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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 181 records · Page 10Linked to original sources
PubMed1996

Syntheses and some applications of chemically defined multivalent glycoconjugates.

Classical multivalent neoglycoproteins have been widely used to study a great number of carbohydrate-protein interactions. The synthesis of other neoglycoconjugates with various shapes, valencies, and conformations has reached considerable levels of sophistication and holds promise as a new tool for glycobiology and biomedical applications. Within the last few years, advances have been made towards both the syntheses and understanding of the antigenic properties of water-soluble glycopolymers. Some of these glycopolymers are finding applications as inhibitors of microbial adhesins and as carriers for drug delivery to specific cells. Novel dendritic carbohydrate structures are emerging as potent ligands for carbohydrate-binding proteins.

Glycoconjugates↗
PubMed1996

Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers.

The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described. The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures. An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures. The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined. Analogous mannosylated copolyacrylamides were also prepared for comparison. The IC50 values were also plotted as a function of dendrimer valencies. The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively. Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins. These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.

Carbohydrate Conformation↗
PubMed1996

Aflatoxin B1 contamination in groundnut samples collected from different geographical regions of India: a multicentre study.

Under a multicentre study conducted by the Indian Council of Medical Research, 2062 samples of groundnut were collected from rural and urban areas of 11 states representing different geographical regions of the country. These samples were analysed for aflatoxin B1 using the AOAC method. Analytical quality assurance between various participating laboratories was ensured through analysis of check-samples. Twenty-one per cent of groundnut samples collected from 11 states exceeded the permissible Indian regulatory limit of 30 micrograms/kg. No statistically significant difference in percentage of samples contaminated (> 30 micrograms/kg) were observed between pooled rural (22.9%) and urban (19.9%) data. Amongst the 11 states, the minimum and maximum per cent contamination respectively (> 30 micrograms/kg) was observed to be 15.2 in the state of Andhra Pradesh and 28.3 in the state of Haryana. The maximum level of contamination of 833 micrograms/kg was observed in the state of Gujarat. The median level of < 5 micrograms/kg was observed in the states of Gujarat, Haryana, Punjab, Andhra Pradesh and Karnataka. In all other states studied, the median level was found to vary between 10 and 20 micrograms/kg. The 90th percentile values were high in Andhra Pradesh (125 micrograms/kg), Gujarat (111 micrograms/kg) and Haryana (110 micrograms/kg). In the remaining states the 90th percentile value ranged from 60 to 93 micrograms/kg. Analysis of pooled data showed the percentages of samples exceeding the level of contamination of 5 micrograms/kg and 15 micrograms/kg, respectively were 44.9% and 37.4% which therefore, showed a marked increase when compared with the per cent of samples exceeding 30 micrograms/kg in the overall data.

Aflatoxin B1↗
PubMed1996

Distinct substrate preference of human and mouse N-methylpurine-DNA glycosylases.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein, removes several N-alkylpurine adducts, hypoxanthine, cyclic ethenoadducts of adenine, guanine and cytosine and 8-oxoguanine from DNA. The recombinant human and mouse MPGs, purified from Escherichia coli, show a significant difference in substrate preference. While both proteins prefer 3-methyladenine over other N-alkylpurines in DNA, the mouse MPG removes 7-methylguanine and 3-methylguanine at an approximately 2- to 3-fold higher rate than the human protein when adjusted for equal activity for the release of 3-methyladenine from DNA. Hybrid recombinant proteins containing N-terminal and C-terminal halves of the human and mouse glycosylases were partially purified from MPG-negative E.coli. Their substrate preferences suggest that the N-terminal half is more critical for the recognition of 3-methylguanine and 7-methylguanine.

Animals↗
PubMed1996

Myoblast transplantation in monkeys: control of immune response by FK506.

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing beta-gal were observed 1 week after the transplantation, but no cells expressing beta-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-beta) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (beta-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.

Animals↗
PubMed1996

Producing STR locus patterns from bloodstains and other forensic samples using an infrared fluorescent automated DNA sequencer.

Short tandem repeat (STR) analysis is increasingly being used in forensic case analysis because of the large number of STR loci in the human genome and their highly polymorphic nature. An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from simulated forensic samples. The amplification strategy used a 19 base pair extension on the 5' end of one of the PCR primers. This sequence is identical to the sequence of a universal M13 Forward sequencing primer which is included in the amplification reaction. Allelic bands were detected by incorporation of the M13 primer-fluorescent dye conjugate into PCR products thus eliminating the need for direct conjugation of fluorescent dye to individual STR primers. By using an IR-based automated DNA sequencer and Tth DNA polymerase, polymorphic STR alleles were detected on-line rapidly and efficiently from bloodstains using only a high temperature incubation to extract DNA from blood cells. Five STR loci were also amplified using Chelex extracted DNA from simulated forensic samples. Multiplexing of three primer pairs in a single PCR mixture for amplification was accomplished using Taq polymerase. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with silver staining and fluor detection systems. Real-time detection permits immediate visualization of the data and STR alleles are displayed as familiar autoradiogramlike images that can be analyzed by computer. By loading a 64 lane gel twice and multiplexing with three primer pairs, forensic scientists can type at least three loci from 120 samples in one day.

Alleles↗
PubMed1996

Dehydroepiandrosterone-fatty acid esters in human plasma: formation, transport and delivery to steroid target tissues.

In addition to dehydroepiandrosterone (DHEA) sulfate (S), the human plasma also contains a second form of DHEA ester: DHEA-fatty acid esters (DHEA-FA). In the human adult, the plasma concentrations of DHEA-FA, DHEA and DHEAS are in the range of 6, 12 and 2000 nM respectively. Although the adrenal is responsible for almost all production of DHEAS in the circulation, DHEA-FA is formed from DHEA by an enzyme present in the circulation. Our work has clearly demonstrated that lecithin-cholesterol acyltransferase, localized on high density lipoprotein, is responsible for DHEA-FA production. Once DHEA-FA is formed, it is subsequently transferred to very low density lipoprotein (VLDL) and low density lipoprotein (LDL), like cholesteryl esters. Plasma lipoproteins contain at least 90% of circulating DHEA-FA of which 40% are found in the LDL fraction. Analysis of the fatty acid composition of tritiated DHEA-FA-labelled LDL ([3H]DHEA-FA-LDL) indicated the prevalence of DHEA-linoleate/palmitoleate and DHEA-oleate. Treatment of [3H]steroid-FA-LDL with charcoal does not remove radioactivity, thus suggesting that the non-polar steroid is incorporated into the central non-polar core of the lipoproteins. Incubation of [3H]DHEA-FA-LDL with ZR-75-1 breast cancer cells produced a time-dependent increase in labeled non-conjugated steroids in the cell culture medium, whereas the levels of tritiated DHEA-FA decreased. Lipoidal radioactivity in cells increased with time, but non-conjugated radioactivity associated with the cells showed no such increase. HPLC analysis of the culture medium indicated the presence of tritiated DHEA and androst-5-one-3 beta, 17 beta-diol. Our study indicates that circulating DHEA-FA incorporated into lipoproteins may indeed act as a substrate for potent steroid formation following their entry into steroid target cells.

Adrenal Glands↗
PubMed1996

Selective spectrophotometric determination of glucose and fructose in the presence of aldoses using phenol-acetone reagent and cerium(III) chloride.

Aqueous mixtures of glucose and fructose produce red solutions when treated with 2% (w/v) phenol in 5% (v/v) aqueous acetone in the presence of concentrated sulfuric acid. The color is stable for days, and the red chromophore has an absorbance maximum at 568 nm. When the concentration of phenol is raised to 25%, fructose, but not glucose, produces red solutions, allowing for the selective detection of ketoses. Two complementary methods have been developed to remove the interference of ketoses in solutions containing glucose. The first one relies on the selective reduction of ketoses with sodium borohydride in the presence of cerium(III) chloride prior to the addition of the phenol-acetone reagents. The second method is based on the differential specific determination of glucose using 2% versus 25% levels of phenol. The relative sensitivities of different sugars are also presented as well as the applicability of the methods using bacterial polysaccharides for immunochemical analyses. The quantitative determination of glucose or ketoses in the polysaccharides does not require hydrolysis prior to the estimation.

Acetone↗
PubMed1995

Increased granzyme B mRNA after alloincompatible myoblast transplantation.

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.

Animals↗
PubMed1995

The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase.

Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Genetic linkage for Darier disease (keratosis follicularis).

Darier disease is an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion. Recent data have provided evidence for linkage of the Darier disease locus to 12q23-24.1 in British families. We have carried out linkage analysis using the 12q markers D12S58, D12S84, D12S79, D12S86, PLA2, and D12S63 in 6 Canadian families. Pairwise linkage analysis generated positive lod scores at all 6 markers at various recombination fractions, and each family showed positive lod scores with more than one marker. The peak lod score in the multipoint analysis (Zmax) was 5.5 in the interval between markers D12S58 and D12S84. These positive lod scores in North American families of varied European ancestry confirm the location of the Darier disease gene, and suggest genetic homogeneity. The future identification and sequencing of the gene responsible for Darier disease should lead to improved understanding of the disease and of keratinocyte adhesion in general.

Adolescent↗
PubMed1995

Lymphocyte infiltration following allo- and xenomyoblast transplantation in mdx mice.

Human and mouse (C57BL/10SnJ+/+) myoblasts were injected separately in the muscles of C57BL/10ScSn mdx/mdx mice. Mouse myoblasts (C57BL/10SnJ+/+) were also injected in normal mice (C57BL/10SnJ+/+ and BALB/c+/+). Some muscles that received a xenotransplantation (i.e., human myoblasts) were previously injected with a myotoxin, i.e., notexin. This treatment was not used for the allografts (i.e., mouse myoblasts). Human myoblast injections did not increase the number of dystrophin-positive cells above the background level due to backmutation. Moreover, the human myoblasts detected with an anti-HLA antibody decreased rapidly during the 6-week follow-up. The injection of normal mouse myoblasts in mdx mice did, however, increase the number of dystrophin-positive fibers. Moreover, numerous cells expressing mouse MHC class II, macrophages, granulocytes, neutrophils, natural killer cells, and a subset of T lymphocytes were detected by immunohistochemistry in cryostat sections of myoblast-injected muscles. These cells were present within 1 week of the myoblast injection in the muscle regions containing injected human or mouse myoblasts, and progressively decreased during the 6-week follow-up in the human myoblast transplantation. Lymphocyte infiltration reached a significant level following xeno- and alloincompatible transplantations. Antibodies against the human myoblasts and against alloincompatible myoblasts were also detected in the serum of the recipients. These results suggest that humoral and cellular immune reactions are responsible for the poor outcome of myoblast transplantation in mice and could be involved in failure of transplantation in Duchenne muscular dystrophy patients. These results indicate that adequate immunosuppression must be used in these patients.

Animals↗