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B S Parekh

Publications and source records attributed to B S Parekh.

At least 37 records · Page 2Linked to original sources
PubMed1996

Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.

Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.

Bacterial Proteins↗
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

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential.

OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies.

AIDS Serodiagnosis↗
PubMed1994

Differential replication and pathogenic effects of HIV-1 and HIV-2 in Macaca nemestrina.

OBJECTIVE: HIV-1 and HIV-2 isolates representing various geographic regions and distinct viral subtypes were examined for their ability to establish both in vitro and in vivo productive infections of Macaca nemestrina (pigtail macaque) peripheral blood mononuclear cells. METHODS: Animals were inoculated with either autologous cell-associated or cell-free viral preparations of selected isolates. HIV-specific immune responsiveness, hematologic changes, genetic variation, and virus burden were monitored as delineators of HIV pathogenesis. RESULTS: HIV-2 replication in vitro and in vivo correlated with nascent antigen production and rising viral titers as determined by infectious center assays. Infection was detectable by polymerase chain reaction amplification of proviral sequences in macaque cells as early as 1 week postinoculation. Two distinct patterns of CD4+ cell depletion induced by HIV-2 infection were observed during the first month postinoculation and characterized by a moderate loss sustained through 20 weeks postinoculation or a substantial loss maintained long-term (> 90 weeks). Identity between inoculating viral stocks and subsequent viral isolates from animals was established comparatively by limited sequence analysis of specific domains within the HIV-2 pol and env genes. In contrast, replication of HIV-1 isolates was limited or only semipermissive in vitro. Intravenous inoculation of HIV-1 field isolates, using conditions successful for HIV-2 (for example, identical viral titers), failed to establish a productive viral infection leading to seroconversion of fluctuations in hematologic cell markers. Infection with a high-titer inoculum of a laboratory-adapted HIV-1 strain in vivo, as demonstrated by polymerase chain reaction analysis, produced seroconversion in the absence of overt viral replication or hematologic variations in one out of four animals. CONCLUSIONS: This system provides for multifaceted modeling of HIV pathogenesis, primarily with HIV-2 and potentially with HIV-1/-2 chimerics, in support of immunotherapeutic developments and critical evaluation of intervention practices.

Amino Acid Sequence↗
PubMed1993

Teratogenic effects of neonatal arenavirus infection on the developing rat cerebellum are abrogated by passive immunotherapy.

The effects of viral infection on the developing nervous system and the potential of passive immunotherapy to protect against infection were examined. When 4-day-old Lewis rats were injected intracerebrally with lymphocytic choriomeningitis virus (LCMV) the majority of stem cells within the external granular layer of the developing cerebellum became infected. The infection progressed to the molecular layer, internal granular layer, and the Purkinje cells. By 15 days postinfection the molecular and internal granular layers of LCMV-infected cerebella were noticeably thinner than those in the controls and the individual folia were smaller. Neurons remained infected for up to 40 days as determined by immunohistochemistry. However, in rats treated with rat monoclonal anti-LCMV antibodies the staining was limited to the cells of ependyma and choroid plexus and was not detectable by 15 days postinfection. Macroscopically the infection resulted in pronounced hypoplasia, with the cerebella of 21-day-old LCMV-infected rats weighing 52 +/- 10 mg compared with 159 +/- 30 mg for control rats. Antibody-treated rats exhibited normal cerebellar size and development. Neutralizing antibodies specific for the viral GP-1 glycoprotein were protective but nucleoprotein-specific antibodies were not. Furthermore, suckling rat pups born of and nursed by LCMV-immune mothers were spared from cerebellar disease following neonatal infection. These results suggest that passive immunotherapy of neonates can provide effective protection against teratogenic effects of neonatal viral infection on the developing CNS.

Animals↗
PubMed1993

Dynamics of maternal IgG antibody decay and HIV-specific antibody synthesis in infants born to seropositive mothers. The NYC Perinatal HIV Transmission Study Group.

We have used a human immunodeficiency virus type 1 (HIV-1)-specific IgG-Fc capture enzyme immunoassay (IgG-CEIA) to elucidate the dynamics of HIV-1 maternal antibody decay and de novo synthesis of HIV-1 antibodies in infants. Two hundred and thirty-nine serum specimens from 77 infants were analyzed by the IgG-CEIA and by two different conventional EIAs. With the IgG-CEIA, IgG was captured by an anti-human IgG monoclonal antibody (3C8) that reacts with all subclasses and was detected by recombinant HIV-1 envelope protein (CBre3)-peroxidase conjugate. Unlike the conventional EIAs, the IgG-CEIA showed a rapid decay of HIV-1-specific antibody in uninfected infants, with decline to background levels by 6 months (T1/2 [half-life] = 28-30 days). All 69 specimens collected from 39 uninfected infants between 6 and 15 months of age were negative by IgG-CEIA. However, HIV-1 antibodies remained high in infected infants; 20/22 infants (90.9%) with specimens between the ages of 6 to 23 months were positive by IgG-CEIA. Subtracting mean IgG-CEIA optical density values of seroreverting infants from those of HIV-1-infected infants in corresponding age groups provided a model for seroconversion in infected infants, with detectable IgG antibody synthesis starting about 3 months after birth. The IgG-CEIA may be a simple and important tool for early diagnosis of HIV-1 infection in infants at 6 months of age.

Female↗
PubMed1993

Highly specific V3 peptide enzyme immunoassay for serotyping HIV-1 specimens from Thailand.

OBJECTIVE: To develop and evaluate a simple V3 peptide-based enzyme immunoassay (EIA) for large-scale serotyping of HIV-1 specimens from Thailand. DESIGN: Serologic reactivities with synthetic peptides derived from the V3 loop of gp120 were used for typing HIV-1 specimens. METHODS: Synthetic peptides PND-A and PND-B, derived from the consensus amino-acid sequences of the V3 loop of gp120 from two major genomic variants of HIV-1 in Thailand (A and B), were evaluated in an EIA on 61 Thai HIV-1 sera for which genotypes had been determined by polymerase chain reaction. The peptide EIA was then applied to sera from 188 HIV-1-infected patients, selected in non-random, convenience samples of known risk groups from four geographic regions of Thailand. RESULTS: The sensitivities and specificities of PND-A and PND-B were 86% (30 out of 35) and 96% (25 out of 26) and 92% (24 out of 26) and 94% (33 out of 35), respectively, with 100% predictive values of a monoreactive positive test for both peptides. The assay classified 101 specimens as serotype A, 39 as serotype B, eight as serotype AB (dually reactive), and 40 as untypable (non-reactive). Excluding dual reactors and non-reactors, 92% (77 out of 84) of specimens from patients probably infected by sexual contact were serotype A; conversely, 76% (28 out of 37) of injecting drug users were serotype B. CONCLUSION: The serologic results corroborated previous findings, in a smaller subset of samples, of an apparent segregation of viral subtypes by mode of transmission, suggesting two separate HIV-1 epidemics in Thailand. This peptide EIA could be a valuable epidemiologic tool in determining the dynamics of the rapid spread of HIV-1 in Thailand.

Comorbidity↗
PubMed1993

Human immunodeficiency virus 1-specific IgA capture enzyme immunoassay for early diagnosis of human immunodeficiency virus 1 infection in infants. NYC Perinatal HIV Transmission Study Group.

A simplified human immunodeficiency virus 1 (HIV-1)-specific IgA capture enzyme immunoassay (IgA-CEIA) was evaluated and compared with IgA-Western blot assay for early diagnosis of HIV-1 infection in infants born to seropositive women. A total of 232 coded sera collected prospectively from 70 infants were tested. All 25 sera from 10 HIV-1-negative infants born to seronegative mothers (negative controls) were negative by both assays. All 111 sera from 37 seroreverting, uninfected infants were negative by IgA-CEIA (specificity, 100%), whereas 110 of 111 sera were negative by IgA-Western blot assay (specificity, > 99%). Overall IgA-CEIA detected HIV-IgA in 20 (87%) of 23 infected infants, and IgA-Western blot assay detected HIV-IgA in 21 (91.3%) of 23 infants; specimen-wise agreement between the 2 assays was > 80%. Analysis of results by age group indicated that after 2 months of age both assays were equivalent with sensitivity ranging from 60 to 80%. Quantitative data provided by IgA-CEIA suggests that the bulk of HIV-1 IgA synthesis in most HIV-1-infected infants occurs after 2 months of age.

AIDS Serodiagnosis↗
PubMed1992

Simian immunodeficiency virus needlestick accident in a laboratory worker.

The macaque monkey infected with simian immunodeficiency virus (SIV) is an animal model of the acquired immunodeficiency syndrome. We investigated a laboratory worker who was exposed by needlestick accident to blood from an SIV-infected macaque. Seroreactivity to SIV developed within 3 months of exposure, with antibody titres peaking from the third to the fifth month and declining thereafter. Polymerase chain reaction for SIV sequences and cultures of peripheral-blood mononuclear cells failed to show infection. Inoculation of an SIV-negative monkey with blood from the worker did not cause infection. Animal-care and laboratory workers should adhere strictly to recommended procedures to avoid accidental exposures when working with SIV-infected animals or specimens.

Animals↗
PubMed1991

Lack of correlation between maternal antibodies to V3 loop peptides of gp120 and perinatal HIV-1 transmission. The NYC Perinatal HIV Transmission Collaborative Study.

Recent reports have suggested that maternal antibodies to specific epitopes of the variable region 3 (V3 loop) of gp120 of HIV-1 might protect against perinatal transmission. In an attempt to confirm these findings, sera from 34 HIV-1-seropositive mothers, representing 13 episodes of mother-to-infant transmission and 23 episodes of non-transmission (two mothers had two pregnancies each during the study period), were tested for the presence of antibodies to various regions of the gp120 V3 loop. Synthetic peptides were generated from HIV-1MN. Of the four peptides tested by enzyme-linked immunosorbent assay (ELISA), only antibody to the C53 peptide (Env310-322, principal neutralizing determinant) was present in maternal sera. Antibody to the C53 sequence was present in 11 specimens from transmitting mothers and 21 from non-transmitting mothers (84.6 and 91.3%, respectively, P = 0.6). No reactivity was detected against the C51, C57, or C58 peptide sequences, located on the sides of the V3 loop. In an antigen-limited ELISA, only two specimens from transmitting mothers and two specimens from non-transmitting mothers had detectable 'high-affinity' antibodies to C53 at low antigen concentrations (15.4 and 8.7%, respectively; P = 0.6). Our results do not support previous reports that epitope-specific antibodies to the V3 loop peptides protect against perinatal transmission. Further research is required to determine whether any specific maternal humoral response might influence HIV-1 perinatal transmission.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Oligomeric nature of transmembrane glycoproteins of HIV-2: procedures for their efficient dissociation and preparation of Western blots for diagnosis.

Western blot (WB) analysis of various strains of HIV-2 indicated that transmembrane glycoprotein (TMP) of HIV-2 exists as trimers. These trimers have molecular weights and electrophoretic mobilities in the region of the major external glycoprotein, gp120, resulting in WB misidentification during diagnosis. A simple and rapid procedure was developed using trichloroacetic acid (TCA) to efficiently dissociate oligomeric forms of the TMP to monomers prior to the preparation of WB. This procedure permitted the unambiguous identification of antibodies to gp120 and to the TMP. Use of HIV-2 WB strips without any oligomeric forms of the TMP demonstrated (1) that cross reactivity of HIV-1-positive specimens on HIV-2 WB was mainly directed to Gag and Pol proteins, with some reactivity to gp36/gp41 TMP, but none to gp120; (2) that these strips can substantially reduce the number of specimens falsely identified as dually (HIV-1 and HIV-2) reactive; and (3) that HIV-2-positive specimens reacted to viral gp120 in a strain-specific manner, demonstrating high antigenic variation in this glycoprotein. It is recommended that this general procedure of viral protein dissociation be used for HIV-2 WB preparation.

Blotting, Western↗
PubMed1987

Site-specific antibodies define a cleavage site conserved among arenavirus GP-C glycoproteins.

Arenaviruses share a common strategy for glycoprotein synthesis and processing in which a mannose-rich precursor glycoprotein, termed GP-C in lymphocytic choriomeningitis virus (LCMV), is posttranslationally processed by oligosaccharide trimming and proteolytic cleavage to yield two structural glycoproteins, GP-1 and GP-2. Mapping the orientation and proteolytic cleavage site(s) in such polyproteins has traditionally required direct protein sequencing of one or more of the cleaved products. This technique requires rigorous purification of the products for sequencing and may be complicated by amino-terminal modifications which interfere with sequence analysis. We used an alternative approach in which synthetic peptides corresponding to sequences bracketing a potential protease cleavage site were used to raise antisera which define the boundaries of the cleaved products. We found that cleavage of LCMV GP-C to yield GP-1 and GP-2 occurs within a 9-amino-acid stretch of GP-C which contains a paired basic amino acid group -Arg-Arg-, corresponding to amino acids 262 to 263 in the LCMV GP-C sequence. By comparison with the predicted amino acid sequences of a second LCMV strain, LCMV-WE, as well as with the deduced amino acid sequences of the New World arenavirus Pichinde and the Old World virus Lassa, we observed similar conservation of paired basic and flanking amino acid sequences among these viruses.

Amino Acid Sequence↗
PubMed1986

Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II.

In contrast to reticulocyte polypeptide chain initiation factor 2 (eIF-2), the Artemia factor retains activity in the presence of Mg2+ or after phosphorylation of its alpha-subunit by rabbit reticulocyte heme-controlled repressor (Mehta, H. B., Woodley, C. L., and Wahba, A. J. (1983) J. Biol. Chem. 258, 3438-3441). Furthermore, we have so far been unable to demonstrate a requirement for a GDP/GTP nucleotide exchange factor with Artemia eIF-2. In order to explain these differences we compared the structure of eIF-2 from Artemia and rabbit reticulocytes by using one- and two-dimensional phosphopeptide and iodopeptide maps. Partial trypsin digestion of the alpha-subunit of Artemia eIF-2 after phosphorylation by the heme-controlled repressor generates a 4000 Mr phosphopeptide. Upon extensive trypsin digestion, the two-dimensional phosphopeptide maps of the alpha-subunits for the reticulocyte and Artemia factors are indistinguishable, whereas the iodopeptide maps are different. In addition, immunoblotting indicates that there is no consistent cross-reactivity of the reticulocyte subunits with antibodies prepared in rabbits against the Artemia eIF-2 subunits. A casein kinase II activity was isolated from Artemia embryos that phosphorylates the beta-subunit of reticulocyte eIF-2, but specifically phosphorylates the alpha-subunit of eIF-2 preparations from several non-mammalian sources, including Artemia, yeast, and wheat germ embryos. Since this kinase phosphorylates a site distinct from that recognized by the heme-controlled repressor, and this phosphorylation does not alter the ability of Artemia eIF-2 to undergo nucleotide exchange, caution must be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in non-mammalian cells.

Animals↗
PubMed1986

Proteins of lymphocytic choriomeningitis virus: antigenic topography of the viral glycoproteins.

Topographical relationships among antigenic sites on the envelope glycoproteins of lymphocytic choriomeningitis virus (LCMV) were established using a panel of monoclonal antibodies (MAb) directed against viral GP-1 and GP-2. Purified MAb were radioiodinated and used as probes in a solid phase competitive binding assay. Epitopes on LCMV GP-1 were found to cluster in four antigenic sites. Five neutralizing MAb raised by immunization with the WE strain of LCMV reacted with a single topographic site, termed GP-1A, which was present on four strains of LCMV examined in this study. A second site, GP-1B, was characterized by two MAb which partially competed with one another and with a subset of neutralizing antibodies. This site appeared to be close to site A and was found to be nonneutralizing. The third site, GP-1C, contained sequential epitopes and was also nonneutralizing. Antibodies binding to site B enhanced the binding of MAb at site C, presumably through a conformational change. In addition to the common neutralizing site A, LCMV Armstrong strain (LCMV-Arm) GP-1 contained a second topographically related neutralizing site, GP-1D, which was specific for LCMV-Arm, absent in WE, and appeared to be the major immunogenic epitope on GP-1 of this virus. Analysis of MAb binding to LCMV GP-2 demonstrated the presence of three overlapping binding sites. GP-2A was defined by two antibodies while GP-2B and C represented binding sites of one antibody each. Guinea pigs primed with LCMV-Arm and challenged with LCMV-WE developed a significant immune response which was directed toward the common major neutralizing site, GP-1A, but had poor responses to the LCMV-Arm specific neutralizing site GP-1D. Immune sera contained antibody to site GP-1B but lacked detectable antibody to GP-1C.

Animals↗
PubMed1985

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.

Acetonitriles↗