Community involvement in assessing health needs of immigrant Salvadoreans living in Washington, D.C. area.
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Adult↗
Biomedical subjects
S Shankar
Publications and source records attributed to S Shankar.
Complex formation of the elongation factor Tu from Pseudomonas aeruginosa with nucleoside diphosphate kinase modulates ribosomal GTP synthesis and peptide chain elongation.
The elongation factor Tu (EF-Tu) from Pseudomonas aeruginosa was purified as a 45-kDa polypeptide that forms a complex with both the 12- and 16-kDa forms of nucleoside-diphosphate kinase (Ndk) and predominantly synthesizes GTP. 70 S ribosomes of P. aeruginosa predominantly synthesize GTP, which is inhibited in presence of anti-Ndk antibodies. Anti-EF-Tu antibodies change the specificity of ribosomal GTP synthesis to all nucleoside triphosphate synthesis. Ndk has been shown to be a part of 30 S ribosomes, whereas EF-Tu is found to be associated with the 50 S ribosomal subunit. These data indicate that GTP synthesis in the ribosome is modulated both by Ndk and by EF-Tu. Peptide chain elongation as measured by polymerization of Phe-tRNA on a poly(U) template in presence of GDP can be inhibited by anti-Ndk antibodies and restored by the addition of GTP. Anti-EF-Tu antibodies similarly inhibit peptide chain elongation by P. aeruginosa ribosomes in the in vitro translation assay; however, this inhibition cannot be overcome by adding back GTP. Because the purified EF-Tu.16-kDa Ndk complex predominantly synthesizes GTP, it seems likely that this complex is a significant source of GTP for translational elongation in protein biosynthesis.
Antibodies↗
Intraoperative ultrasonographic troubleshooting after the arterial switch operation.
BACKGROUND: Less than perfect coronary artery translocation accounts for the majority of perioperative deaths after the arterial switch procedure for transposition of the great arteries. Some types of coronary arterial anatomy are associated with a higher risk of death. METHODS: Prospective epicardial ultrasound examination of all neonates with failing left ventricle or difficulty in weaning off cardiopulmonary bypass was performed after completion of the arterial switch operation during a 2-year period from March 1994 to February 1996. The aim was to identify any mechanical, and potentially remediable, factors accounting for ventricular failure. RESULTS: Four neonates fulfilling the above criteria were identified during a 2-year period when epicardial echocardiography was routinely applied. In 2 patients coronary arterial problems in the form of kinking of the proximal left coronary artery (1 patient) and extrinsic compression of the artery by the neo-pulmonary trunk (1 patient) were identified and successfully corrected. In 2 other patients, supravalvar aortic stenosis was recognized, leading to prompt revision. CONCLUSIONS: Epicardial echocardiography has an important "troubleshooting" role in the subgroup of patients with a suboptimal hemodynamic result after the arterial switch operation. Patients with unusual coronary anatomy should routinely be candidates for such studies.
Cardiopulmonary Bypass↗
Neurologic complications of treatment of childhood acute immune thrombocytopenic purpura with intravenously administered immunoglobulin G.
We studied the incidence, associated morbidity, and impact on health care charges of neurologic complications in 38 children with acute immune thrombocytopenic purpura (ITP) treated with intravenously administered IgG. Thirteen patients (34%) had transient neurologic complications, manifested by severe headache, nausea, and, rarely, aseptic meningitis. Computed tomography was performed in nine patients. Twelve patients were hospitalized longer than was required for their ITP alone. Neurologic complications caused by the IgG preparations used in the treatment of childhood ITP occur more frequently than has previously been suggested and may substantially increase the cost of treatment.
Acute Disease↗
The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme.
We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4 B matrix and passing the total cell extract through it revealed four proteins (P70, P65, P60, and P50, respectively) of M(r) 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M(r) 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.
Amino Acid Sequence↗
Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells.
Mammalian heterotrimeric GTP-binding protein (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the alpha-, beta- or the gamma-subunits of heterotrimeric G proteins. Such antibodies also after the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk-G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian alpha- and beta-G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the alpha-, beta- and gamma-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the alpha- and gamma-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.
Amino Acid Sequence↗
Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.
We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.
Amino Acid Sequence↗
How safe is Biers Block in the accident and emergency department?
We report on the use of Biers Block in an accident and emergency department carried out during the period 1987 to 1994. A total of 915 procedures were carried out, of which 815 (98%) involved emergency procedures. The age of patients ranged between 17 and 92 years. Acceptability by the patients was uniformly good. We feel it is a useful method of producing analgesia in the limb that does require training but no extensive experience or anaesthetists' expertise.
Adolescent↗
Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.
We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.
Amino Acid Sequence↗
Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA.
Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients. One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P. aeruginosa chromosomal map. The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family. On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy. Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P. aeruginosa strains isolated from cystic fibrosis patients. We expressed and purified the protein products from the mucA and mucB open reading frames. The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo. Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.
Bacterial Proteins↗
Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.
We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.
Alginates↗
Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme.
Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.
Cytoplasm↗
Characterization of a phosphoprotein phosphatase for the phosphorylated form of nucleoside-diphosphate kinase from Pseudomonas aeruginosa.
Nucleoside-diphosphate kinase (ATP:nucleoside-diphosphate phosphotransferase, EC 2.7.4.6; NDP kinase) is an important enzyme for the maintenance of the correct cellular levels of nucleoside triphosphates (NTPs) and their deoxy derivatives (dNTPs) and is involved in the regulation of cellular development. The enzyme is under the dual control of algR2 and algH in Pseudomonas aeruginosa. We report here the purification and characterization of a protein that dephosphorylates the phosphorylated intermediate form of the P. aeruginosa NDP kinase (Ndk). Dephosphorylation of Ndk phosphate leads to loss of its enzymatic activity. The 10.1-kDa polypeptide shows 77% homology at the N terminus with the Spo0E phosphatase, identified as a negative regulator of sporulation in Bacillus subtilis and 66% with the human Bax protein, identified as an effector of programmed cell death. The phosphatase termed Npp showed varied specificity toward phosphorylated Ndks from different sources including human erythrocyte Ndk phosphate. Its activity toward other histidine phosphates such as CheA or the alpha-subunit of succinyl-CoA synthetase or phosphoesters such as p-nitrophenyl phosphate was quite limited. Npp was stable at room temperature up to 2 h and required Mg2+ for activity. The presence of a phosphatase capable of dephosphorylating the phosphorylated form of P. aeruginosa Ndk represents an interesting and efficient mode of post-translational modification of an enzyme crucial to cellular development.
Adenosine Triphosphate↗
Exopolysaccharide alginate synthesis in Pseudomonas aeruginosa: enzymology and regulation of gene expression.
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Alginates↗
Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation.
We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa. AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa. One of the E. coli genes, rnk, encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA, encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli. While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli. We find that a mutation in rnk drastically reduces the level of Ndk in E. coli. A mutation in sspA, however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.
Adhesins, Bacterial↗
The Escherichia coli genes sspA and rnk can functionally replace the Pseudomonas aeruginosa alginate regulatory gene algR2.
The algR2 (also known as algQ) gene of Pseudomonas aeruginosa has previously been identified as being necessary for alginate production at 37 degrees C. We have cloned two genes, from a cosmid library of Escherichia coli, which can restore mucoidy to an algR2 mutant of P. aeruginosa. The complementing regions of both cosmids were localized by subcloning restriction fragments. One of the E. coli genes identified here has not previously been described; we have named this gene rnk (regulator of nucleoside diphosphate kinase). It encodes a 14.9 kDa protein with no homology to any other protein. The other gene, sspA, is a regulator involved in stationary-phase regulation in E. coli. Either gene will restore mucoidy to an algR2-deficient strain of P. aeruginosa. AlgR2 has been shown to regulate at least two enzymes, succinyl-CoA synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa. When we examined the ability of the E. coli analogues to regulate Ndk, we found that rnk but not sspA was able to restore Ndk activity to the P. aeruginosa algR2 mutant. Furthermore, rnk was able to restore growth of the algR2 mutant in the presence of Tween 20, which inhibits other Ndk-like activities.
Alginates↗
Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100.
Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy. The genes encoding the proteins involved in the first step (tftA and tftB [previously designated tftA1 and tftA2, respectively]) have been cloned and sequenced. The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays. The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group. These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134. The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension. The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100. The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters.
Base Sequence↗
Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH.
Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37 degrees C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30 degrees C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30 degrees C, and it is fully defective in Ndk production.
Alginates↗