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S Barik

Publications and source records attributed to S Barik.

At least 19 recordsLinked to original sources
PubMed2006

Immunophilins: for the love of proteins.

Immunophilins are chaperones that may also exhibit peptidylprolyl isomerase (PPIase) activity. This review summarizes our knowledge of the two largest families of immunophilins, namely cyclophilin and FK506-binding protein, and a novel chimeric dual-family immunophilin, named FK506- and cyclosporin-binding protein (FCBP). The larger members of each family are modular in nature, consisting of multiple PPIase and/or protein-protein interaction domains. Despite the apparent difference in their sequence and three-dimensional structure, the three families encode similar enzymatic and biological functions. Recent studies have revealed that many immunophilins possess a chaperone function independent of PPIase activity. Knockout animal studies have confirmed multiple essential roles of immunophilins in physiology and development. An immunophilin is indeed a natural 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper folding and assembly.

Amino Acid Sequence↗
PubMed2003

Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis: potential role of endopeptidase O in the invasion of host cells.

Porphyromonas gingivalis has been isolated from lesions of advanced adult periodontitis, and implicated as a periodontal pathogen. We have previously cloned a novel endopeptidase, designated PepO, from P. gingivalis 381, which exhibited significant homology to human endothelin-converting enzyme (ECE)-1. In order to determine the nature and function of the PepO in the host, a pepO gene-deficient mutant strain was constructed by allelic exchange mutagenesis using the ermF-ermAM cassette. No endopeptidase activity was detected in the pepO-deficient mutant. In addition, adherent HeLa (HEp-2) cells were infected with the mutant and the two wild-type strains for assessment of bacterial invasion by an antibiotic protection assay. The invasion efficiency of the mutant strain was about a quarter of the wild type strains. These results suggest that PepO is involved in the first step, i.e. invasion/lysis of mammalian cell membrane, which affects the kinetics of rate of invasion.

Adult↗
PubMed2001

Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses.

BACKGROUND: Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes. RESULTS: Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. CONCLUSIONS: Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Gene Silencing↗
PubMed2001

A novel tetratricopeptide repeat (TPR) containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum.

BACKGROUND: The malarial parasite, Plasmodium falciparum (Pf), is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined. RESULTS: We provide biochemical and sequence evidence for a protein serine/threonine phosphatase type PP5 in Plasmodium falciparum, and named it PfPP5. The 594-amino acid polypeptide was encoded by a 1785 nucleotide long intronless gene in the parasite. The recombinant protein, expressed in bacteria, was indistinguishable from native PfPP5. Sequencing comparison indicated that the extra-long N-terminus of PfPP5 outside the catalytic core contained four tetratricopeptide repeats (TPRs), compared to three such repeats in other PP5 phosphatases. The PfPP5 N-terminus was required for stimulation of the phosphatase activity by polyunsaturated fatty acids. Co-immunoprecipitation demonstrated an interaction between native PfPP5 and Pf heat shock protein 90 (hsp90). PfPP5 was expressed in all the asexual erythrocytic stages of the parasite, and was moderately sensitive to okadaic acid. CONCLUSIONS: This is the first example of a TPR-domain protein in the Apicomplexa family of parasites. Since TPR domains play important roles in protein-protein interaction, especially relevant to the regulation of PP5 phosphatases, PfPP5 is destined to have a definitive role in parasitic growth and signaling pathways. This is exemplified by the interaction between PfPP5 and the cognate chaperone hsp90.

Amino Acid Sequence↗
PubMed2001

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.

Apoptosis↗
PubMed2001

Comparison of thyroid function in pregnant and non-pregnant Asian and western Caucasian women.

BACKGROUND: Gestational thyrotoxicosis may be more prevalent in Asian women. METHODS: We have measured thyroid function, ferritin and bone specific alkaline phosphatase (BALP) as peripheral markers of thyroid function and hCG in Asian and western Caucasian women in non-pregnant and early pregnancy. RESULTS: TSH was lower in Asian women in non-pregnancy but not during normal pregnancy and this may reflect increased sensitivity of the thyroid gland to thyroid stimulation in the Asian population. No ethnic difference was found in FT3, FT4 or hCG but ferritin was lower and BALP higher in Asian women whether pregnant or not and this may be a reflection of iron balance and vitamin D status. CONCLUSIONS: We found during normal pregnancy that dynamic patterns of change for thyroid hormones and hCG are not different in Asian and western Caucasian women. We have developed gestation related reference intervals, which are a pre-requisite to the study of ethnic differences in gestation thyrotoxicosis in pregnancy.

Alkaline Phosphatase↗
PubMed2001

Characterization of a novel serine/threonine protein phosphatase (PfPPJ) from the malaria parasite, Plasmodium falciparum.

A novel protein phosphatase cDNA of the PPP superfamily was identified from the malaria parasite, Plasmodium falciparum (Pf), and tentatively named PfPPJ. The predicted primary structure of the phosphatase contained all the known conserved motifs of the PPP superfamily essential for catalytic activity. The enzyme was specific for dephosphorylation of phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. However, the sequence at its C-terminal end was unique, and was consistent with its resistance to the classical PP2A-specific inhibitors such as okadaic acid and microcystin-LR, and the PP1-specific inhibitor, mammalian heat-stable inhibitor-2 (I-2). Even the catalytic core of PfPPJ had a sequence substantially different from the other PPPs such that PfPPJ could be placed in an apparently separate phylogenetic branch. At 294 amino acids residues, PfPPJ was one of the smallest okadaic acid-resistant PPP phosphatases known. By Northern blot analysis, the expression of the PfPPJ mRNA showed the following pattern: schizont > ring > trophozoite, which closely paralleled the expression of the protein, as determined by immunofluorescence. Together, these results suggested a parasitic stage-specific transcriptional regulation of this novel and potentially unique protozoan phosphatase.

Amino Acid Sequence↗
PubMed2000

PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum.

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T(173)PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn(2+) over Mg(2+) ions as a cofactor. The Lys(38)-->Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

Alternative Splicing↗
PubMed2000

Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA.

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-L-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.

Actins↗
PubMed1999

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.

Alkenes↗
PubMed1999

Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex.

Two major protein phosphatase (PP) activities were purified from cytosolic extracts of the erythrocytic stage of the malaria parasite, Plasmodium falciparum. Both enzymes were specific for phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. The biochemical properties of the enzymes suggested their strong similarity with eukaryotic PP2A and PP2B protein phosphatases. Both enzymes preferentially dephosphorylated the alpha subunit of phosphorylase kinase, and were resistant to inhibitor-1. The PP2A-like enzyme required Mn2+ for activity and was inhibited by nanomolar concentrations of okadaic acid (OA). The cDNA sequence of the PP2A-like enzyme was identified through a match of its predicted amino acid sequence with the N-terminal sequence of the catalytic subunit. The PP2B-like (calcineurin) enzyme was stimulated by calmodulin and Ca2+ or Ni2+, but was resistant to OA. Malarial calcineurin was strongly and specifically inhibited by cyclosporin A (CsA) only in the presence of wild type P. falciparum cyclophilin but not a mutant cyclophilin. The inhibition was noncompetitive, and provides a potential explanation for the cyclosporin-sensitivity of the parasite. There was no significant quantitative difference in the total protein Ser/Thr phosphatase activity among the ring, trophozoite, and schizont stages.

Amino Acid Sequence↗
PubMed1999

Differential effect of common laboratory treatments on hypoosmotic swelling responses of human spermatozoa.

PURPOSE: The impact of some of the common laboratory interventions on the hypoosmotic swelling (HOS) responses of human-spermatozoa was investigated. METHODS: The semen samples underwent different laboratory treatments prior to the standard HOS test, which involved incubation of sperm in the hypoosmotic solution. Fresh semen served as a control for all treatment groups and underwent the same HOS procedure. The HOS-reactive spermatozoa and the type of HOS reactions (swelling types) in each group were identified under a phase-contrast microscope for comparison. RESULTS: All the seven types of HOS responses documented in fresh semen sperm also occurred in the laboratory-processed sperm. The total HOS responses of sperm that underwent cryopreservation, heat shock, and Percoll wash were significantly different from those of the corresponding control. Percoll washing of semen influenced HOS subtypes a and g; cryopreservation affected subtypes a, b, c, and d; and heat shock altered subtypes a, f, and g. In contrast, prolonged postejaculation and cold shock did not affect any of the HOS responses. None of the treatments influenced the d and e responses. CONCLUSIONS: These results indicate that the total HOS response value and specific response subtypes are significantly affected by some of the laboratory treatments but not others.

Cold Temperature↗
PubMed1999

Analysis of in vitro migration patterns of human spermatozoa by a petri dish-based horizontal column.

Spermatozoa are required to travel a considerable distance in vivo to meet the oocyte at the fertilization site. However, none of the existing in vitro tests critically evaluates migration of sperm to assess their potential of reaching the oocyte. On the other hand, an in vivo model is not suitable for this type of study because of ethical and technical constraints. In the present study we utilized a horizontal column technique to analyze sperm migration. Migratory characteristics of fresh, unwashed semen sperm and sperm undergoing various treatments were examined in vitro using a Petri dish-based horizontal fluid column. The procedure involved loading a sperm sample into the column and determining sperm concentration, motility, and viability at different column segments for different migration durations (6, 12, 24, 48, and 72 h). All sperm samples produced an exponential migration pattern in all durations of migration. Propagation along the column edge, tendency to exit from the column, and hiding in the blind pouches were some of the important characteristic features exhibited by the migratory sperm. Variations in migration patterns were documented among semen donors, between fresh and frozen semen, and between washed and unwashed sperm. Prolonged postejaculation time diminished migratory potential. The recovery of sperm in the column end was independent of seminal variables with the exception of oligozoospermia. These observations suggest that the Petri dish-based horizontal column is effective for analyzing sperm migration characteristics for prolonged periods. The potential of this migration assay in predicting the in vivo potential of spermatozoa to reach the fertilization site will be worth exploring.

Humans↗
PubMed1999

Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia.

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M(r) of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.

Acid Phosphatase↗
PubMed1999

Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphoprotein P is essential for the transcription elongation activity of the viral polymerase; phosphorylation by casein kinase 1 occurs mainly at Ser(215) and is without effect.

The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser(232) in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser(232) was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser(215). In vitro, P protein could be phosphorylated by purified CK1 at Ser(215) but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser(215), Ser(232), and Ser(237) were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.

Animals↗