Search PubMed⌕ Search

Biomedical subjects

R Anand

Publications and source records attributed to R Anand.

At least 145 records · Page 8Linked to original sources
PubMed1992

Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.

Base Sequence↗
PubMed1992

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Base Sequence↗
PubMed1992

Intravitreal ganciclovir pharmacokinetics in rabbits and man.

Cytomegalovirus (CMV) retinitis occurs in immunocompromised patients and can be treated by repeated intravenous or intravitreal injections of ganciclovir (GCV) or foscarnet. Due to toxicity and complications these modalities are not ideal. The development of alternative administration routes is hindered by a lack of pharmacokinetic data. Devices giving pseudo zero order release of GCV were implanted intravitreally first in rabbits and then in patients with AIDS associated CMV retinitis as part of a Phase I clinical trial. Steady state intravitreal GCV levels were obtained immediately after death and the elimination rate constants were calculated assuming first order pharmacokinetics. Normalizing for retinal surface area, distribution volume and anatomic volume, the retinal elimination rate constants were calculated. These were found to be 0.017 cm-2hr-1 in rabbits and 0.015 cm-2hr-1 in man. This indicates that the rabbit eye is a good model for studying intravitreal pharmacokinetics of ganciclovir and suggests a common elimination mechanism which may be trans-retinal.

Animals↗
PubMed1992

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.

Chromosomes, Fungal↗
PubMed1992

Human immunodeficiency virus type 1 nef quasispecies in pathological tissue.

The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-1-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nef quasispecies in pathologic tissues during HIV-1 infection of the human host.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Macular pseudoholes. Clinical features and accuracy of diagnosis.

BACKGROUND: Epimacular membrane with pseudohole is an important vitreomacular disorder that belongs in the differential diagnosis of impending and established macular hole. To better characterize this lesion, the authors attempted to identify various features of eyes with epimacular membrane and pseudohole. METHODS: Demographic, clinical, photographic, and fluorescein angiographic data for 14 eyes with epimacular membrane and pseudohole were reviewed. Horizontal and vertical diameters of the pseudoholes were measured, and the original diagnosis was recorded for each eye. Fluorescein angiography was performed in 11 eyes. RESULTS: The mean age of patients with macular pseudoholes was 61.6 years, and median visual acuity for pseudohole eyes was 20/30. Mean horizontal and vertical diameters of the pseudoholes were 384 and 410 microns, respectively. None of the eyes with pseudoholes had the characteristic ophthalmoscopic features associated with full-thickness macular holes or impending macular holes. Results of fluorescein angiography showed three eyes with increased tortuosity or abnormal straightening of the perifoveal vessels; three eyes with a foveal window defect; and three eyes with late leakage from the perifoveal vessels. The original diagnosis of the initial examining physician was correct in only 43% of eyes with epimacular membrane and pseudohole. CONCLUSION: Epimacular membrane with pseudohole may be an underdiagnosed lesion and commonly mistaken for impending macular hole, full-thickness hole, or lamellar hole. These data may be of use as more patients are being considered for recently advocated surgical treatments for impending and established macular hole.

Adult↗
PubMed1992

Variable infections with and serologic responses of rabbits to five different HIV-1 strains, including one neural tissue isolate.

The infectivity of different strains of HIV-1 in rabbits was investigated. The HIV-1RF and HIV-1MN inocula induced anti-envelope antibodies detectable by Western blot, and in the case of HIV-1RF, these antibodies were also detectable by ELISA. The peripheral blood lymphocytes (PBL) and lymph nodes from rabbits inoculated with HIV-1IIIB, HIV-1MN and HIV-1Z3, were positive for virus by culture and by polymerase chain reaction (PCR). HIV-1BRVA, originally isolated from a patient with AIDS dementia, infected the brain of the inoculated rabbit, as indicated by both virus culture and PCR. In this case PCR was positive using four different primer pairs. Throughout the study, rabbits showed no clinical signs of HIV-1 infection and no remarkable histopathology was observed in the tissues examined. The apparent differences in infectivity and tissue tropism of the five HIV-1 strains demonstrated here provide additional evidence that the rabbit may serve as a useful model for studying HIV-1 infection and pathogenesis.

Animals↗
PubMed1991

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.

Animals↗
PubMed1991

Identification of CpG islands in a physical map encompassing the Friedreich's ataxia locus.

The Friedreich's ataxia locus has been previously assigned to chromosome 9q 13-21.1 by the demonstration of tight linkage to two anonymous DNA markers. MCT112 (Z greater than 80, theta = 0) and DR47 (Z greater than 50, theta = 0). The absence of recombination between these three loci has prevented the resolution of gene/probe order in this region, impeding strategies for gene isolation. We report physical mapping over a 4-Mb genomic interval, linking the markers MCT112 and DR47 on a common 460-kb NotI fragment and identifying 11 CpG islands in the 1.7-Mb interval most likely to contain the Friedreich's ataxia locus. Four of these islands were detected only by analysis of three YAC clones spanning a 700-kb interval including the MCT112/DR47 cluster. Without clear evidence of the precise location of the disease locus from recombination events, each of these regions must be considered as specifying a potential "candidate" sequence for the mutated gene.

Chromosome Mapping↗
PubMed1991

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.

Base Sequence↗
PubMed1991

Ribosomal inhibitory proteins from plants inhibit HIV-1 replication in acutely infected peripheral blood mononuclear cells.

Peripheral blood mononuclear cells from seronegative donors were stimulated with phytohemagglutinin and then infected with human immunodeficiency virus (HIV-1). Using this experimental system, the antiviral activity of two translation inhibitory proteins (pokeweed antiviral protein, PAP-S, and Luffa ribosomal inhibitory protein, LRIP-I) isolated from plants and a recombinant form of ricin A chain were studied. Previously, it had been shown that toxin polypeptides linked to monoclonal antibodies could inhibit HIV-infected cells. In the present study, the free, unconjugated, proteins were found to inhibit HIV replication at doses in which they were nontoxic to uninfected peripheral blood mononuclear cells. Among the inhibitory proteins, PAP-S and recombinant ricin A chain markedly reduced the reverse transcriptase activity and the expression of p24 core protein in infected cultures. Dose response studies indicate that the anti-HIV activity of PAP-S was comparable to AZT. The other ribosome inhibitory proteins (RIPs) showed moderate but significant antiviral activity.

Cell Survival↗