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G Gupta

Publications and source records attributed to G Gupta.

At least 127 records · Page 7Linked to original sources
PubMed1995

Local and global structural properties of the HIV-MN V3 loop.

Studies of the feasibility of a subunit vaccine to protect against human immunodeficiency virus (HIV) infection have principally focused on the third variable (V3) loop. The principal neutralizing determinant (PND) of HIV-1 is located inside the V3 loop of the surface envelope glycoprotein, gp120. However, progress toward a PND-based vaccine has been impeded by the amino acid sequence variability in the V3 loops of different HIV isolates. Theoretical studies revealed that the variability in sequence and structure of the V3 loop is confined to the N- and C-terminal sides of the conserved GPG crest. This leaves three regions of the V3 loop conserved both in sequence and secondary structure. We present the results of NMR studies that test the validity of our theoretical predictions. Structural studies are reported for the HIV-V3 loop (HIV-MN) in the linear and cyclic (S-S-bridged) forms. For the V3 loop sequence of the HIV-MN isolate, the three conserved secondary structural elements are as underlined below: turns turn helix CTRPNYNKRKRIHIGPGRAFYTTKNIIGTIROAHC Finally, the conformational requirement of the PND in the V3 loop-antibody interaction is tested by monitoring the monoclonal antibody binding to the HIV-MN V3 loop in the linear and cyclic forms by enzyme-linked immunosorbent assay. The binding data reveal that the cyclic V3 loop is a better ligand for the monoclonal antibodies than the linear form although the latter has the same sequence. This means that the monoclonal antibodies recognize the PNDs as conformational epitopes.

Amino Acid Sequence↗
PubMed1995

Human immunodeficiency virus (HIV) antigens: structure and serology of multivalent human mucin MUC1-HIV V3 chimeric proteins.

Molecular modeling and two-dimensional NMR techniques enable us to identify structural features in the third variable region (V3) loop of the human immunodeficiency virus (HIV) surface glycoprotein gp120, in particular the principal neutralizing determinant (PND), that remain conserved despite the sequence variation. The conserved structure of the PND is a solvent-accessible protruding motif or a knob, structurally isomorphous with the immunodominant knobs in the tandem repeat protein of human mucin 1 (MUC1) (a tumor antigen for breast, pancreatic, and ovarian cancer). We have replaced the mucin antigenic knobs by the PND knobs of the HIV MN isolate in a set of chimeric human MUC1/HIV V3 antigens. This produced multivalent HIV antigens in which PNDs are located at regular intervals and separated by extended mucin spacers. In this article we show by two-dimensional NMR spectroscopy that the multivalent antigens preserve the PNDs in their native structure. We also demonstrate by ELISA that the antigens correctly present the PNDs for binding to monoclonal antibodies or polyclonal antisera from HIV-infected patients.

Amino Acid Sequence↗
PubMed1995

Structure of a tumor associated antigen containing a tandemly repeated immunodominant epitope.

Human mucins are T or S glycosylated tandem repeat proteins. In breast cancer, mucins become under or unglycosylated. Two-dimensional nuclear magnetic resonance experiments are performed on chemically synthesized mucin tandem repeat polypeptides, (PDTRPAPGST-APPAHGVTSA)n the unglycosylated form for n=1,3 where (APDTR) constitutes the antigenic sites for the antibodies isolated form the tumors in the breast cancer patients. These studies demonstrate how the tandem repeats assemble in space giving rise to the overall tertiary structure, and the local structure and presentation of the antigenic site(APDTR) at the junction of two neighboring repeats. The NMR data reveal repeating knob-like structures connected by extended spacers. The knobs protrude away from the long-axis of Muc-1 and the predominant antigenic site (APDTR) forms the accessible tip of the knob. Multiple tandem repeats enhance the rigidity and presentation of the knob-like structures.

Amino Acid Sequence↗
PubMed1995

Activities and androgenic regulation of lysosomal enzymes in the epididymis of rhesus monkey.

The activities and androgenic regulation of seven lysosomal enzymes viz. acid phosphatase, N-acetyl hexosaminidase, alpha-mannosidase, beta-glucuronidase, DNase II, RNase II and phospholipase A was established in caput, corpus and cauda segments of monkey epididymis. Estimation of enzyme activities in the the epididymis of control, castrated and castrated-androgen replaced monkeys revealed that all the enzymes except RNase II showed higher activity in caput and corpus as compared to cauda. The enzymes were reduced markedly after castration and on subsequent androgen replacement there was a significant stimulation of the repressed activities, but the control levels were not restored. RNase II showed highest activity in cauda which was further elevated after castration. The possible role of these enzymes in sperm maturation and disposal is discussed.

Androgens↗
PubMed1994

Unusual structures of the tandem repetitive DNA sequences located at human centromeres.

The presence of the highly conserved repetitive DNA sequence d(AATGG)n.d(CCATT)n in human centromeres argues for a special role for this sequence in recognition, most probably through the formation of an unusual structure during mitosis. Quantitative one- and two-dimensional nuclear magnetic resonance (1D/2D NMR) spectroscopic studies reveal that the Watson-Crick duplex d(AATGG)n.d(CCATT)n adopts the usual B-DNA conformation as illustrated by taking d(AATGG)3.d(CCATT)3 as an example, whereas the d(CCATT)n strand is essentially a random coil. In contrast, the d(AATGG)n strand adopts an unusual stem-loop motif for repeat lengths n = 2, 3, 4, and 6. In addition to normal Watson-Crick A.T pairs, the stem-loop structures are stabilized by mismatched A.G and G.G pairs in the stem and G-G-A stacking in the loop. Stem-loop structures of d(AATGG)n are independently verified by gel electrophoresis and nuclease digestion studies and were also previously shown to be as stable as the corresponding Watson-Crick duplex d(AATGG)n.d(CCATT)n [Grady et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1695-1699]. Therefore, the sequence d(AATGG)n can, indeed, nucleate a stem-loop structure at little free energy cost, and if, during mitosis, it is located on the chromosome surface, it can provide specific recognition sites for kinetochore function.

Base Sequence↗
PubMed1994

Activities and androgenic regulation of kreb cycle enzymes in the epididymis and vas deferens of rhesus monkey.

The activities of nine enzymes of the TCA cycle were estimated in the initial segment, caput, corpus and cauda segments of epididymis and vas deferens of adult rhesus monkey and expressed as units per mg DNA. These enzymes were also estimated in epididymal segments and vas deferens of castrated and castrated-androgen replaced monkeys as well. Results indicated higher activities of most of the enzymes in vas deferens as compared to epididymal segments. All the enzymes showed marked reduction in epididymis and vas deferens after castration, the effect being much more pronounced in the epididymis, than in the vas. Androgen replacement in castrated monkeys stimulated most of the enzymes markedly in epididymis and in the vas deferens as compared to their castrated values. The response of cauda and vas deferens to exogenous androgen treatment was however moderate, as compared to the other epididymal segments. The studies indicate that energy metabolism in the epididymis (as well as in the vas deferens) is strictly androgen dependent and the energy charge of these target organs is likely to fall appreciably after castration, which may in turn affect many energy dependent processes of these organs (e.g. absorption, secretion of specific substances etc.) which have been considered important for sperm maturation and survival.

Androgens↗
PubMed1994

Beta-adrenergic modulation of rat brain insulin receptor activity in normal and hyperglycaemic conditions.

The [125I]Insulin binding and receptor kinase activity were assessed in rat brain in the presence of 10 microM concentrations of the beta agonist isoproterenol. While insulin binding remained unaltered, beta agonist treatment enhanced significantly receptor kinase activity in control and hyperglycaemic conditions. Antihyperglycaemic effects of isoproterenol were discussed in relation to adrenergic effects on insulin action in brain.

Animals↗
PubMed1994

Fine needle aspiration cytology of cervical lymphadenopathy with special reference to tuberculosis.

One hundred eighty cases of cervical lymphadenopathy have been studied by fine needle aspiration cytological examination followed by histopathologic examination of the excised lymph nodes. The diagnostic accuracy was 84.4% for tuberculous lymphadenitis by fine needle aspiration cytological examination. Observation of caseous necrosis (84.2%) and epithelioid cells (73.6%) were the most characteristic diagnostic features in the aspirated smears. Acid-fast bacilli were observed in 45.6% cases. Metastatic carcinoma also yielded a high diagnostic accuracy ie, 89%. Fine needle aspiration cytology has been found to be safe, quick, inexpensive with high diagnostic accuracy in cervical lymphadenopathy.

Adolescent↗
PubMed1993

Loss of a neutralizing epitope by a spontaneous point mutation in the V3 loop of HIV-1 isolated from an infected laboratory worker.

The third hypervariable region, or V3 loop, represents the principal neutralizing domain of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequential viral isolates from a laboratory worker (LW) accidentally infected with HIV-1IIIB in 1985 were analyzed using type-specific neutralizing monoclonal antibodies directed to the V3 loop. A single amino acid substitution, Ala-->Thr at position 21 in the V3 loop of HIV-1LW isolated in 1987, was shown to determine the loss of the neutralizing epitope recognized by one of the monoclonal antibodies (M77). However, this antibody efficiently recognized linear V3 loop peptides containing either the Ala or Thr residue at position 21, indicating that a local change in conformation was responsible for the epitope loss in the native gp120. Molecular modeling studies, experimentally supported by different amino acid replacements at position 21, indicated that the Ala-->Thr substitution leads to a drastic change in the domain of the V3 loop, which contains the complementary surface for antibody binding. These results provide evidence for the first time that a conformation-dependent epitope within the V3 loop of HIV-1 is involved in the generation of neutralization escape mutants in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Structure of a parallel-stranded tetramer of the Oxytricha telomeric DNA sequence dT4G4.

We report here the determination of the solution structure of the single-copy tetramer of the sequence dT4G4, the unit repeat in telomeres of the ciliated protozoan Oxytricha, in the presence of potassium ions. This is accomplished by a combination of sequential assignments and distance determinations from 2D proton NMR with model building, based on conformational analysis of the structure using a full-matrix NOESY simulation and molecular dynamics. Each strand in this tetramer structure has an identical environment and conformation: a parallel-stranded, right-handed helix, with all nucleotides in the C2'-endo, anti configuration. The T flanking the G cluster stacks in a 4-fold symmetrical helical array, while the remaining T's become increasingly flexible and sample multiple stacked configurations.

Animals↗
PubMed1993

Sampling of the conformations of the d(CGCTGCGGC) hairpin in solution by two-dimensional nuclear magnetic resonance and theoretical methods.

Most NMR studies of DNA oligomers have focused on rigid structures that show a strong preference for one or a small set of ground-state conformations. There is an increasing interest in extending NMR methods to investigate DNA systems in which this preference does not exist. A DNA hairpin is one such system where a large number of low-energy structures coexist in solution. In this article we show how 1D/2D NMR data of the d(C1-G2-C3-T4-G5-C6-G7-G8-C9) hairpin are used to map the conformational space of this molecule. First, we characterize the gross morphology of the hairpin by monitoring the exchangeable imino signals in the molecule. Second, we extract a set of inter-proton distances (i.e., the average values and the associated dispersions) for various pairwise interactions by performing full-matrix NOESY simulation with respect to the observed NOESY data for mixing times of 250 and 100 ms. Third, we use these distances as structural constraints to perform a 300-ps molecular dynamics simulation at 500 K. Fourth, we extract 600 snapshots (one after every 0.5 ps) from the MD trajectory and perform constrained energy minimization to map local minima on the sampled energy surface (we call this the rapid temperature quenching step). Fifth, we assign 600 structures to 14 disjoint clusters such that conformationally similar hairpins belong to the same cluster while conformationally distinct hairpins belong to different clusters. Finally, we interpret the NOESY data in terms of conformationally distinct structures by recalculating NOESY contributions taken from representative structures of different clusters. Our analyses clearly demonstrate that the NMR data correspond to an ensemble of distinct structures, i.e., a set of energetically stable but conformationally distinct structures that satisfies the constraints of loop folding in the d(C1-G2-C3-T4-G5-C6-G7-G8-C9) hairpin. Two types of loop folding consistent with NMR data are obtained: (i) a hairpin with two G.C pairs in the stem and four residues in the loop and (ii) a hairpin with two G.C pairs and a reverse wobble G.T pair in the stem plus two residues in the loop.

Base Sequence↗
PubMed1993

Solution structure of the V3 loop of a Thailand HIV isolate.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV) is located inside the third variable loop (designated the V3 loop) of the envelope glycoprotein gp120. The V3 loop is typically 35 amino-acids long, and the 1st and the 35th residues in the loop are invariant cystines involved in a disulfide-bridge. Although PNDs from different HIV isolates contain a conserved GPG-sequence, the amino acids flanking the conserved sequence show hypervariability among HIV isolates; the GPG and the two flanking regions are collectively referred to as the GPG-crest or the PND. The amino acid sequence variability in the GPG-crest gives rise to different antigenic specificities for different PNDs from different HIV isolates. By combining two-dimensional nuclear magnetic resonance (2D NMR) and molecular modeling techniques, we have developed a method to study (1) the global tertiary fold of the V3 loops of HIV and (2) the local structure of the PND at the tip of the V3 loop. In this article, we report the results of our structural studies on the V3 loop of a Thailand HIV isolate. The sequential assignment is made by combining DQF-COSY, TOCSY, and NOESY/ROESY experiments. Various intra- and inter-residue inter-proton distances are estimated by full-matrix analyses of the NOESY data at 100 and 400 ms of mixing times and of the ROESY data at 60 and 200 ms of mixing times. 100 inter-residue distances are used as structural constraints in a simulated annealing procedure to derive energetically stable structures. Two functional motifs in the V3 loop, i.e., the glycosylation site and the GPG-crest, form defined structures: a turn is located at the glycosylation site, and the GPG-crest forms a protruding domain with a type-II GPGQ turn. The other regions of the V3 loop are rather flexible--especially the C-terminal DIRKAYC-stretch. These flexible regions of the V3 loop lead to conformational flexure of the entire V3 loop without altering the local structures of the glycosylation site or the GPG-crest. However, the ROESY experiments revealed no slow exchange among different V3 loop conformations, and therefore the flexible conformations are in fast exchange within the NMR time scale. The extent of this conformational flexibility is also discussed.

Amino Acid Sequence↗
PubMed1993

Viral pathogenesis and opportunistic infections.

Knowledge about the pathogenesis and management of HIV infection has increased dramatically over the last decades. Because of advances in drugs, diagnosis and management, HIV-infected patients are living longer. This paper provides current concepts of HIV pathogenesis and antiviral therapy, and an overview of AIDS-related opportunistic infections.

AIDS-Related Opportunistic Infections↗