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Biomedical subjects

G Roy

Publications and source records attributed to G Roy.

At least 19 recordsLinked to original sources
PubMed1999

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.

Base Sequence↗
PubMed1998

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.

Alleles↗
PubMed1998

Flow cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease.

Intraepithelial lymphocytes (IEL) represent a heterogeneous cellular compartment of unknown functions and controversial ontogeny. Previous observations in humans indicate that the majority of IEL subsets express the CD3 complex associated with either the alphabeta or the gammadelta T-cell receptor components, and describe the characteristic increase of CD3+TCRgammadelta+ IEL in coeliac disease. In the present work, we analyze the surface antigen expression of intraepithelial lymphocytes isolated from duodenal biopsies of control subjects and coeliac disease patients. We describe a CD3-CD7 + IEL subset frequently found in control subjects (41.41+/-21.8), with the following features: 1) most of these cells are CD45R0+ CD103+ and CD44- CD28- CD5-; 2) a significant percentage express CD56 (44.7%+/-21.3), CD2 (55.1%+/-16.2), and CD94 (16.2%+/-7.3). Furthermore, they are CD122+ and CD25-; 3) this CD3- IEL subset exhibit an activated phenotype expressing higher levels of CD69, CD103, and CD38 than the CD3+ subset. Interestingly, this CD3- subset is drastically reduced in CD patients (2.2+/-2.9 in active disease, 6.3+/-4.6 in treated patients versus 41.4+/-21.8 in control subjects). The imbalanced ratio "increased TCRgammadelta versus decreased CD3- CD7+" is a permanent finding in CD patients following clinical and histological remission. This parameter might provide helpful diagnostic information (easily obtained by 3-color FCM from diagnostic biopsies), and suggest a potential implication in the pathogenesis of coeliac disease.

Antigens, CD7↗
PubMed1998

Halide and alkyl phenols block volume-sensitive chloride channels in human glial cells (U-138MG).

Swelling of cells in hypotonic media activates a volume-sensitive Cl channel with well-known characteristics, but its structure and its regulation are still largely undetermined. It also has many inhibitors and most of them are also blocking other types of Cl channels. The numerous inhibitors of Cl channels have apparently no structural relationship among them. The purpose of this study was to try to determine the most simple molecules that could block these channels and identify some common properties among inhibitors. From the 37 new molecules that were studied, it was found that simple halide phenols like trichloro and triiodophenols could block these channels in the micromolar range. Also alkyl phenols, like butylphenols, are very sensitive blockers, comparable to other well-known blockers. But acidic halide phenols or nitrophenols are poor blockers. Also neutral polyphenols are more sensitive than acidic polyphenols. All these results indicate that the common basis for blocking these Cl channels is a phenol with hydrophobic groups, like short alkyl chains or an additional phenyl ring, attached to some of its sites, preferably sites 3-4-5. These results identify a new family of Cl channel blockers and hopefully improve our understanding of the blocking mechanism.

Chloride Channels↗
PubMed1998

Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation.

The effect of gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters (methyl, propyl, octyl, and lauryl) has been studied on several tumoral and nontumoral cells. Three types of behavior have been observed; the first type is represented by the mouse B cell lymphoma Wehi 231 cell line in which death occurs according to the biochemical characteristics of classical apoptosis showing the DNA ladder fragmentation pattern. The second type is represented by the mouse fibroblast L929 cell line in which morphological characteristics such as cell shrinkage, chromatin condensation, and appearance of apoptotic bodies can be evidenced by microscopical observation. However, the typical DNA fragmentation is absent. Peripheral blood lymphocytes are representative of a third type of behavior. In a resting state they can withstand higher concentrations of these compounds. If the drug is washed, they proliferate normally upon the addition of the mitogen phytohemagglutinin (PHA). However, if the drug is added in the presence of PHA, a clear antiproliferative effect can be demonstrated. A special interest for these compounds stems from the fact that some of them are currently used as antioxidant food additives with the European Community codes E-310 (propylgallate), E-311 (octylgallate), and E-312 (laurylgallate).

Animals↗
PubMed1998

Specific interaction of wild-type and truncated mouse N-methylpurine-DNA glycosylase with ethenoadenine-containing DNA.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, is responsible for the removal of a wide variety of alkylated base lesions in DNA, e.g., N-alkylpurines and cyclic ethenoadducts of adenine, guanine, and cytosine. These lesions, some of which are mutagenic and toxic, are generated endogenously or by genotoxic agents such as N-alkylnitrosamines and vinyl chloride. Wild-type mouse MPG, expressed from recombinant baculovirus, was purified to near homogeneity for studying its specific interaction with substrate, 1,N6-ethenoadenine- (epsilonA-) containing DNA. Electrophoretic mobility shift assays (EMSA) indicated that MPG formed a specific complex with a 50-mer epsilonA-containing duplex oligonucleotide. This complex was shown to be a transient reaction intermediate, because it could be formed only with the unreacted substrate and contained active enzyme molecules. DNA footprinting studies confirmed the specific binding of the protein to the epsilonA-containing duplex oligonucleotide; eight nucleotides on the epsilonA-containing strand and 16-17 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion. A systematic deletion analysis of MPG was carried out in order to determine the minimally sized polypeptide capable of forming a stable substrate complex that is also suitable for characterization by NMR spectroscopy and X-ray crystallography. A truncated polypeptide (NDelta100CDelta18) lacking 100 and 18 amino acid residues from the amino and carboxyl termini, respectively, was found to be the minimal size that retained activity. The truncated and wild-type enzymes have similar kinetic properties. Moreover, both EMSA and DNase I footprinting studies indicated identical pattern of specific binding by the truncated and full-length polypeptides. Removal of five and nine additional residues from the amino- and carboxyl-termini of this polypeptide, respectively, resulted in a complete loss of activity. These results suggest that minimal structural change occured as a result of truncation in the NDelta100CDelta18 mutant, which may thus be suitable for elucidating the structure and mechanism of MPG.

Adenine↗
PubMed1998

Helicobacter pylori infection and insulin-dependent diabetes mellitus.

Helicobacter pylori is associated with different diseases: duodenal ulcer, rosacea, ischaemic heart disease and gastric cancer. Given the abnormal immunological response and the high prevalence of gastrointestinal symptoms in diabetic patients, we conducted a study on H. pylori prevalence among these patients. We designed a case control study of a population-based cohort. Eighty insulin-dependent diabetes mellitus (IDDM) patients with an average age (24.05 +/- 8.3 years), and 100 control subjects (25 +/- 7.1 years) were selected to verify the seroprevalence of Helicobacter pylori in these populations. One serum sample was obtained from each subject for evaluation of antibodies against Helicobacter pylori, parietal cells (APA) and pancreatic islets cells (ICA). The seroprevalence of H. pylori among IDDM patients aged less than 24 years was significantly higher than among control subjects; the corresponding rate among IDDM aged greater than 24 years was significantly lower than among control subjects. Antibodies against parietal cells (APA) and islet cells (ICA) among H. pylori positive diabetic patients were significantly higher than among H. pylori negative diabetic patients. IDDM patients were subdivided on the basis of the evolutive course of diabetes. Seroprevalence of H. pylori as well as prevalence of ICAs decreased with IDDM duration. Nevertheless, no variation in the prevalence of APAs during the course of diabetes was observed. We observed an association between the seroprevalence of Helicobacter pylori and the duration of IDDM. The seroprevalence of H. pylori and ICA decreased with the evolutive course of diabetes mellitus among IDDM. The prevalence of ICA and APA in IDDM H. pylori positive subjects was higher than among controls.

Adolescent↗
PubMed1998

Induction of thyroid tumours in (C57BL/6N x C3H/N)F1 mice by oral administration of kojic acid.

The tumorigenicity of kojic acid (KA), which is widely used for food and cosmetics in Japan, was examined in B6C3F1 mice. Female and male animals were divided into three groups and given 0, 1.5 and 3.0% KA containing food from the age of 6 weeks. At sacrifice after 20 months, thyroid weights were significantly increased in both sexes of mice receiving KA, especially in the male groups. The enlarged thyroid glands histologically featured diffuse hyperplasia and follicular adenomas, the incidences of the latter being 65% and 87%, respectively in 1.5% and 3.0% KA-treated males, significantly higher than the control value of 2%. In the females, the figures were 2%, 8% and 80% in the 0%, 1.5% and 3.0% KA groups, respectively. The serum free T3 levels in the 3.0% KA animals of both sexes at month 6 were significantly lower than in the controls. On the other hand, their serum TSH levels were higher, although the differences disappeared at later time points. In conclusion, continuous administration of high dose of KA induces thyroid adenomas in male and female B6C3F, mice, presumably by a mechanism involving decrease in serum free T3 levels and increased TSH.

Adenoma↗
PubMed1997

Characterization of a chloride-selective channel from rough endoplasmic reticulum membranes of rat hepatocytes: evidence for a block by phosphate.

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 +/- 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl- than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at -20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br- > Cl- > I- approximately F-. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br- and Cl- have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 microM DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl-. Our results suggest that channel block involves the interaction of HPO2-4 with a site located at 70% of the membrane span.

Animals↗
PubMed1997

Quantitative reverse transcriptase polymerase chain reaction for measuring the N-methylpurine-DNA glycosylase mRNA level in rodent cells.

A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of the N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal of N-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative amounts of PCR products of the experimental mRNA and a known amount of an exogenous reference RNA which is nearly identical to the experimental RNA. However, unlike in the earlier procedures in which deletion or insertion sequences were added to the reference RNA template, which may affect the efficiency of PCR but are needed to generate different size PCR products, experimental and reference RNAs yield PCR products of the same size in the new method. However, prior digestion with EcoRI allows separation of the two products because a unique EcoRI site was created in the reference RNA vector by point mutations. The QRT-PCR procedure is particularly useful for studying expression of the MPG gene whose mRNA level is very low and difficult to quantitate by Northern blot analysis. The number of MPG mRNA molecules/cell in late log-phase cultures varied from about 6 to 30 in several rodent lines. The SSV-NRK rat cell line has 6 +/- 0. 2 molecules/cell, while mouse NIH3T3 cells have about 30 +/- 1 molecules/cell. If the mRNA level is indicative of the level of the active MPG enzyme, these results may imply a variation in the capacity of various lines to remove the cytotoxic and mutagenic adducts from DNA.

3T3 Cells↗
PubMed1997

Efflux systems and increased trypanothione levels in arsenite-resistant Leishmania.

The mechanism of resistance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mechanism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania.

ATP-Binding Cassette Transporters↗
PubMed1997

Evaluation of donor skin disinfection methods.

BACKGROUND: Because most bacteria isolated from contaminated platelet concentrates are thought to originate from the donor's skin, the efficacy of four methods of skin disinfection was compared. STUDY DESIGN AND METHODS: Contact plates were used for antecubital skin cultures after they were demonstrated to be easier to use and at least as sensitive as a swab system. One antecubital fossa of each subject was disinfected by a standard method, the use of a povidone-iodine swabstick containing 0.75-percent available iodine followed by the use of a povidone-iodine swabstick containing 1-percent available iodine. The other arm was disinfected with either a 70-percent isopropyl alcohol scrub followed by an ampoule of 2-percent iodine tincture (Group 1; n = 126); a green-soap sponge followed by a 70-percent isopropyl alcohol swab, used for donors who are allergic to iodine (Group 2; n = 30); or a 0.5-percent chlorhexidine gluconate and 70-percent isopropyl alcohol sponge followed by an ampoule of 0.5-percent chlorhexidine gluconate and 70-percent isopropyl alcohol (Group 3; n = 40). Contact plate cultures were done before and after disinfection, and colonies counted after a 48-hour 37 degrees C incubation period. RESULTS: Similar numbers of bacteria grew from both antecubital fossae of the same subject before disinfection (p = 0.71). Compared to the standard povidoneiodine method, isopropyl alcohol and tincture of iodine resulted in significantly less bacterial growth (p < 0.001), the green soap and isopropyl alcohol method resulted in significantly more bacterial growth (p < 0.001), and the chlorhexidine gluconate and isopropyl alcohol method resulted in similar amounts of bacterial growth (p > 0.3). CONCLUSION: Isopropyl alcohol scrub followed by iodine tincture is more efficacious than povidone-iodine as measured by contact plate cultures. For donors who are allergic to iodine, chlorhexidine gluconate and isopropyl alcohol is more efficacious than green soap and isopropyl alcohol.

1-Propanol↗
PubMed1997

Characterization of stable RNAs from the resected intestinal tissues of individuals with either Crohn's disease or ulcerative colitis.

Circular RNAs reminiscent of viroids and the human hepatitis delta virus have been proposed as possible nonconventional pathogens responsible for Crohn's disease and ulcerative colitis, two inflammatory bowel diseases. Consequently, RNA was extracted from various areas of intestinal tissues from individuals with either Crohn's disease or ulcerative colitis as well as several appropriate control diseases, and analyzed by two-dimensional gel electrophoresis. No circular viroid-like RNAs (< 1500 nucleotides) were detected, confirming a previous report that was limited to the investigation of small RNAs (< 300 nucleotides). However, three small, unusually stable, linear RNAs were shown to be associated to both Crohn's disease and ulcerative colitis tissues: a specific 28S ribosomal RNA cleavage product characterized previously; a 5.8S ribosomal RNA conformer; and a fragment homologous to transcripts from DNA CpG islands. The two last RNAs were detected prior to visible morphological tissue alterations, suggesting that they are produced early during the inflammation and that they have value as molecular diagnostic tools for the inflammatory bowel diseases. The potential cellular mechanisms producing these RNAs and their involvement in inflammatory bowel disease are discussed.

Colitis, Ulcerative↗
PubMed1997

Effect of 17 beta-estradiol, retinoic acid and tamoxifen upon primary and transplanted thyroid tumor in B6C3F1 mice fed an iodine deficient diet.

This study was aimed to establish TSH dependent, transplantable thyroid tumor (TT) in B6C3F1 (BCF1) mice. In addition, transplanted TT was examined for its growth in mice given 17 beta-estradiol (E2), retinoic acid (RA), tamoxifen (TAM), T3 and T4. Both sexes of BCF1 mice were observed for 12 months under IDD and distilled water (DW), starting at 4 weeks of age. Groups of mice received an i.p. injection of radioactive iodine (131I) once at a dose of 60 mu Ci/head and/or given 0.25 mg E2 pellet s.c. One piece of induced pituitary or thyroid tumor was individually dissected aseptically and s.c. grafted under the fat pad of one site of the neck in the same strain of mice at 5 weeks of age. All mice were sacrificed between 7.5 to 13.5 months after grafting the tumors depending on the experiments. The transplantability of both pituitary and thyroid tumor was 100% in IDD mice, but TT was about 50% with a combined treatment of IDD plus E2. A supplement of thyroid hormones of T3 or T4 in mice with IDD completely inhibited the growth of in situ or grafted thyroid tumors. The growth of in situ thyroid gland was significantly promoted by the oral administration of RA in both sexes, whereas the growth of transplanted TT was significantly increased by RA in the female, but not in the male. Oral administration of TAM proved inhibitory upon in in situ and transplanted TT in the male, but not in the female. Thyroid tumor induced by IDD could grow only in mice with IDD and was partially regulated of its growth by RA and TAM.

Animals↗
PubMed1996

Gene disruption of the P-glycoprotein related gene pgpa of Leishmania tarentolae.

Transfection of pgpA into Leishmania confers resistance to arsenite and antimonials. By gene targeting mediated by homologous recombination the two alleles of the pgpA gene of a L. tarentolae wild-type cell were disrupted sequentially with the neomycin and hygromycin phosphotransferase genes. This pgpA null mutant showed an increased sensitivity to arsenite and antimonite. In addition, the L. tarentolae pgpA null mutant exhibited a decreased intracellular survival inside murine macrophages. The observed phenotypes were reverted to levels not statistically different than wild-type when an intact pgpA gene was introduced into the null mutant. Disruption of the pgpA chromosomal locus in an arsenite resistant mutant indicated that PgpA is not essential for resistance to oxyanions, although it might be required in the early steps of selection when resistance is being established.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling.

The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation.

Animals↗