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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 127 records · Page 7Linked to original sources
PubMed1998

In vitro cellular response to polypyrrole-coated woven polyester fabrics: potential benefits of electrical conductivity.

Electrically conducting polypyrrole-treated films have recently been shown to influence the morphology and function of mammalian cells in vitro. This type of polymer represents a possible alternative biomaterial for use in vascular implantation. The present study compared the in vitro biocompatibility of the five different polyester woven fabrics having increasing levels of electrical conductivity ranging from 4.5 x 10(4) to 123 omega/square with that of low density polyethylene and polydimethylsiloxane primary reference materials. Biocompatibility was measured in terms of four different types of in vitro cellular response, including (a) an indirect and (b) a direct control organotypic culture assay using endothelial cells, (c) a polymorphonuclear (PMN) cell activation study using flow-cytometric measurements of CD11/CD18 integrin molecule expression, and (d) a semiquantification of interleukin (IL)-6 mRNA expression on monocytes/macrophages using reverse-transcriptase polymerase chain reaction. The organotypic culture study revealed that the fabrics with high levels of conductivity exhibited lower cell migration, proliferation, and viability. The PMN activation study of blood from 10 healthy adult donors demonstrated that the two most conductive fabrics were able to identify the more reactive donors. The levels of IL-6 mRNA expression by monocytes/macrophages decreased as the conductivity level of the fabrics increased. The results of the present study therefore indicate that high levels of conductivity (< 200 omega/square) on polyester fabrics are detrimental to the growth, migration, and viability of endothelial cells; induce elevated PMN activation; and affect the intracellular metabolism of monocytes. They also point to a specific range of conductivity (10(3) < 10(4) omega/square) which is associated with an optimum in vitro cellular response.

Adult↗
PubMed1998

Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. Direct identification of Lys-212 as the active nucleophilic residue.

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 x 10(4) and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the Km was 47 nM, and kcat was approximately 0.6/min, independent of whether DHU paired with G or A. The enzyme carries out beta-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5'-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.

Base Sequence↗
PubMed1998

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin.

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.

Antigens, Viral, Tumor↗
PubMed1998

Diversity of C-linked neoglycopeptides for the exploration of subsite-assisted carbohydrate binding interactions.

Diversity of alpha-galactose based C-linked neoglycopeptides (1b, 2b, 3c, 4d, and 5d) has been developed to explore the importance of subsite-assisted carbohydrate binding interactions. Deprotected C-linked neoglycopeptides (1b, 2b, 3c, 4d, and 5d) were synthesized and tested in competitive inhibition assays using a model enzyme-linked lectin (e.g., Maclura pomifera). Compound 2b, with two alpha-galactoside units on the side chain of the lysine residue of the dipeptide backbone, exhibited a remarkable effect with a 2.82-fold increase in its inhibitory properties (IC50 1.48 mM) in comparison to 1b (IC50 4.18 mM).

Binding Sites↗
PubMed1998

Topoisomerase I stimulates SV40 T antigen-mediated DNA replication and inhibits T antigen's ability to unwind DNA at nonorigin sites.

We have previously found that purified SV40 T antigen and topoisomerase I (topo I) bind to one another in vitro. In this report, we determined the effects of human topo I on T antigen-mediated DNA replication and investigated whether it altered T antigen's biochemical activities. Topo I stimulates DNA replication and especially increases the amounts of finished circular molecules. This protein had no effect on T antigen's ability to bind, distort, or unwind the origin of replication. However, unwinding of DNA by T antigen was strongly inhibited by topo I when it was initiated at sites other than the origin. We demonstrate that the presence of T antigen binding sites in DNA interfere with inhibition of unwinding by topo I. These results indicate that topo I may increase the specificity of unwinding by inhibiting the reaction at non-origin sites. Fragments of T antigen that bind to topo I abrogate topo I's inhibition of non-origin-dependent unwinding, indicating that topo I inhibits unwinding through a direct interaction with T antigen. We propose a model whereby T antigen and topo I function together at the origin to specifically unwind it and initiate DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1998

Communication with chaotic lasers

Recent experiments with chaotic electronic circuits have shown the possibility of communication with chaos. The experimental demonstration of chaotic communication with an optical system is described. An erbium-doped fiber ring laser (EDFRL) was used to produce chaotic light with a wavelength of 1.53 micrometers. A small 10-megahertz message was embedded in the larger chaotic carrier and transmitted to a receiver system where the message was recovered from the chaos. Chaotic optical waveforms can thus be used to communicate masked information at high bandwidths.

Journal Article↗
PubMed1998

Monitoring of cytomegalovirus infections by the CD8+CD38+ T-cell subset in kidney transplant recipients.

BACKGROUND: The CD8+CD38+ T-cell subset can predict progression to acquired immune deficiency syndrome among human immunodeficiency virus-positive subjects. This T-cell subset usually increases during other active viral infections (cytomegalovirus [CMV], Epstein Barr virus). We report on its usefulness in the early detection of CMV infection in kidney transplant recipients. METHODS: Quantitation of CD8+CD38+ T cells was monitored by dual-color flow cytometry analysis on 77 patients during the posttransplantation period. Seventeen of the 52 patients at risk for CMV disease (33%) had primary infection or reactivation and three patients had herpes simplex virus infection only. RESULTS: In every patient with CMV disease, high values for the CD8+CD38+ subset were detected with a 90% positive predictive value for the primary infections. Elevated values were observed at the very first clinical signs of the viral disease or within the few preceeding days. Acute rejection episodes did not provoke false-positive results. CONCLUSION: This immunologic marker is sensitive and easily obtainable on a daily basis. It may help to direct therapy during rejection or serve as a tool for early detection of clinical viral diseases.

ADP-ribosyl Cyclase↗
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

Conventional and hypobaric activation of an ultrasound contrast agent.

Hypobaric activation is a new injection technique for use with the contrast agent EchoGen and, in this study, the agent's ability to produce parenchymal enhancement in vivo, with and without prior hypobaric activation, was investigated. Injections, ranging in dose from 0.05 to 0.5 mL/kg, were administrated through a peripheral vein to eight woodchucks with multiple hepatomas. At the 0.10 mL/kg dose level, seven of eight injections following hypobaric activation (88%) resulted in definite parenchymal enhancement. Conversely, dosages of 0.10 mL/kg without prior hypobaric activation produced no grey-scale changes. Only at the 0.4 and 0.5 mL/kg dosage level did the conventional administration technique obtain similar results (4 of 5 injections increased the echogenicity for a 0.4 mL/kg dose). These differences were statistically significant (p = 0.031). In vitro experiments were conducted to establish the physical mechanisms behind hypobaric activation. Relative measurements of contrast microbubble sizes were performed with a phase Doppler particle analyzer after hypobaric and after conventional (bolus) activation. Hypobaric activation produced approximately 20 times more microbubbles per unit volume than the conventional method. In conclusion, this investigation has demonstrated the benefits of prior hypobaric activation when performing in vivo contrast studies with EchoGen and determined the physical mechanisms behind this new injection technique. Hypobaric activation of EchoGen increases contrast enhancement and reduces dose size.

Animals↗
PubMed1998

Myoblast transplantation in non-dystrophic dog.

Dog myoblasts obtained from muscle biopsies were infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-Gal) gene. These myoblasts were initially transplanted in the irradiated muscles of SCID mice and beta-Gal positive muscle fibers were observed. beta-Gal myoblasts were also transplanted back either in the donor dogs (autotransplantation model) or in unrelated recipient dogs (allotransplantation model). Following these myoblast injections, a rapid inflammatory reaction developed within the muscle as indicated by an expression of P-selectin and of pro-inflammatory cytokine mRNAs (interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta), and by a neutrophil infiltration. Following either auto- or allotransplantation in inadequately or non-immunosuppressed dogs, a specific immune reaction also developed within 2 weeks as indicated by the infiltration of CD4+ and of CD8+ lymphocytes, the increased expression of IL-10 and granzyme B mRNAs and the presence of antibodies reacting with the injected cells. Some dogs were immunosuppressed with several combinations of FK506, cyclosporine (CsA) and RS-61443. In dogs immunosuppressed with CsA combined with RS-61443, only a few myoblasts and myotubes expressing beta-Gal were observed 1-2 weeks after the transplantation, but no muscle fibers expressing beta-Gal were observed after 4 weeks, and antibodies against the injected cells were formed. In dogs immunosuppressed with FK506 alone, although no antibodies against the injected cells were produced, there were no small cells and no muscle fibers expressing beta-Gal 1 month after the transplantation. However, FK506 triggered diarrhea and vomiting in dogs. When the dogs were immunosuppressed with FK506 combined with CsA and RS-61443, muscle fibers expressing beta-Gal were present 4 weeks after the transplantation and no antibodies reacting with donor myoblasts were detected. These results indicate that the combination of three immunosuppressive agents (i.e., FK506, CsA and RS-61443) is effective in controlling the specific immune reactions following myoblast transplantation in dogs and they underline that the outcome of myoblast transplantation is dependent in part on an adequate immunosuppression. These results obtained here in normal dogs may justify myoblast transplantation in dystrophic dogs despite the side effects of FK506.

Animals↗
PubMed1998

Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins.

Clusters of di-, tri-, and tetra-antennary alpha-D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide [Man alpha(1 --> 6)[Man alpha(1 --> 3)]Man] haptens to poly-isocyanate or -isothiocyanate tethering cores. The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively. Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses. In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl alpha-D-mannopyranoside against pea lectin and Con A, respectively. Compounds 30 and 31 were thus 35- and 289-fold more potent than the reference monosaccharide based on their hapten contents. As a general observation, the ligands bearing the Man alpha(1 --> 6)[Man alpha(1 --> 3)]Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands.

Binding Sites↗
PubMed1998

High temperature conjugation of proteins with carbohydrates.

A new procedure was used to conjugate lactose and dextran with BSA without using coupling or activating reagents. The method is simple, rapid and cheap. Reducing sugars covalently bind to proteins when lyophilized together and briefly heated to a high temperature.

Animals↗
PubMed1998

Development and characterization of monoclonal antibody to T-antigen: (gal beta1-3GalNAc-alpha-O).

The saccharide antigen, Gal beta1-3GalNAc or T antigen, is of biologic importance in many systems. It is a tumor-associated carbohydrate antigen, a temporally expressed antigen in germinal center B cells and cortical T cells, a parasite-associated antigen, a spermatozoa vitality marker and an antigen on aged red blood cells. It may play a role in normal cellular adhesion and in tumor cell metastasis. Well characterized monoclonal antibodies (MAb) to Gal beta1-3GalNAc will be useful for further studies in these areas. We developed an IgG3 MAb to Gal beta1-3GalNAc by immunizing the mice with a synthetic Gal beta1-3GalNAc-BSA conjugate. The MAb was analyzed using inhibition enzyme immunoassays with related synthetically prepared sugars to determine the restrictions involved in the antibody binding. Use of synthetic sugars as competitors enabled us to delineate the epitope restrictions on the binding activity of this monoclonal and will enable use of this MAb in studies concerning the biologic importance of this disaccharide.

Adenocarcinoma↗
PubMed1998

Role of presensitization and donor-recipient crossmatching in corneal graft outcome.

PURPOSE: A positive donor-recipient crossmatch (CM) due to preexisting recipient lymphocytotoxic antibodies is known to be an important factor in allograft failure in the majority of organ transplantations. However, the effect of positive CM on corneal graft outcome is less known. METHOD: Between 1982 and 1994, CM was performed by the microlymphocytotoxicity method using donor lymphocytes and recipient pretransplant serum in 759 consecutive corneal transplantations (maximal follow-up, 36 months). Patients were evaluated regarding the type of allospecificity of antibodies involved and their role on corneal graft outcome (rejection and failure). RESULTS: A positive CM was found in 61 patients (8%) and a negative CM in 698 patients (92%). The positive and negative CM groups had similar graft rejection rates at 36 months. Patients with a positive CM due to antibodies directed against donor human leukocyte antigen (HLA) (as defined on the basis of private and public or CREG HLA allele specificities) did not have an increased risk of rejection. However, patients with positive CM and presensitization (previous graft or rejection history) had a statistically significant increase in risk of corneal endothelial rejection. CONCLUSION: This study shows that donor-recipient CM could be a useful procedure for the selection of recipients for corneal transplantation in patients presensitized by anterior graft or previous corneal rejection.

Adult↗
PubMed1998

Role of in vivo proton magnetic resonance spectroscopy in the diagnosis and management of brain abscesses.

OBJECTIVE: In vivo proton magnetic resonance spectroscopy was performed for 24 patients with pyogenic brain abscesses, to examine the consistency of the spectral patterns and to observe the changes in metabolites with treatment. METHODS: Localized proton spectra were obtained from 4- to 8-ml volumes in the abscesses, using stimulated echo acquisition mode and spin echo sequences. Twenty-two patients were treated with combined surgical and medical therapy, and two patients were treated conservatively. High-resolution magnetic resonance spectroscopy was performed for 15 samples of abscesses obtained from these patients, to confirm the assignments of resonances seen in vivo. Postaspiration studies were performed for 12 patients treated with combined medical and surgical therapy and 2 patients treated medically. RESULTS: Lactate and amino acids were seen in spectra for all patients, irrespective of the time of spectroscopy after the onset of combined medical and surgical therapy. Acetate and pyruvate disappeared after 1 week of combined treatment. CONCLUSION: It was concluded that spectral patterns for brain abscesses are consistent and specific and can assist in the noninvasive diagnosis of abscesses. Responses to combined treatment could be monitored by showing the changes in metabolite patterns in serial spectroscopic studies.

Adolescent↗
PubMed1998

Self-assembling bis-dendritic peptides: design, synthesis and characterization of oxalyl-linked bis-glutamyl peptides [Glu(n)(CO2Me)n + 1-CO-]2; n = 1,3,7.

Three generations of glutamic acid dendrons [Glu(n),(CO2Me)n + 1; n = 1, 3, 7] have been joined together head-to-head by an oxalyl unit to form highly sterically congested bis-Glu-dendritic peptides with gelling properties. The single crystal X-ray structure of the first generation bis-dendritic peptide showed an extended hydrogen-bonded chiral tape with modest nonlinear optical activity.

Crystallography, X-Ray↗
PubMed1998

Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans.

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.

Animals↗