumulation, arose early during the selection process and were stable even when cells were grown in absence of the drug for prolonged period.
Animals↗