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Biomedical subjects

A Thakur

Publications and source records attributed to A Thakur.

At least 73 records · Page 4Linked to original sources
PubMed1995

Cloning of the gene for phosphoglycerate kinase from Schistosoma mansoni and characterization of its gene product.

As molecules on the surface or associated with the outer covering (tegument) of Schistosoma mansoni are a major focus as potential vaccine candidates, affinity purified antibodies which are specific to the tegumental antigens were used to immunoscreen a lambda gt11 S. mansoni cercarial cDNA library. One of the identified clones was found to encode the glycolytic enzyme phosphoglycerate kinase (PGK, EC 2.7.2.3). The 1.5-kb cDNA clone has a single open reading frame encoding 416 amino acids and exhibits over 60% identity to PGKs from a number of eukaryotic species. Recombinant S. mansoni PGK (SmPGK) was overexpressed in Escherichia coli, purified, and shown to have PGK enzyme activity. Native protein affinity purified from S. mansoni adult worms was shown by microsequencing to have the same amino-acid sequence as deduced from the cDNA sequence, thus confirming the cDNA clone we identified encodes S. mansoni phosphoglycerate kinase. Antibodies localize the native SmPGK to various tissues including the tegument of 3-h schistosomula and 42-day adult worms.

Amino Acid Sequence↗
PubMed1995

Rhabdomyosarcoma of the oral cavity. Report of four cases.

Clinical and pathologic findings of four cases of rhabdomyosarcoma of the oral soft tissues are described that include findings from immunohistochemistry and electron microscopy. Three cases occurred in children under 16 years of age and one in a 22-year-old. Included is a brief discussion on reported gene abnormalities that may contribute to neoplastic development.

Adolescent↗
PubMed1995

Immune response to Schistosoma mansoni phosphoglycerate kinase during natural and experimental infection: identification of a schistosome-specific B-cell epitope.

A cDNA clone encoding Schistosoma mansoni phosphoglycerate kinase (SmPGK) was previously identified by affinity-purified antibodies which are specific for 3-h-old schistosomula tegumental antigens. Antibodies to the recombinant SmPGK which has enzymatic activity were localized to various tissues including the tegument of the 3-h-old schistosomula and 42-day-old adult worms. In this study, we show that SmPGK is an immunogenic molecule in both natural infection in humans and experimental vaccination in animals. To understand the role that a highly conserved molecule like SmPGK played during schistosome infection, we affinity purified antibodies to SmPGK from patients with chronic schistosomiasis and demonstrated that they did not cross-react with human PGK. However, affinity-purified rabbit anti-SmPGK antibodies did show immunoreactivity to both human PGK and rabbit PGK. Thus, during natural infection antibodies that cross-react with human PGK are not produced; however, as a result of active immunization with an intact conserved molecule, such cross-reacting antibodies are produced. Immunological analysis of cyanogen bromide digests of SmPGK with monoclonal antibodies that recognize SmPGK but not human PGK identifies a B-cell epitope on a 12.2-kDa fragment represented by amino acids 61 to 174.

Animals↗
PubMed1994

Invertebrate host-parasite relationships: convergent evolution of a tropomyosin epitope between Schistosoma sp., Fasciola hepatica, and certain pulmonate snails.

Monoclonal antibodies (mAb) directed against Schistosoma mansoni tropomyosin isoform, SMTM (Xu et al. Experimental Parasitology 69, 373-392, 1989), were used to test for cross-reactivity with Biomphalaria glabrata antigens. One mAb (1F10) recognized antigens of 39, 41, and 80 kDa in a snail head/foot antigen preparation but not a hepatopancreas antigen preparation. Another mAb (1C1) cross-reacted with a 39-kDa antigen in the head/foot extract but not in the hepatopancreas extract. Epitope mapping revealed the 1F10 epitope to be between amino acids 135 and 188 of both Bg39 (Dissous et al. Molecular and Biochemical Parasitology 43, 245-256, 1990) and BgTMII (Weston and Kemp, Experimental Parasitology 76, 358-370, 1993), while the 1C1 epitope was located between amino acids 189 and 213 of BgTMII. Various invertebrate species, including members from Trematoda, Pulmonata, Annelida, and Arthropoda, were tested for cross-reactivity with the monoclonal antibodies. While the 1F10 mAb displayed broad invertebrate cross-reactivity, the 1C1 mAb cross-reactivity was restricted to schistosomes, F. hepatica, and the pulmonate snails B. glabrata and Physa sp.

Amino Acid Sequence↗
PubMed1993

Mimicry of the a determinant of hepatitis B surface antigen by an antiidiotypic antibody. I. Evaluation in hepatitis B surface antigen responder and nonresponder strains.

B and T cell responses of several strains of mice, immunized with a monoclonal antiidiotype (anti-Id) that mimics the a determinant of hepatitis B surface antigen (HBsAg), were studied to determine if the immune response to the anti-Id was regulated by H-2-linked immune response genes as has been previously observed for HBsAg. We report that immunization with anti-Id could elicit HBsAg-specific antibodies in mice of the H-2d,q, or f haplotype and in an outbred wild mouse strain (Mus spretus), thus circumventing the H-2 haplotype restriction pattern observed when immunizing with HBsAg in H-2f mice. Purified lymph node T cells from mice of the H-2d or q haplotype and M. spretus that were primed in vivo with HBsAg or anti-Id could be stimulated in vitro with either HBsAg or anti-Id but not with an irrelevant antibody of the same subclass as the anti-Id. However, purified lymph node T cells from H-2f mice that were primed in vivo with the anti-Id could only be stimulated in vitro with anti-Id. No in vitro stimulation whatsoever was observed in H-2f mice immunized with HBsAg. The effect of processing and presentation of the anti-Id by antigen-presenting cells (APC) was studied in mice of the H-2d haplotype. Stimulation of purified lymph node T cells by HBsAg and anti-Id was shown to be strictly dependent on APC and restricted by major histocompatibility complex class II antigens at the I-A locus. Treatment of APC with paraformaldehyde or chloroquine abrogated the T cell response to all antigens except for a nine-amino acid synthetic peptide representing a partial analogue of the group a determinant of HBsAg S(139-147). The significance of these results is discussed in the context of understanding the mechanism of mimicry elicited by the anti-Id.

Amino Acid Sequence↗
PubMed1993

Schistosoma mansoni: a Cu/Zn superoxide dismutase is glycosylated when expressed in mammalian cells and localizes to a subtegumental region in adult schistosomes.

We previously isolated a gene from Schistosoma mansoni with the capacity to encode a 20-kDa polypeptide that had homology to Cu/Zn superoxide dismutase from 19 other species. The predicted protein sequence contained a hydrophobic N-terminus as well as a site for N-linked glycosylation, suggesting that the protein was a secreted or membrane-associated form of the enzyme. To study this protein further, we first expressed it in a prokaryotic system and used the gene product to make both monoclonal and polyclonal antibodies. We then expressed the protein in CMT3 cells, a monkey kidney cell line, to investigate possible post-translational modifications. Our results demonstrated that the schistosomal protein expressed in CMT3 cells migrated on an SDS-polyacrylamide gel as multiple glycosylated species with molecular masses of 20, 22, and 23 kDa. Glycosylation was inhibited by tunicamycin, resulting in the expression of an unglycosylated product which migrated with a molecular mass of 18 kDa. The CMT3-cell expressed protein eluted from a gel filtration column with a molecular mass larger than 200 kDa, suggesting that it was membrane-associated or bound to a high-molecular-weight component. The product could not be detected in the medium of the CMT3 cell culture and apparently was not secreted. Comparison between the protein expressed in CMT3 cells and that found in adult schistosomes showed that the parasite-derived gene product was also heterogeneous but had different molecular masses (20, 23, and 25 kDa). The protein was localized in frozen sections of adult worms to the subtegumental area as detected by indirect immunofluorescence using monoclonal antibodies. Since the protein was glycosylated but not secreted we suggest it be called signal peptide-containing superoxide dismutase.

Animals↗
PubMed1993

Serum protein polymorphisms and malaria in Madya Pradesh, India.

The relationship of serum protein polymorphisms to the presence of malaria antibodies was studied in 473 muria gond tribal subjects from Bastar district, Central India, an area endemic for both P. falciparum and P. vivax infection. A control group of 100 subjects in Delhi, which has a low prevalence of malaria, was also studied. Serum proteins (transferrin, haptoglobin and albumin) were analyzed for polymorphic variants by starch gel electrophoresis. Malarial antibodies were assayed by enzyme linked immunosorbent assay (ELISA), while thin blood films were screened for the presence of malaria parasites. Among serum proteins transferrin CD variant showed significant correlation with malarial infection. There were no significant differences observed between Hp1 and Hp2 variants of haptoglobin in relation to presence of malarial antibodies. Statistical analysis for albumin variants was not attempted because the number of individuals showing abnormal bands was small.

Adolescent↗
PubMed1992

Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.

Amino Acid Sequence↗
PubMed1992

Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni.

Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.

Amino Acid Sequence↗
PubMed1992

Malaria and ABO blood groups.

Subjects from Muria gond tribal community (n = 258) as well as from Delhi (n = 100) were classified according to ABO blood groups, and were also assayed for malarial antibodies by ELISA technique. The distribution of ABO blood groups did not differ significantly in Muria gonds and Delhi subjects. Within Muria gonds the observed frequency of ABO blood groups did not differ significantly from the expected values. No significant difference was observed in the rate of seropositivity for malarial antibodies among subjects with different blood groups. Malarial parasitaemia, although observed more in individuals with blood group A, did not differ significantly as compared with other blood groups. We conclude that ABO blood groups do not show differential susceptibility to malaria.

ABO Blood-Group System↗
PubMed1992

Interaction of malarial infection and glucose-6-phosphate dehydrogenase deficiency in Muria gonds of district Bastar, central India.

Muria gond tribals (n = 473) from district Bastar, central India, an area known to be hyperendemic for malaria, were investigated for malarial infection and glucose-6-phosphate dehydrogenase deficiency. The frequency of G-6-PD deficiency was 21.3% among male subjects and 3.7% among females. Assay of malarial antibodies showed that seropositivity as well as the level of antibodies was significantly higher in male subjects with normal enzyme levels as compared with males G-6-PD deficiency. Females with normal G-6-PD enzyme levels too had higher seropositivity as well as level of antibodies against malaria as compared with females having G-6-PD deficiency. This suggests that G-6-PD deficiency correlates with a higher degree of resistance.

Antibodies, Protozoan↗
PubMed1990

Rejection of mouse sarcoma cells after transfection of MHC class II genes.

Th cells are stimulated by peptide Ag presented in the context of MHC class II molecules. We have reasoned that immune responses against tumors may be more efficient if tumor cells were class II Ag positive, and thereby able to directly function as APC to stimulate tumor-specific Th cell proliferation. We have tested this hypothesis by using DNA-mediated gene transfer to generate syngeneic MHC class II Ag-expressing mouse Sal sarcoma cells (Sal/Ak transfectants). Autologous A/J mice challenged i.p. or s.c. with Sal/Ak transfectants do not develop tumors, whereas A/J mice challenged with the class II negative parental Sal tumor have a high tumor incidence. Furthermore, immunization of the autologous host with Sal/Ak transfectants completely protects against subsequent challenge with wild-type Sal cells. MHC class II-expressing tumor cells, therefore, stimulate an improved tumor-specific immune response, and the immunity is cross-reactive with the class II negative tumor. Inasmuch as the transfected MHC class II gene product is not functioning as a target molecule for autologous tumor rejection, the improved immunogenicity of the Sal/Ak cells is probably due to stimulation of a tumor-specific Th cell population. The increased immunogenicity of Sal/Ak cells is, therefore, probably the result of direct presentation of Sal tumor-associated Ag in the context of tumor cell MHC class II molecules to Th lymphocytes. These studies demonstrate that induction of tumor cell MHC class II Ag expression is a potential strategy for tumor-specific immunotherapy, and suggest that tumor immunity may be enhanced by improved Th cell generation.

Animals↗
PubMed1989

Interferons as gene activators. Indications for repeated gene duplication during the evolution of a cluster of interferon-activatable genes on murine chromosome 1.

We have described earlier a gene cluster, including at least six interferon-activatable genes closely linked to the erythroid alpha spectrin locus and the serum amyloid P-component locus on murine chromosome 1. Here, we report that sequences of three genes from the cluster (the 201, 202, and 204 genes) are very similar in a segment extending from at least 550 nucleotides upstream of the 3' end of the transcription initiation region to beyond the first exon intron border (96% similarity between the 202 and 204 genes and 89% similarity between the 201 and 204 genes). This region contains the following two types of interferon-responsive enhancers: a GA box and a Friedman Stark sequence. The proteins coded for by the 202 gene (51 kDa) and the 204 gene (72 kDa) are hydrophilic. The amino acids have been conserved in the two proteins in 47% of the sequence. Each protein includes two apparently contiguous, approximately 200-amino acid long segments with much sequence similarity (27% in the 202 protein and 34% in the 204 protein). These segments are preceded in the 204 protein only by a segment including four perfect and three imperfect repeats of a 7 amino acid sequence. These and other data suggest that the evolution of the gene cluster involved the duplication of a DNA segment generating a double length transcription unit and subsequent divergence and duplication of this unit giving rise to at least two interferon-activatable genes (the 202 and 204 genes).

Amino Acid Sequence↗
PubMed1986

Interferons as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene.

Treatment of cells with interferons induces various mRNAs and the corresponding proteins. We have described previously the isolation of a mouse cDNA clone (cDNA clone 202) which specifies an mRNA whose level is increased 20-fold in beta-interferon-treated Ehrlich ascites tumor cells. The increase is a consequence of an increased rate of transcription. The mRNA encodes a 56,000-dalton protein. We report here the isolation of a genomic clone including the 5' terminus of the 202 gene with the interferon-responsive region. Experiments involving primer extension and protection from cleavage by S1 nuclease revealed the existence of multiple 5' termini of 202 mRNAs in Ehrlich ascites tumor and Ltk- cells. Treatment with beta-interferon increased the level of these 202 mRNAs with different 5' termini nonuniformly. A 0.8-kilobase DNA segment from the 202 gene (including its 5' flanking region and its 5'-terminal exon) was ligated to the chloramphenicol acetyltransferase gene, and the resulting construct was transfected into mouse Ltk- cells. Treatment of these cells with beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Within the first, untranslated exon of the 202 gene, we found a 29-nucleotide long sequence that is partially homologous to sequences which occur upstream from interferon-inducible human HLA and metallothionein IIA genes (Friedman, R. L., and Stark, G. R. (1985) Nature 314, 637-639).

Acetyltransferases↗
PubMedDate not supplied

Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-gamma-interferon antibody.

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.

Amino Acid Sequence↗
PubMedDate not supplied

Comparison of potential pathogenic traits of staphylococci that may contribute to corneal ulceration and inflammation.

Staphylococcus epidermidis and Staphylococcus aureus are two of the commonest bacteria isolated from corneal ulcers. The aim of the current investigation was to determine the frequency of potentially pathogenic traits in the two staphylococcal species. Strains of both species, some isolated from eyes during active corneal inflammation, were screened for their ability to degrade a variety of proteins and hyaluronic acid and the production of cytotoxins. S. aureus produced more tissue-destructive enzymes than the S. epidermidis. S. aureus strains more commonly synthesized the cytotoxin, alpha-toxin. The production of elastase was more common among S. aureus strains isolated from ulcerative events. The production of alpha-toxin was inversely correlated with isolation from keratitis. This study has demonstrated that S. aureus is better equipped with a range of potentially damaging enzymes and toxins than S. epidermidis and that S. aureus elastase may be involved in the production of corneal ulcers.

Bacterial Toxins↗