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Biomedical subjects

N Sarver

Publications and source records attributed to N Sarver.

At least 19 recordsLinked to original sources
PubMed1999

Bone marrow stromal cell-mediated gene therapy for hemophilia A: in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648.

To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for hemophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following transfection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10(6) cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (approximately 70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10(6) cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed > or = 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.

Animals↗
PubMed1996

Conference summary: novel HIV therapies--from discovery to clinical proof of concept.

In this report we have highlighted only a few examples of the extensive efforts underway to better understand the process of HIV pathogenesis, to develop new therapeutic agents to inhibit virus replication, and to identify strategies to restore damage done to the immune system during HIV disease progression. It is expected that progress in these areas will continue to advance, and that development of more effective therapies will lead to comprehensive multifaceted, multipronged treatment regimens.

HIV Infections↗
PubMed1995

Nucleic acid vaccines.

The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines. Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native conformation, glycosylation profiles, and other posttranslational modifications that mimic antigen produced during natural viral infection. Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocyte responses to diverse protein antigens. Advantages of nucleic acid-based vaccines include the simplicity of the vector, the ease of delivery, the duration of expression, and, to date, the lack of evidence of integration. Further studies are needed to assess the feasibility, safety, and efficacy of this new and promising technology.

Animals↗
PubMed1994

Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Frontiers in HIV-1 Therapy: fourth conference of the NIAID National Cooperative Drug Discovery Groups-HIV.

This summary describes current studies in antiviral targeting as reported at the Frontiers in HIV Therapy conference, November 3-7, 1991, in San Diego, California. In parallel with the progressive steps in HIV-1 replication, the meeting covered potential antiviral targets starting from the time HIV-1 docks with the CD4 receptor to virus release. The summary concludes with current research trends to block HIV-1 growth.

Animals↗
PubMed1991

EGF induces increased ligand binding affinity and dimerization of soluble epidermal growth factor (EGF) receptor extracellular domain.

The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF.

Animals↗
PubMed1991

The potential use of catalytic RNAs in therapy of HIV infection and other diseases.

This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.

Base Sequence↗