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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 145 records · Page 8Linked to original sources
PubMed1998

Sequence analysis of mitochondrial DNA hypervariable regions using infrared fluorescence detection.

The non-coding region of the mitochondrial genome provides an attractive target for human forensic identification studies. Two hypervariable (HV) regions, each approximately 250-350 bp in length, contain the majority of mitochondrial DNA (mtDNA) sequence variability among different individuals. Various approaches to determine mtDNA sequence were evaluated utilizing highly sensitive infrared (IR) fluorescence detection. HV regions were amplified either together or separately and cycle-sequenced using a Thermo Sequenase protocol. An M13 universal primer sequence tail covalently attached to the 5' terminus of an amplification primer facilitated electrophoretic analysis and direct sequencing of the amplification products using IR detection.

DNA Primers↗
PubMed1997

Prevention by anti-LFA-1 of acute myoblast death following transplantation.

Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. One of the problems possibly responsible for the limited success of clinical trials is the rapid death of the myoblasts after transplantation. To investigate this problem, myoblasts expressing beta-galactosidase were injected in the tibialis anterior muscles of mice. Beta-galactosidase activity was reduced by 74.7% after 3 days. Myoblast death observed at 3 days was reduced to 57.2% when the hosts were irradiated. This result suggested that host cells were contributing to this phenomenon. Transplantation in SCID and FK506-treated mice did not reduce cell death, indicating that mortality was not due to an acute specific reaction. In contrast, administration of the anti-LFA-1 (TIB-213) mAb markedly reduced myoblast death at 3 days without altering leukocyte tissue infiltration. We postulated that neutrophils were mediating myoblast mortality by an LFA-1-dependent mechanism. To test this hypothesis, IL-1beta-activated myoblasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethylester) (DCFH), a marker for oxidative stress. Addition of neutrophils and zymosan-activated serum resulted in a time-dependent DCFH fluorescence; this neutrophil-induced oxidation was considerably inhibited by TIB-213. These results indicate that an effective control of the inflammatory reaction will be necessary for any new clinical trials of myoblast transplantation and suggest that neutrophil-mediated myoblast injury occurs by an LFA-1-dependent pathway.

Animals↗
PubMed1997

Infrared fluorescent detection of D1S80 alleles.

A genetic locus D1S80 containing a variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, the D1S80 locus was amplified using polymerase chain reaction (PCR) technology and the alleles detected using a high sensitivity infrared (IR) fluorescence automated DNA sequencer. IR-labeled amplification products were generated using oligonucleotide primers which were covalently linked to an infrared fluorescent dye (IRD41) at the 5'-end. Human genomic DNA (1.0 ng or less) isolated from blood and various simulated forensic samples was successfully amplified using this technology. Allelic bands were detected by incorporation of the IR fluorescent dye into PCR products. Both Long Ranger and polyacrylamide denaturing gels permitted clear resolution of individual alleles that differ by only one repeat unit. In the smaller gels a separation distance of only 15 cm allowed separation of the alleles in less than 2 h from sample loading to visualization. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for post-electrophoretic gel handling for the detection of D1S80 alleles. Real-time detection is valuable for immediate visualization of the data and the alleles are displayed as familiar autoradiogram-like images which can also be analyzed by computer. By loading a 64-lane gel twice it is possible to type at least 120 samples in 1 day using a single gel.

Alleles↗
PubMed1997

Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6-alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spetroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.

Binding Sites↗
PubMed1997

Role of non-major histocompatibility complex antigens in the rejection of transplanted myoblasts.

Myoblasts obtained from donors histoincompatible for several non-major histocompatibility complex antigens (i.e., including minor histocompatibility antigens) and from syngeneic donors were transplanted without any immunosuppression into the muscles of male dystrophic C57BL/10J mdx/mdx mice. Myoblasts from syngeneic mice resulted in the formation of a high percentage of dystrophin-positive fibers 16 weeks after the transplantation. There was no evidence of a cellular immune reaction against the donor myoblasts, i.e., no infiltration by CD4 or CD8 lymphocytes and no increased expression of granzyme B and interferon-gamma mRNAs. Transplantation of myoblasts obtained from donors histoincompatible only for non- major histocompatibility complex antigens produced a transient increase of dystrophin-positive fibers at 4 weeks after transplantation for some donor strains but not for others. For donor strains that did produce an increase at 4 weeks, the number of dystrophin-positive fibers was reduced 16 weeks after the transplantation. There was evidence of a cellular immune reaction-infiltration by CD4 and by CD8 lymphocytes and increased expression of granzyme B and interferon-gamma mRNAs. Transplantation of myoblasts obtained from male C57BL/10J +/+ mice into female C57BL/10J mdx/mdx mice also led to the presence of only a few dystrophin-positive fibers with the same signs of cellular immune reaction. In this later case, the cellular immune response was attributed to the H-Y minor antigens. Finally, antibodies against fetal calf serum were detected after both syngeneic and nonsyngeneic transplantations, indicating that the culture medium may also be a source of antigens. In mice, the presence of these antibodies against culture medium did not reduce the success of a first syngeneic transplantation.

Actins↗
PubMed1997

Multiplex amplification of STR loci with gender alleles using infrared fluorescence detection.

The analysis of short tandem repeat (STR) polymorphisms has proven extremely useful for gene mapping, paternity testing, and forensic analysis. Several commercial products are currently available for performing amplification and analysis of STRs. We have adapted Promega Geneprint Systems for use with a high sensitivity infrared (IR) fluorescent automated DNA sequencer. IR-labeled amplification products are generated by including a small quantity of IR-labeled dATP in the reaction. Several Geneprint STR loci can be multiplexed together with the amelogenin sex identification locus in a single amplification reaction. We have successfully amplified up to five Geneprint STR loci together with the amelogenin locus thus improving the throughput of analysis. Purified genomic DNA as well as simulated forensic samples have been utilized for these multiplex amplifications.

Alleles↗
PubMed1997

Cloning and characterization of p52, the fifth subunit of the core of the transcription/DNA repair factor TFIIH.

TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.

Amino Acid Sequence↗
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

Regulation of membrane lipid bilayer structure during seasonal variation: a study on the brain membranes of Clarias batrachus.

(1) A significant seasonal variation in the membrane fluidity (as sensed by DPH-fluorescence polarization), membrane lipid components (phospholipid and neutral lipid), fatty acid composition of membrane phospholipid (phosphatidylcholine, phosphatidylethanolamine and sphingomyelin), positional distribution of fatty acids at Sn-1 and Sn-2 position of phosphatidyl-choline and -ethanolamine is noticed in the brain membranes (myelin, synaptosomes, and mitochondria) of a tropical air breathing teleost, Clarias batrachus. (2) A 'partial compensation' of membrane fluidity during seasonal adaptation is observed in myelin and mitochondria membrane fractions. Synaptosomes membrane fraction exhibits a different response. Depletion (about 15-70%) of membrane lipid components (phospholipid, cholesterol, diacylglycerol and triacylglycerol) per unit of membrane protein is the characteristic feature of summer adaptation. An increase (about 20-100%) in the level of oleic acid and decrease (about 20-60%) in the level of stearic acid are almost common features in membrane phospholipid fractions of winter-adapted Clarias (3) From the tissue slice experiment it is evident that there is an activation of cellular phospholipase A2 at lower growth temperature and of cellular phospholipase A1 at higher growth temperature and this suggests the reorganization of molecular architecture of the membrane during seasonal adaptation. (4) Accumulation of oleic acid in Sn-1 position and polyunsaturated fatty acids in Sn-2 position of phosphatidylcholine and -ethanolamine during winter indicates an increase in the concentration of 1-monoenoic, 2-polyenoic molecular species of phospholipid in order to maintain the stability of membrane lipid bilayer.

Acclimatization↗
PubMed1997

The Dialine II graft: a new collagen-impregnated warp-knitted polyester arterial prosthesis.

The Dialine graft, a new prototype of knitted vascular prosthesis that uses a different brand of polyester fibers as an alternative to Dacron fibers, has been shown to offer excellent in vitro physical performance and in vivo healing. Although it still requires preclotting, the Dialine prosthesis was made impervious by impregnation of bovine type I collagen cross-linked with vapors of formalin. The purpose of the present investigation was to compare the in vitro physical characteristics of the Dialine II graft with those of the collagen-impregnated Hemashield graft. In addition, we studied the healing performance as a thoracoabdominal bypass in dogs for prescheduled periods of implantation ranging from 4 hours to 6 months. In vitro, the bursting strength, resistance to dilatation, and suture retention strength properties of the Dialine II prosthesis were all shown to exceed those of the Hemashield control graft. In the first weeks after implantation, the Dialine II grafts induced a discrete inflammatory response, as shown by the constant leukocyte counts observed both before implantation and when the animals were killed, as well as by the histologic observation of a few inflammatory cells in contact with the collagen. Consequently, the Dialine II grafts showed a slow rate of bioresorption of cross-linked collagen. At 1 month, a thin internal collagenous capsule was present at both anastomoses, laying over the original collagen coating. At 3 and 6 months, areas of thrombotic deposits and endothelialized areas were observed on the luminal surface. Because results of early clinical trials have been highly satisfactory, this prosthesis may be recommended for use without restriction as a medium- and large-diameter blood conduit.

Animals↗
PubMed1997

Chemoenzymatic synthesis of dendritic sialyl Lewis(x).

Traditional structure activity relationship studies (SAR) have led to the development of numerous sialyl Lewis(x) analogs in the search for potential antiinflammatory agents. However, these methods do not take into account cluster or multivalent effects. Reported herein is the chemoenzymatic synthesis of di-, tetra-, and octa-valent sLe(x) ligands scaffolded on dendrimers. Hypervalent L-lysine cores with covalently attached 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) residues were chemically prepared and enzymatically transformed into sLe(x) containing dendrimers so that multivalency, and its role in selectin-sLe(x) interactions may be evaluated. This work constitutes another successful enzymatic synthesis of sLe(x) and represents the first example of GlcNAc elongation on a synthetic dendrimer scaffold. These sLe(x) dendrimers are currently being investigated as selectin antagonists.

Acetylglucosamine↗
PubMed1997

Role of ABO and Lewis blood group antigens in donor-recipient compatibility of corneal transplantation rejection.

PURPOSE: There are conflicting results regarding the role of human leukocyte antigen (HLA) matching and ABO compatibility in corneal graft rejection for low- and high-risk patients. Lewis blood group antigens could be an important histocompatibility system. Beneficial effects of Lewis antigens matching have been reported in renal transplantation, but its effect is still unknown in corneal allografting. METHODS: Between 1987 and 1993, ABO, Lewis and HLA phenotypes were determined in 697 consecutive grafts of corneal transplantations. The effect of Lewis matching on corneal endothelial rejection was evaluated over a 3-year period. Data analysis was done by plotting survival curves with the Kaplan-Meier method for survivorship data and performing statistical analysis with the log-rank test (Mantel-Haenszel test) for curve comparison. RESULTS: In vascularized recipients, the ABO, Lewis, and HLA systems did not influence the graft outcome. However, for the unvascularized recipients, the endothelial 3-year rejection rate was significantly lower for both Lewis compatible patients (84% vs. 68%; log rank = 0.03) and HLA compatible patients (86% vs 72%; log rank = 0.001), but not for the ABO-matched patients (82% vs. 79%; log rank = 0.56). CONCLUSIONS: The authors' study suggests that Lewis antigens and HLA matching could positively influence corneal graft survival for the unvascularized recipients, but it did not seem to have any effect in vascularized recipients.

ABO Blood-Group System↗
PubMed1997

Liver hemostasis using high-intensity focused ultrasound.

Liver hemorrhage, the major cause of death in hepatic trauma, is notoriously difficult to control. We report on the use of high-intensity focused ultrasound (HIFU) to arrest the bleeding from incisions made in rabbit livers. A HIFU transducer, with a spherically curved aperture of 6.34 cm2 area, a focal length of 4 cm and a frequency of 3.3 MHz was used. In approximately 94% of the incisions, the hemorrhage was reduced to a slow oozing of blood in less than 2 min. The maximum temperature of liver tissue around the incision area, during HIFU application, was measured to be 86 degrees C. The mechanism of hemostasis, confirmed by histological examination, appears to be coagulative necrosis of a volume of liver tissue around the incision. We believe that acoustic hemostasis, with the unique characteristic of "volume cauterization," offers a novel method for the management of liver hemorrhage and, thus, has major clinical implications.

Animals↗
PubMed1997

1H magnetic resonance imaging and 31P magnetic resonance spectroscopy in experimental filariasis.

1H Magnetic resonance imaging and 31P magnetic resonance spectroscopy (MRS) have been carried out in experimental rodent filariasis, i.e., Acanthocheilonema viteae infection in the rodent host, Mastomys coucha. The T2-weighted image of the infected host shows fine hyperintense thread like structures of adult filariid nests in the cervical region. 31P MRS of normal and infected hosts, localized over the same region of interest, show seven major peaks corresponding to phosphomonoesters (including glucose-6-phosphate, fructose-6-phosphate, fructose-1-6-diphosphate, phosphorylcholine, and adenine monophosphate or AMP), inorganic phosphate, glycerophosphorylcholine, phosphoenolpyruvate, phosphocreatine and nucleoside di- and tri-phosphates. Concentrations of phosphomonoesters (PMEs) are higher in the normal rodent compared with the infected ones. In vivo 31P MRS provides a non-invasive assessment of tissue bioenergetics and phospholipid metabolism.

Animals↗
PubMed1997

Optimizing lectin-carbohydrate interactions: improved binding of divalent alpha-mannosylated ligands towards Concanavalin A.

The synthesis and binding properties to Jack bean phytohaemagglutinin in (Concanavalin A, Con A) of a new family of divalent alpha-D-mannopyranoside ligands are described. The synthesis of these ligands is based on the coupling of commercially available diamines to p-isothiocyanatophenyl 2,3,4,6 tetra-O-acetyl-alpha-D-mannopyranoside (4). The resulting dimers 6, 15 to 22 and 30 were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as standard. Divalent mannosylated ligand 35 bearing a non-aromatic aglycon was also tested for comparison purposes. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohaemagglutinin (Con A) were determined. The inhibitions showed dimers to be approximately 10- to 90-fold more potent than methyl alpha-D-mannopyranoside. Variations in the intra-mannosyl distance proved to be an important factor for optimum binding.

Binding Sites↗