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Biomedical subjects

R Pal

Publications and source records attributed to R Pal.

At least 109 records · Page 6Linked to original sources
PubMed1989

Monoclonal antibodies to HTLV-III451 gp41: delineation of an immunoreactive conserved epitope in the transmembrane region of divergent isolates of HIV-1.

We report on the development of monoclonal antibodies directed against the transmembrane portion of the envelope of HTLV-III451 gp41. One of these monoclonal antibodies, designated M71/2B4, was found to cross-react with transmembrane proteins from other independent isolates of HIV-1, namely IIIB, MN, and RF. Thus, this monoclonal antibody identifies an epitope located in a region of gp41 that is conserved among all these isolates. To identify this conserved region a series of E. coli recombinant proteins were screened in immunoblot with M71/2B4. From these results the epitope recognized by this antibody appears to map at the amino terminus of gp41, in the region indicated between the cleavage site with gp120 (aa 508) and the HindIII site (aa647).

Animals↗
PubMed1989

Effect of cadmium on lipid peroxidation and antioxidant enzymes in undernourished weanling rat brain.

The effect of early postnatal cadmium exposure on lipid peroxidation and antioxidant enzymes in undernourished weanling rat brain has been studied. The results suggest that undernutrition makes the weanling rat brain more susceptible to the neurotoxic effects of cadmium. Cadmium at a low dose of 1 mg/kg body weight did not produce any changes in lipid peroxidation and antioxidant enzymes in normal weanling rat brain, but caused a significant increase in lipid peroxidation and markedly decreased the activities of antioxidant enzymes like glutathione peroxidase, superoxide dismutase and catalase when subjected to undernutrition.

Animal Nutritional Physiological Phenomena↗
PubMed1989

Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin.

The processing and secretion of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected cells treated with the trimming glucosidase inhibitor deoxynojirimycin (DNM). In Molt3 cells infected with human T-lymphotropic virus type III (HTLV-IIIB), DNM inhibited the intracellular proteolytic processing of gp160 to gp120 and gp41. A clone of the HUT78 cell line called 6D5, when chronically infected with the HIV-1 isolate HTLV-III451 was shown to release both gp160 and gp120 into the culture medium. The secretion of envelope glycoproteins from these infected cells was not inhibited by DNM treatment. The secreted proteins had higher molecular weights than gp160 and gp120 from cultures not treated with DNM, presumably due to the presence of unprocessed carbohydrate residues on the polypeptide chain. These secreted glycoproteins from DNM-treated cells exhibited specific interaction with the CD4 molecule on the surface of target cells. However, the syncytium formation induced by HIV-1-infected cells on CD4+ cells was significantly inhibited in the presence of the glucosidase inhibitor. The minimal cytotoxicity of the DNM coupled with its strong inhibitory effect on the cell-to-cell spread of the virus suggest that it may be potentially useful in antiviral drug therapy of HIV-1 infection.

1-Deoxynojirimycin↗
PubMed1989

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Adolescent↗
PubMed1989

Vaccine for control of fertility.

Birth control vaccines constitute a new category of vaccines. Immunization with the objective of selectively blocking a physiological process differs in many respects from immunizing against pathogens. Conceptually, these widen the orbit of therapeutic intervention by immunological methods. Success recorded in making vaccines regulating fertility offers models to regulate any other physiological process in the body. At a practical level, the task of making such vaccines is beset with inherent difficulties and with new challenges. This article, dedicated to Avrion Mitchison, aims to discuss these problems and to record successes wherever achieved.

Family Planning Services↗
PubMed1988

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes.

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.

Cell Line↗
PubMed1988

Processing of the structural proteins of human immunodeficiency virus type 1 in the presence of monensin and cerulenin.

The synthesis and processing of structural proteins of human immunodeficiency virus type 1 (HIV-1) were studied in infected cells treated with monensin and cerulenin. In MOLT-3 cells chronically infected with HTLV-IIIB, monensin inhibited the proteolytic cleavage of the env-coded polyprotein gp160 to gp120, leading to the accumulation of the precursor gp160. The formation of syncytia normally observed when CEM cells are cocultivated with HIV-1-infected MOLT-3 cells was significantly inhibited in the presence of monensin. The effect of the ionophore on the culture was reversible, as withdrawal of monensin from the medium restored the ability of the cells to form syncytia with CEM cells and led to the resumption of the processing of gp160 to gp120. Monensin did not affect the synthesis and processing of gag-coded proteins and regulatory proteins. Cerulenin, an inhibitor of de novo fatty acid biosynthesis, inhibited the myristoylation and the proteolytic cleavage of the gag-coded polyprotein Pr53gag to p24 but did not affect the processing of gp160. However, use for monensin and cerulenin as antiviral agents for treatment of HIV-1 infection cannot be foreseen because of the pronounced in vitro toxicity observed.

Antifungal Agents↗
PubMed1988

A unique human immunodeficiency virus culture secreting soluble gp160.

A clone of the HUT78 cell line, chronically infected with the HIV-1 isolate HTLV-III451, has been demonstrated to secrete unprocessed HIV-1 envelope precursor protein gp160 as well as mature gp120. Further, when grown in serum-free defined medium these cells released approximately five times the amount of virus compared with cultures in normal medium. These proteins corresponded in their immunologic reactivities with the respective envelope proteins of the HTLV-IIIB isolate. They formed high-affinity soluble complexes with the CD4 antigen and inhibited the syncytium formation induced by HTLV-IIIB on CD4-positive cells. This is the first description of an HIV-1 culture system capable of shedding into the medium native gp160 that is soluble in the absence of detergents.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Antigenicity, function, and conformation of synthetic oligopeptides corresponding to amino-terminal sequences of wild-type and mutant matrix proteins of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) has a major antigenic determinant (epitope 1) that maps to a region extending from amino acids 19 through 43 and transcription-inhibition activity that maps to the first 43 N-terminal amino acids (J.R. Ogden, R. Pal, and R. R. Wagner, J. Virol. 58:860-868, 1986). The M protein of temperature-sensitive mutant tsO23(III) is devoid of epitope 1 and transcription-inhibition activity and substitutes glutamic acid for glycine at amino acid 21 as well as having amino acid substitutions at positions 111 and 227 (K. Morita, R. Vanderoef, and J. Lenard, J. Virol. 61:256-263, 1987). We undertook to map more precisely epitope 1 and the transcription-inhibition region of VSV M protein by means of synthetic oligopeptides generated by an automated solid-phase protein synthesizer. A pentadecapeptide designated PI(wt, Gly21), corresponding to amino acids 17 to 31 of wild-type (wt) M protein, strongly bound monoclonal antibody MAb2 (directed to epitope 1); however, an analogous pentadecapeptide with glutamic acid substituted for glycine at position 21, designated PII(tsO23, Glu21), completely failed to recognize MAb2. Polyclonal antibody raised in rabbits immunized with PI(wt, Gly21) reacted strongly with wt M protein, the homologous pentadecapeptide, and, to a lesser extent, PII(tsO23, Glu21). Anti-PII(tsO23, Glu21) failed to recognize PI(wt, Gly21) or wt M protein. Anti-PI(wt, Gly21) competed efficiently for binding of MAb2 to wt M protein and was as effective as MAb2 in reversing inhibition of VSV transcription by wt M protein. Neither PI(wt, Gly21) nor PII(tsO23, Glu21) exhibited any ability to inhibit VSV transcription. However, a lysine-rich oligopeptide, PII(Met1-Leu20), corresponding to the first 20 N-terminal amino acids of wt M protein, and polylysine itself did inhibit VSV transcription, albeit much less efficiently than native wt M protein. Monospecific polyclonal antibody directed to the 20-mer oligopeptide PIII(Met1-Leu20) reversed transcription inhibition by M protein in a dose-dependent manner almost identical to that of anti-PI(wt, Gly21) and epitope 1-specific MAb2. Examination by circular dichroism spectropolarimetry revealed significant differences in the conformation of the two pentadecapeptides attributable to the Gly in equilibrium Glu amino acid substitution at position 21.

Amino Acid Sequence↗
PubMed1987

Hemagglutination patterns of Pseudomonas aeruginosa strains of different serogroups, Habs and Fisher types.

Pseudomonas aeruginosa strains of sixteen Habs and three Fisher types were examined for their hemagglutination activity using vertebrate erythrocytes including human ones. Their carbohydrate-binding specificity as determined by hemagglutination-inhibition suggests a possible method of their purification. This study also indicates the possibility of an occurrence of multiple lectins of different specificity.

Hemagglutination Inhibition Tests↗
PubMed1987

Functional and antigenic domains of the matrix (M1) protein of influenza A virus.

The membrane- and ribonucleocapsid (RNP)-binding domains of the matrix (M1) protein of influenza A virus (WSN strain) were partially mapped and characterized by reactivity with monoclonal antibodies (MAb) as well as by proteolytic cleavages and amino acid sequencing of the resulting peptides. Of two peptides formed by formic acid hydrolysis, a 9-kilodalton fragment at the amino-terminal third of the M1 protein was recognized by MAb M2-1C6 (to epitope 1), and a 15-kilodalton fragment at the carboxy-terminal two-thirds was recognized by MAb 289/4 (to epitope 2). Partial cleavage by staphylococcal V8 protease gave rise to a 16-kilodalton peptide, mapping to amino acid 8, which was recognized by MAbs to all three epitopes but rather weakly by MAb 904/6 to epitope 3. These studies suggest that epitope 1 of the M1 protein resides between amino acids 8 and 89, whereas epitopes 2 and possibly 3 are located between amino acids 89 and 141 or somewhat more carboxy distal. The intact M1 protein and its N-terminal 9- and 10-kilodalton peptides generated by formic acid or V8 protease cleavage, respectively, reconstituted with dipalmitoylphosphatidylcholine vesicles, but these N-terminal peptides had little effect on in vitro transcription of the RNP core. In sharp contrast, both intact M1 protein and the C-terminal 15-kilodalton formic acid fragment were able to inhibit viral transcription markedly. Moreover, MAb 289/4 (to epitope 2) reversed this inhibited transcription significantly. These studies suggest that the lipid-binding domain of the M1 protein is located within the amino-terminal third, whereas the site involved in the interaction of the M1 protein with RNP cores is located within the carboxy-terminal two-thirds.

Amino Acid Sequence↗
PubMed1986

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies.

The matrix (M) protein of vesicular stomatitis virus (VSV) appears to function as a bridge between the ribonucleocapsid (RNP) core and the envelope in assembly of the virion. Two such properties would necessitate at least one site for interaction with the nucleocapsid and one with the envelope. In this study M protein was found to mediate the in vitro binding to RNP cores of phospholipid vesicles, representing membrane structures. The M protein could bind initially to either the vesicles or the RNP cores to promote RNP-vesicle association. A trypsin-resistant fragment (MT) of M protein, missing the initial 43 amino acids from its amino terminus, reconstituted with acidic phospholipid vesicles with the same binding efficiency as did whole M protein, suggesting that the carboxy-terminal 81% retained those regions of the M protein which interact with a lipid bilayer. The MT protein, however, was considerably less efficient than intact M protein as an inhibitor of in vitro virus transcription; almost 2.5-fold more MT protein than intact M protein was required for 50% inhibition of VSV transcription, indicating that a site for interaction with the RNP core may have been lost. A monoclonal antibody which is able to reverse the in vitro inhibition of transcription by M protein did not react by immunoblotting with MT protein. Partial tryptic digests of the M protein probed with this monoclonal antibody indicated that epitope 1 lies between amino acid residues 18 and 43. This region appears to be a site that promotes interaction of the M protein with the RNP core of VSV. Monoclonal antibodies to epitopes 2 and 3, which exhibit some overlap in binding to M protein but do not reverse transcription inhibition, were mapped by cleavage with N-chlorosuccinimide at regions in a carboxy direction from epitope 1.

Animals↗
PubMed1985

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues.

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid Bilayers↗
PubMed1985

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes.

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Monoclonal antibodies to the matrix protein of vesicular stomatitis virus (New Jersey serotype) and their effects on viral transcription.

Of 33 hybridomas raised by immunization of BALB/c mice with the matrix (M) protein of the New Jersey serotype of vesicular stomatitis virus (VSV), 17 secreted monoclonal antibodies (mAb) of the IgG isotype and, unexpectedly, 16 of the IgM isotype. All these monoclonal antibodies bound strongly to VSV-New Jersey M protein by ELISA, immunoprecipitation, and immunoblotting assays, but exhibited only slight or no cross-reactivity with the M protein of VSV-Indiana. Four antigenic determinants of VSV-New Jersey M protein could be identified by competitive binding of 125I-labeled monoclonal antibodies but three of these epitopes exhibited partial overlap. Monoclonal antibodies to two epitopes reversed the inhibitory effect of M protein on in vitro transcription of VSV-New Jersey ribonucleoprotein. However, monoclonal antibodies to the other two epitopes had little effect on M-protein transcription inhibition but actually increased significantly the transcriptional inhibitory effect of M protein under certain experimental conditions. Monoclonal antibodies to all four epitopes reacted strongly with the M protein of the tsC1 mutant of VSV-New Jersey which is restricted in transcription inhibition.

Animals↗
PubMed1985

Monoclonal antibodies to the M protein of vesicular stomatitis virus (Indiana serotype) and to a cDNA M gene expression product.

Twenty-nine independent hybridomas producing monoclonal antibodies to the matrix (M) protein of vesicular stomatitis virus (Indiana serotype) were prepared by fusion of SP2/0 myeloma cells with spleen lymphocytes obtained from BALB/c mice which had been immunized with the purified M protein. The specific reactivity of each monoclonal antibody was determined by an enzyme-linked immunosorbent assay and a competitive binding assay. Most of the antibodies were of the immunoglobulin G2a and G2b isotypes, although some were immunoglobulin M. By measuring the competitive binding of 125I-antibody, we identified four antigenic determinants in the M protein of the virus; two of these determinants, however, exhibited a large degree of overlap. Western blot analysis revealed little or no cross-reactivity of the antibodies with other viral proteins or with the M protein of the New Jersey serotype. Prolonged trypsin proteolysis removed the first 43 amino acids from the amino-terminal region of the M protein, but it retained its reactivity with monoclonal antibodies to each epitope, except for diminished reactivity with one. To aid in future mapping of these epitopes, we inserted a cDNA clone of the mRNA encoding the M protein of vesicular stomatitis virus into an inducible lac expression vector; the M protein produced in the JM103 strain of Escherichia coli under induced conditions was found to be approximately the same size as native M protein and was recognized by the monoclonal antibodies. These monoclonal antibodies and the cDNA clone should be useful for studying the role of M protein in virus maturation and the regulation of viral transcription.

Animals↗
PubMed1985

Regulation of viral transcription by the matrix protein of vesicular stomatitis virus probed by monoclonal antibodies and temperature-sensitive mutants.

The ability of the matrix (M) protein of wild-type vesicular stomatitis virus (VSV) to regulate viral transcription was studied with monoclonal antibodies and temperature-sensitive (ts) mutants in complementation group III, the M proteins of which are restricted in transcription inhibition. The marked inhibition of transcription by VSV ribonucleoprotein (RNP) cores complexed with M protein (RNP/M) was reversed by antibody to epitope 1. Antibodies to epitopes 2 and 3 not only failed to reverse the transcription-inhibitory activity of isolated M protein but actually increased M-protein inhibition of transcription in a reconstituted system. Monoclonal antibodies to epitopes 2 and 3 strongly bound to M proteins from all wild-type and ts-mutant virions, but monoclonal antibody to epitope 1 completely failed to bind to the M protein of ts023(III) even though it reacted strongly with M proteins of mutants tsG31(III) and tsG33(III). The M protein of a tsO23 revertant (R11) completely recovered its capacity to inhibit transcription and to bind monoclonal antibody to epitope 1, whereas the M proteins of three other revertants remained restricted in their capacity to inhibit transcription and to bind monoclonal antibody to epitope 1. These studies indicate that exposure of epitope 1 on the surface of M protein is essential for inhibiting transcription by VSV RNP cores.

Antibodies, Monoclonal↗