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A M Karim

Publications and source records attributed to A M Karim.

18 recordsLinked to original sources
PubMed1998

Characterization of Sm20.8, a member of a family of schistosome tegumental antigens.

Two cDNA clones each encoding a 20.8-kDa protein (Sm20.8) were identified from the human blood fluke Schistosoma mansoni sporocyst and adult worm cDNA expression libraries by antibodies derived from rabbits vaccinated with irradiated cercariae and purified over an NP-40 extract of 3h schistosomula. Each identified cDNA has an open reading frame encoding a protein of 181 amino acids and shows homology (29-30%) with Sm21.7, Sm22.6, and Sj22.6, previously identified as belonging to a family of soluble schistosome tegumental antigens. An EF-hand calcium-binding motif is found in Sm20.8 protein in two different positions. However, neither motif binds 45calcium (45Ca) Recombinant Sm20.8 showed immunoreactivity with sera from infected humans and rabbits vaccinated with irradiated cercariae. Polyclonal rabbit sera against the Sm20.8 recognized the native protein in an extract of infected snail (sporocyst), cercariae, 3 hour schistosomules (3 h NP-40) and an adult worm preparation but not in uninfected snail tissue or eggs. Further demonstration that Sm20.8 was expressed in the different developmental stages of the parasite was by RT-PCR. Confocal microscopy demonstrates that Sm20.8 localizes to the tegument of adult worms and 3 h np-40. The IgG fraction specific to Sm20.8 mediated complement killing of schistosomules in vitro by 34%. Vaccination of mice with naked DNA containing the Sm20.8 gene and subsequently challenged with cercariae showed 30% reduction in worm burden compared to controls.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme.

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.

Amino Acid Sequence↗
PubMed1997

Reduced effects of L-carnitine on glucose and fatty acid metabolism in myocytes isolated from diabetic rats.

Depressed glucose utilization and over-reliance of muscle tissues on fat represents a major metabolic disturbance in diabetes. This study was designed to investigate the relationship between fatty acid oxidation and glucose utilization in diabetic hearts and to examine the role of L-Carnitine on the utilization of these substrates in diabetes. 14CO2 release from [1-14C]pyruvate (an index of PDH activity), [2-14C]pyruvate and [6-14C]glucose (an index of acetyl-CoA flux through the Krebs cycle), [U-14C]glucose (an index of both PDH and acetyl-CoA flux through the Krebs cycle), and [1-14C]palmitate oxidation were studied in cardiac myocystes isolated from normal and streptozotocin-injected rats. Palmitate oxidation was increased twofold in diabetic myocytes compared to normal cells (5.4 +/- 1.45 vs 2.35 +/- 0.055 nmol/mg protein/30 min, p > 0.05). L-Carnitine (5 mM) significantly increased palmitate oxidation (60-70%) in normal cells but had no effect on diabetic cells. The activity of PDH and acetyl-CoA flux through the Krebs cycle was severely depressed in diabetes (58.14 +/- 20.27 and 8.63 +/- 0.62 in diabetes vs 128.75 +/- 11.47 and 24.84 +/- 7.81 nmol/mg protein/30 min in controls, p > 0.05, respectively). The efflux of acetylcarnitine, a by-product of PDH activity was also much lower in diabetic cells than in normal cells but had no effect in diabetes. L-Carnitine also had no effect on 14CO2 release from [U-14C]glucose but significantly decreased that from [6-14C]glucose, which reflects oxidative metabolism suggesting that L-Carnitine decreases oxidative glucose utilization. Thus, these data suggest that the overreliance on fat in diabetes may be in part secondary to a reduction of carbohydrate-generated acetyl-CoA through the Krebs cycle.

Acetyl Coenzyme A↗
PubMed1996

Cloning the gene encoding Schistosoma mansoni p50, an immunophilin.

A 2.2-kb fragment of genomic DNA encoding Schistosoma mansoni immunophilin p50 (Smp50) was identified on a 14-kb genomic clone. The sequence of Smp50 reveals seven exons interrupted by six small introns ranging from 28-35 bp in size. The transcription start point, defined by primer extension analysis of schistosome RNA, begins at 30 bp upstream from the start AUG codon. Smp50 lacks a TATA box and appears to be a single-copy gene.

Animals↗
PubMed1996

Synteny mapping in river buffalo.

The cosegregation of ten coding loci has been investigated, in a panel of 37 somatic cell hybrids resulting from the fusion of a hamster cell line and river buffalo lymphocytes, by use of Southern hybridization technique. Five syntenic groups, TCRB-PGY3, ASS-ABL, FUCA1P-CRYG, MBP-YES1, and CGN1-ACTA1, previously assigned to cattle as U13, U16, U17, U28, and U29 respectively, were also found to be syntenic in buffalo. Based on the extensive syntenic conservation and banding homology between cattle and river buffalo, comparative mapping predicts the localization of these syntenic groups on river buffalo Chromosomes (Chrs) :BBU7, BBU12, BBU2q, BBU22, and BBU4q respectively as they have been previously localized on cattle Chrs BTA4, BTA11, BTA2, BTA24 & BTA28.

Animals↗
PubMed1995

Schistosoma mansoni: characterization of p50, an immunophilin.

A 1.4-kb cDNA clone encoding a 50-kDa homologue of p59, a heat shock binding immunophilin, has been isolated from a Schistosoma mansoni cercariae cDNA library. We have designated this clone S. mansoni p50 (Smp50). From the sequence comparison, we speculate that Smp50 is similar in structural organization to other p59 proteins. As with other p59s, Smp50 also shows homology to various FK binding proteins (FKBPs). The amino acids in human FKBP12 which are proposed to be important for FK506 interaction are conserved in the schistosome protein. We have expressed and purified the recombinant protein (rSmp50) from Escherichia coli. rSmp50 demonstrated peptidyl-prolyl cis-transisomerase (PPIase) activity, typical of immunophilins. Western blot analysis of extracts from S. mansoni adult worms probed with rabbit polyclonal antisera, generated against the recombinant polypeptide, has indicated the presence of Smp50 in the parasite. The antisera did not cross-react with proteins from E. coli or HeLa cell extracts. All of the p59s characterized to date have been from vertebrate species. Therefore, our finding represents the first identification of the protein in an invertebrate system.

Amino Acid Isomerases↗
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

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↗
PubMed1986

The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis.

The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA. By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA. These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA. We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions. These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation. The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome. These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.

Escherichia coli↗
PubMed1986

Guanosine 5'-O-(3-thiotriphosphate) as an analog of GTP in protein biosynthesis. The effects of temperature and polycations on the accuracy of initial recognition of aminoacyl-tRNA ternary complexes by ribosomes.

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) is a good analog of GTP in the reactions leading to the formation of a peptide bond in protein biosynthesis. It forms binary and ternary complexes with elongation factor Tu (EF-Tu), and with EF-Tu and aminoacyl-tRNA (aa-tRNA). In addition, it stimulates aa-tRNA binding to ribosomes. Although GTP gamma S hydrolysis is more than three orders of magnitude slower than GTP hydrolysis, both reactions are dependent on the formation of a noncovalent complex (RS X TC) between mRNA-programmed ribosomes and ternary complex, and the complexes resulting from that hydrolysis are intermediates in peptide formation. The rate of dissociation of the ribosome X EF-Tu X GTP gamma S X aa-tRNA complex was determined from the rate of labeled peptide formation in the presence of an unlabeled ternary complex chase. This rate (2.2 X 10(-3) s-1) is similar to that determined previously (Thompson, R.C., and Karim, A.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4922-4926) from the progress of GTP gamma S hydrolysis. The effects of temperature and polycation concentration on this rate constant and that for GTP gamma S hydrolysis are reported. The rate constants measured are consistent with a kinetic rather than thermodynamic limit on the accuracy of the aa-tRNA selection in vivo.

Escherichia coli↗
PubMed1984

Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine.

Mucosal and systemic immune responses to a new oral cholera vaccine, consisting of the B subunit plus killed vibrios, were studied in Bangladeshi volunteers and compared with those to clinical cholera. A single peroral dose of vaccine induced a local IgA antitoxin response in intestinal-lavage fluid of seven of eight vaccinees; the response closely mimicked that of patients convalescing from cholera, and evidence of the induction of local immunologic memory was found as well. Two peroral doses were needed for stimulation of an intestinal IgA immune response to the lipopolysaccharide of Vibrio cholerae that was comparable to the response obtained after clinical cholera. This response to peroral immunization was considerably stronger than that to parenteral vaccination, although the intramuscular route gave rise to the strongest IgG antitoxin and antilipolysaccharide responses in serum. The results suggest that B subunit-whole cell vaccine, when given in at least two oral doses, may be a good candidate for use in cholera prophylaxis.

Administration, Oral↗
PubMed1982

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S].

Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programmed ribosome. However, the rate constant for the subsequent hydrolysis of the beta-gamma pyrophosphate bond (3.9 X 10(-3) s-1 at 5 degrees C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage of this low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, Phe-tRNAPhe, and GTP[gamma S] (2.7 X 10(-3) s-1) and the second-order rate constant for formation of this complex (3.3 X 10(6) M-1 s-1). Therefore, the Kd of the complex may be calculated to be 8.2 X 10(-10) M. An analogous near-cognate complex with Leu-tRNA2Leu in place of Phe-tRNAPhe has been determined by equilibrium methods to have a Kd greater than 1.7 X 10(-6) M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and near-cognate ternary complexes with great accuracy. Therefore, its failure to show this high specificity with the physiological ternary complexes containing GTP is due to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination of speed and accuracy required in protein biosynthesis.

Biological Evolution↗
PubMed1981

Effect of Mg2+ concentration, polyamines, streptomycin, and mutations in ribosomal proteins on the accuracy of the two-step selection of aminoacyl-tRNAs in protein biosynthesis.

Discrimination against the binding of noncognate aminoacyl (aa)-tRNAs by mRNA-programmed ribosomes is the outcome of two selection steps, one involving an aa-tRNA.EFTu.GTP complex, which occurs prior to and includes GTP hydrolysis, the other involving the aa-tRNA alone, which follows GTP hydrolysis. Conditions which lead to errors in protein synthesis have been found to influence the accuracy of one or both selection steps in a system measuring poly(U)-directed binding of Leu-tRNA2Leu. Streptomycin has a large effect only on the discrimination process following GTP hydrolysis, but the other pertubations of recognition studied, high [Mg2+], polyamines, the strA1 and ram1 mutations, affect both discrimination processes. The general result is consistent with the view that proofreading of aa-tRNA by ribosomes for the most part uses the same specificity determinants used in the initial selection of a ternary complex.

Escherichia coli↗
PubMed1981

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes.

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

Anticodon↗
PubMedDate not supplied

Identification and characterization of Schistosoma mansoni p17.7, a cyclophilin.

Antibodies affinity purified against tegumental components of schistosomula were used to screen a Schistosoma mansoni lambda gt11 adult worm cDNA expression library. One of the reactive clones was determined by sequence analysis to encode a protein homologous to cyclophilins of other species, in particular cyclophilin A. The 0.8-kb cDNA clone contained an open reading frame of 483 nucleotides which corresponds to a translation product of 161 amino acids with a deduced molecular size of 17.7 kDa. We have chosen to designate this clone as S. mansoni p17.7 (Smp17.7). The overexpressed and purified recombinant Smp17.7 (rSmp17.7) was demonstrated to possess peptidylprolyl cis-trans isomerase (PPIase) or rotamase activity typical of cyclophilins. Western blot analysis of Nonidet P-40 and a total soluble extract of adult schistosomes probed with affinity-purified antisera to rSmp17.7, demonstrated the presence of this protein in the parasite. Immunofluorescence studies using the purified antisera indicates a localization in various tissues including the tegument and the gut. As cyclophilin is able to interact with cyclosporin A (CsA), which has been shown to be antischistosomal in mice infected with S. mansoni, the characterization of this S. mansoni cyclophilin homologue may allow a better understanding of the schistosomicidal nature of cyclosporin A and lead to a novel strategy of therapy for schistosomiasis.

Amino Acid Isomerases↗