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D Skrincosky

Publications and source records attributed to D Skrincosky.

7 recordsLinked to original sources
PubMed2001

Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies.

Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.

Amino Acid Sequence↗
PubMed2000

Identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus 7.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.

Amino Acid Motifs↗
PubMed1997

Human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a recently described T-lymphotropic herpesvirus, which infects almost all children by the age of three years and persists lifelong, with the shedding of infectious virus in saliva. HHV-7 is similar to human herpesvirus 6 (HHV-6) in its genetic content and in many of its biological properties, which include the ability to cause at least some cases of exanthem subitum (roseola). Despite these similarities, important differences between HHV-7 and HHV-6 exist, including the fact that HHV-7 binds to the cellular CD4 molecule and uses this protein as a necessary component of its receptor, while HHV-6 binds to a different (and unknown) receptor. Furthermore, the pathogenesis and sequelae of HHV-7 infection remain very poorly understood. This review provides a critical summary of research on HHV-7.

Journal Article↗
PubMed1997

Human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is a T-lymphotropic herpesvirus, which infects almost all children by the age of two years and persists lifelong. Two distinct variants of HHV-6, HHV-6A and HHV-6B, have been described, and the latter has been shown to be a common cause of acute febrile illnesses in young children, including exanthem subitum (roseola). HHV-6 has also been associated with a number of neurological disorders, including encephalitis and seizures, and the virus has been postulated to play a role in acquired immunodeficiency syndrome (AIDS), multiple sclerosis (MS) and chronic fatigue immunodeficiency syndrome (CFIDS). This review provides a critical summary of research conducted on HHV-6.

Journal Article↗
PubMed1997

Altered Golgi localization of core 2 beta-1,6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans.

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.

Animals↗
PubMed1989

2'-Fluorinated isonucleosides. 1. Synthesis and biological activity of some methyl 2'-deoxy-2'-fluoro-2'-pyrimidinyl-D-arabinopyranosides.

New reactions of methyl 2,2-difluoro glycosides are described that were utilized for synthesis of some novel nucleoside derivatives. Thus, treatment of methyl 2-deoxy-2,2-difluoro-3,4-O-isopropylidene-alpha (beta)-D-erythro-pyranoside (2) with anhydrous HCl resulted in selective displacement of one fluorine atom with chlorine to give a 2-deoxy-2-chloro-2-fluoro glycoside 3. Reaction of 3 with silylated uracil in the presence of SnCl4 provided a 2-deoxy-2-fluoro-2-uracil-substituted glycoside 4. 2-Fluoro-2-deoxy glycosides substituted with other pyrimidines at C-2 were prepared similarly by the reaction of acylated 2,2-difluoro or 2-fluoro-2-bromo derivatives (5 and 6, respectively) with silylated pyrimidines. The resulting 2'-fluorinated isonucleosides were evaluated for their antitumor and antiviral activities. Compounds 7a,b, 8a,b, and 10a,b demonstrated 50% tumor cell growth inhibition in vitro (IC50) at 10(-4)-10(-5) M. At similar concentrations no antiviral activity was observed in vitro. Therapeutic activity was obtained with 7a,b and 8a,b in DBA/2 mice with L1210 leukemia. Administration of 7a,b at 500 mg/kg, ip daily, for 5 consecutive days, resulted in a 55% increase in life span (% ILS) while administration of 8a,b in the same manner at 200 mg/kg caused a 29% ILS. Treatment with 7a,b to mice with drug-resistant L1210 sublines (5-FU and araC) resulted in 22 and 57% increases in life span, respectively. Lewis lung carcinoma and M5076 sarcoma in mice also responded to the administration of 7a,b with reductions in tumor growth for both tumors and significant increases in life span in mice with Lewis lung carcinoma. Although the mechanism of action of 7a,b is not known, it has been found to be a relatively fast-acting, cell-cycle nonspecific cytotoxic agent that decreases [3H]deoxyuridine incorporation, blocks L1210 cells at the G2 phase of the cell cycle, and is not reversed by exogenous thymidine. These 2'-fluorinated isonucleosides have demonstrated biological activity and may have potential as antitumor drugs.

Animals↗