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R Pal

Publications and source records attributed to R Pal.

At least 37 records · Page 2Linked to original sources
PubMed2001

Enhanced immunogenicity of a contraceptive vaccine using diverse synthetic carriers with permissible adjuvant.

A vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical testing demonstrating the feasibility of the approach in preventing pregnancy in women. Some individuals, however, did not respond adequately despite employing highly immunogenic bacterial toxoids as carriers. We investigated the potential of three promiscuous pathogen-derived Th peptides as carriers, employing alum as the adjuvant. While conjugation with each peptide improved the antibody response against hCG in mice of different haplotypes, immunisation with a combination of these peptide-conjugates generated anti-hCG responses higher than those achieved with the individual peptides or tetanus toxoid (TT). Antibodies were of high affinity and capable of neutralising the bioactivity of hCG but were devoid of anti-peptide reactivity. These results have implication for the design of hCG vaccine with improved immunogenicity for diverse population.

Adjuvants, Immunologic↗
PubMed2001

Macrophage-derived chemokine gene expression in human and macaque cells: mRNA quantification using NASBA technology.

Macrophage-derived chemokine (MDC) is a CC-chemokine that inhibits infection by both macrophage- and T cell-tropic strains of HIV-1. This suppressor activity has led to great interest in fully characterizing the role of MDC in the pathogenesis of HIV-1 infection. Methods for the quantitation of constitutive levels of MDC protein in vivo are lacking. In this report, we describe the development and performance of a NASBA-based assay for the quantification of MDC mRNA expression in human and macaque cells. Although the constitutive in vivo levels of MDC mRNA in macaque and human T lymphocytes were low, in vitro activation of these cells greatly increased MDC transcription. Levels in the human and macaque cells were comparable under all conditions tested. Positive correlations between MDC transcription and protein expression were observed. The results indicate that this assay is extremely sensitive and reproducible over a five log dynamic range, and effectively quantifies MDC mRNA in resting and activated T cells. This assay may therefore permit characterization of the role of MDC in HIV-1/SIV pathogenesis, and in vaccine-induced immune responses.

Animals↗
PubMed2001

Absence of corpus luteum rescue by chorionic gonadotropin in women immunized with a contraceptive vaccine.

OBJECTIVE: To determine whether antibodies induced by a betahCG vaccine in women are competent to neutralize the luteotropic action of hCG. DESIGN: Prospective clinical study. SETTING: Hospitals and a laboratory at an academic center. PATIENT(S): Six immunized and three control women of reproductive age, participating in a clinical testing of betahCG contraceptive vaccine. INTERVENTION(S): Increasing doses of hCG simulating early pregnancy. MAIN OUTCOME MEASURE(S): Anti-hCG antibody titers, serum progesterone and urinary hCG levels, and onset of menses. RESULT(S): Administration of hCG to mimic early pregnancy sustained serum progesterone concentrations and extended the luteal phase in control women. In contrast, serum progesterone levels declined and the luteal phase was not extended if prevailing antibody titers were > or =40 ng/mL in women who had been immunized with a betahCG based vaccine. No booster effect was seen in anti-hCG titers after hCG challenge. CONCLUSION(S): Antibodies elicited by a betahCG vaccine inactivate hCG and prevent the hormone from rescuing corpus luteum, resulting in progesterone fall and normal menses. Lack of booster in the antibody response confirms the reversibility of the approach.

Adult↗
PubMed2001

Microfabricated reaction and separation systems.

Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated 'lab-on-a-chip' systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.

Automation↗
PubMed2001

Estrogen receptors in rat bone: their interaction with estrogen receptor modulators.

Estrogen receptors (ER) were studied in rat bone cytosol using immunoprecipitation, and Western blot technique. Ligand specificity of bone ER was studied using various known modulators of ER. Competitive experiments were performed under exchange conditions in bone tissue obtained from one day old rats. ER alpha and beta subtypes were identified using immunoblotting experiments compared with that of ovarian and uterine tissues. In competitive binding assay, maximum inhibition in specific 3H-E2 binding was shown by E2 followed by tamoxifen and diethylstilbestrol. 7-Hydroxycentchroman and 85/287 also inhibited specific 3H-E2 binding but were less potent as compared to tamoxifen and diethylstilbestrol. However, 85/287 was less effective (81%) as compared to 7-hydroxycentchroman. Polyacrylamide gel electrophoresis of cytosol and Western blot analysis revealed the presence of 55 kD and 66 kD ER immunoreactive bands corresponding to alpha and beta subtypes, respectively, in bone as well as in uterus. Interestingly, the concentration of 55 kD ER was 3-fold higher than that of 66 kD ER. Ovarian cytosol revealed the presence of a 55 kD band only in Western blot analysis. These studies suggest the action of estrogens/ER modulators on osteoblasts which contain a limited number of classical alpha as well as beta sub types of ER that are known to be structurally different in their hormone-binding domains.

Animals↗
PubMed2001

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

Animals↗
PubMed2001

High expression of human chorionic gonadotrophin beta-subunit using a synthetic vaccinia virus promoter.

We have constructed a recombinant vaccinia virus to express the beta-subunit of human chorionic gonadotrophin (betahCG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine. The cDNA encoding the subunit was cloned under the control of a synthetic promoter that could be recognised by a vaccinia virus RNA polymerase to direct transcription. The peak expression level of betahCG directed by a late synthetic promoter (Psyn) was 11.5 microg/ml, a level that was at least sixfold higher than that directed by the p7.5 early/late promoter. The expressed protein was correctly processed post-translationally such that it attained a conformation with correctly folded discontinuous epitope(s) similar to that seen in native betahCG.

Blotting, Southern↗
PubMed2000

Linear Viscoelastic Behavior of Multiphase Dispersions.

The linear viscoelastic behavior of polymer-thickened oil-in-water emulsions, polymer-thickened solids-in-liquid suspensions, and their blends is investigated using a controlled-stress rheometer. The emulsions exhibit a predominantly viscous behaviour at low values of oil concentration in that the loss modulus (G") exceeds the storage modulus (G') over most of the frequency range. At high values of oil concentration, the emulsions exhibit a predominantly elastic behavior. The ratio of storage modulus to loss modulus (G'/G") increases with the increase in oil concentration. Emulsions follow the theoretical model of J. F. Palierne (1990, Rheol. Acta 29, 204) only at low values of oil volume fraction (</=0.176). At high values of oil volume fraction, the Palierne model underpredicts the linear viscoelastic properties of emulsions. Polymer-thickened suspensions are predominantly viscous in nature; G">/=G' over most of the frequency range. The ratio G'/G" varies only slightly with the increase in solids volume fraction. The Palierne model describes the linear viscoelastic properties of suspensions accurately only at low values of solids volume fraction. At high values of solids concentration, the Parlierne model underpredicts the linear viscoelastic properties of suspensions and the deviation increases with the increase in solids concentration. The blends of emulsions and suspensions exhibit strong synergistic effects at low to moderate values of frequencies; the plots of blend modulus versus emulsion content exhibit a minimum. However, at high values of frequency, the blend modulus generally falls between the moduli of pure suspension and pure emulsion. The high-frequency modulus data of blends of emulsions and suspensions are successfully correlated in terms of the modulus ratio versus volume fraction of solids, where modulus ratio is defined as the ratio of blend modulus to pure emulsion modulus at the same frequency. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Viscosity-Concentration Equation for Emulsions of Nearly Spherical Droplets.

A new modified form of the equation of N. Phan-Thien and D. C. Pham (J. Non-Newtonian Fluid Mech. 72, 305 (1997)) is proposed to describe the viscosity-concentration behavior of emulsions of nearly spherical droplets. The proposed equation, as well as other existing theoretical equations, is evaluated in light of a large body of experimental data on concentrated emulsions, covering a broad range of dispersed-phase to continuous-phase viscosity ratios (4.15x10(-3) to 1.17x10(3)). In general, the experimental data exhibit large deviations from the existing theoretical equations; for example, the theoretical equation of Phan-Thien and Pham underpredicts the relative viscosity of concentrated emulsions by a large amount. The equation proposed in this work describes the experimental viscosity data of different emulsion systems remarkably well. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Quantitative evaluation of simian immunodeficiency virus infection using NASBA technology.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Animals↗
PubMed2000

Humoral hyporesponsiveness to a conjugate contraceptive vaccine and its bypass by diverse carriers using permissible adjuvant.

A contraceptive vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical testing that demonstrated the feasibility of the approach in preventing pregnancy in women. Some immunized volunteers however, did not respond with an adequate anti-hCG antibody response despite employing highly immunogenic bacterial toxoids as carriers. Since there is some evidence that T cell responses to a complex protein typically focus on a few immunodominant epitopes, we investigated the responsiveness to hCG in mice of different haplotypes using the protein carrier diphtheria toxoid (DT). Our data showed a differential carrier effect of DT. With the aim of making a more potent immunogen employing promiscuous pathogen-derived Th peptides as carriers, peptide:antigen stoichiometric ratios were optimized. When tested individually using alum as the adjuvant, three such peptide conjugates improved the anti-hCG response, though not consistently to levels higher than the DT conjugate. Immunization with a combination of these synthetic epitopes generated anti-hCG responses higher than those achieved with DT or with the individual peptides. Antibodies were of high affinity and capable of neutralizing the bioactivity of hCG, but were devoid of anti-peptide reactivity. These results support our view that differential hyporesponsiveness in a diverse population may arise from inadequate carrier effect and that it can be overcome by use of pathogen-derived broadly reactive non-B Th epitopes employing only alum, a permissible adjuvant.

Adjuvants, Immunologic↗
PubMed1999

Intraspinal and behavioral consequences of nerve growth factor-induced nociceptive sprouting and nerve growth factor-induced hyperalgesia compared in adult rats.

Intraspinal and behavioral events were studied in adult rats with nociceptive nerves that were undergoing collateral sprouting into adjacent denervated skin. This sprouting, which is driven by endogenous nerve growth factor (NGF), did not cause hyperalgesia. For comparison, we studied an exogenous NGF administration that induced hyperalgesia but was too brief to evoke sprouting. When nociceptive nerves sprouted in skin, back-labeling with wheat germ agglutinin-conjugated horseradish peroxidase revealed that their projections sprouted in the cord. The sprouted peripheral nerves now activated more c-Fos-containing interneurons, which stimulus-response studies showed was not due to an increased afferent discharge. We attribute the interneuron recruitment to synaptogenesis following the intraspinal sprouting. Nociceptive stimulation of dorsal skin reflexly activates underlying cutaneous trunci muscle (CTM). When a nociceptive field expanded by nerve sprouting, so did the area of the evoked CTM reflex: this implies a recruitment of CTM motoneurons. We interpret this "matching" of response to stimulus as an adaptive phenomenon ensured by an adaptive intraspinal sprouting of the nociceptive projections. Neither the intraspinal changes nor the reflex changes occurred if peripheral sprouting was blocked by systemic anti-NGF treatment, indicating that the role of endogenous NGF was only in that sprouting. No comparable adaptive events occurred during NGF-induced hyperalgesia. Neither nociceptive fields nor CTM reflexes were affected; however there was a recruitment of c-Fos-expressing interneurons. This recruitment was not explained by peripheral sensitization, and, because sprouting was not involved here, we attribute the recruitment to "synaptic unmasking," i.e., an increased effectiveness of the preexisting excitatory circuitry.

Adaptation, Physiological↗
PubMed1999

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-D-aspartate receptor-like properties.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

Animals↗
PubMed1999

PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein.

We recently reported the isolation and partial characterization of two novel proteins, MAP17 and PDZK1. Using in situ hybridization, we demonstrated that MAP17 and PDZK1 mRNAs are markedly up-regulated in human carcinomas. PDZK1, originally isolated as a protein interacting with MAP17, contains four PDZ protein-interaction domains and could potentially interact with as many as four target proteins. In this paper, we confirm the overexpression of PDZK1 in human carcinomas using a specific antibody and demonstrate the localization of the PDZK1 gene to human chromosome 1q21, a region frequently altered in neoplastic conditions. Using the yeast two-hybrid system, we have also determined that PDZK1 interacts with the carboxy-terminal portion of cMOAT (MRP2), the canalicular multispecific organic anion transporter associated with multidrug resistance. This is of particular interest because proteins containing PDZ domains are involved in the clustering and signaling pathways of membrane-associated proteins, including ion channels. Therefore, the protein cluster formed by the association of cMOAT, PDZK1, and MAP17 could play an important role in the cellular mechanisms associated with multidrug resistance, and PDZK1 may represent a new target in cancer cells resistant to chemotherapeutic agents.

ATP-Binding Cassette Transporters↗
PubMed1998

Cloning of a brain N-methyl-D-aspartate- and D, L-epsilon-2-amino-4-propyl-5-phosphono-3-pentanoic acid (CGP 39653)-binding protein.

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.

2-Amino-5-phosphonovalerate↗
PubMed1998

Identification of novel transmembrane gene sequence and its use for cell-surface targeting of beta subunit of human chorionic gonadotropin.

We identified a 685-nucleotide gene fragment that codes for the transmembrane and cytoplasmic domains of glycoprotein of the LEP strain rabies virus and carried out experiments designed to express a novel fusion protein on the cell surface. The cDNA encoding the membrane anchor sequence was fused in the correct reading frame to the 3' end of the cDNA encoding the beta subunit of human chorionic gonadotropin (beta(h)CG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine being developed in our laboratory. The fusion gene cassette was placed under the control of a vaccinia virus early promoter and cloned in a host-restricted fowlpox viral vector. The recombinants, when used to infect mammalian cells that do not allow the replication of fowlpox virus, expressed the N-terminal 135 amino acid residues of beta(h)CG anchored in the cell membrane by the 75-amino acid C-terminal sequence derived from rabies virus glycoprotein. This hybrid protein is correctly processed post-translationally and transported efficiently to the plasma membrane of non-permissive cells such that the anchored beta(h)CG molecule retains the correctly folded native antigenic epitope(s).

Amino Acid Sequence↗
PubMed1997

Inhibition of HIV-1 infection by the beta-chemokine MDC.

CD8(+) T lymphocytes from individuals infected with human immunodeficiency virus-type 1 (HIV-1) secrete a soluble activity that suppresses infection by HIV-1. A protein associated with this activity was purified from the culture supernatant of an immortalized CD8(+) T cell clone and identified as the beta-chemokine macrophage-derived chemokine (MDC). MDC suppressed infection of CD8(+) cell-depleted peripheral blood mononuclear cells by primary non-syncytium-inducing and syncytium-inducing isolates of HIV-1 and the T cell line-adapted isolate HIV-1IIIB. MDC was expressed in activated, but not resting, peripheral blood mononuclear cells and binds a receptor on activated primary T cells. These observations indicate that beta-chemokines are responsible for a major proportion of HIV-1-specific suppressor activity produced by primary T cells.

Amino Acid Sequence↗