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Biomedical subjects

S Krishnan

Publications and source records attributed to S Krishnan.

At least 55 records · Page 3Linked to original sources
PubMed1999

Negative regulation of Par-4 by oncogenic Ras is essential for cellular transformation.

Oncogenic variants of the cellular Ras protein are often associated with different types of human cancers. However, the mechanisms by which oncogenic Ras induces transformation are not fully established. Expression of the transcriptional repressor Par-4 was down-regulated by oncogenic Ras via the Raf-MEK-ERK pathway. Restoration of Par-4 levels by abrogation of the Raf-MEK-ERK pathway with the MEK-inhibitor PD98059 or by ectopic Par-4, that acted to inhibit ERK expression and activation, was sufficient to suppress oncogenic Ras-induced transformation. These findings identify Par-4 as a novel target that has to be down-modulated by oncogenic Ras for successful transformation.

3T3 Cells↗
PubMed1999

Oncogenic Ras sensitizes cells to apoptosis by Par-4.

Certain mutations in the mammalian ras gene are oncogenic and are often detected in human cancers. Oncogenic Ras induces the transcription activity of NF-kappaB that confers cell survival. Oncogenic Ras also down-modulates the expression of Par-4, a transcriptional repressor protein, that is essential but not sufficient on its own to induce apoptosis. Here we show that reintroduction of Par-4 by transient transfection leads to apoptosis in cells expressing oncogenic Ras but not in those that lack oncogenic Ras expression. Par-4 abrogates oncogenic Ras-inducible NF-kappaB transcription activity but does not interfere with cytoplasmic activation, or the DNA binding activity, of NF-kappaB. Because abrogation of NF-kappaB transcription activity is sufficient to cause apoptosis in cells expressing oncogenic Ras, our findings identify Par-4 as a novel example of a pro-apoptotic protein that selectively inhibits oncogenic Ras-dependent NF-kappaB function at the transcription level and suggest a mechanism by which Par-4 expression may selectively induce apoptosis in oncogenic Ras-expressing cells.

3T3 Cells↗
PubMed1999

Chemical modification and site-directed mutagenesis of conserved HXXH and PP-loop motif arginines and histidines in the murine bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase.

The sulfurylase domain of the mouse bifunctional enzyme ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase contains HXXH and PP-loop motifs. To elucidate the functional importance of these motifs and of conserved arginines and histidines, chemical modification and site-directed mutagenesis studies were performed. Chemical modification of arginines and histidines with phenylglyoxal and diethyl pyrocarbonate, respectively, renders the enzyme inactive in sulfurylase, kinase, and overall assays. Data base searches and sequence comparison of bifunctional ATP sulfurylase/APS kinase and monofunctional ATP sulfurylases shows a limited number of highly conserved arginines and histidines within the sulfurylase domain. Of these conserved residues, His-425, His-428, and Arg-421 are present within or near the HXXH motif whereas His-506, Arg-510, and Arg-522 residues are present in and around the PP-loop. The functional role of these conserved residues was further studied by site-directed mutagenesis. In the HXXH motif, none of the alanine mutants (H425A, H428A, and R421A) had sulfurylase or overall activity, whereas they all exhibited normal kinase activity. A slight improvement in reverse sulfurylase activity (<10% residual activity) and complete restoration of forward sulfurylase was observed with R421K. Mutants designed to probe the PP-loop requirements included H506A, R510A, R522A, R522K, and D523A. Of these, R510A exhibited normal sulfurylase and kinase activity, R522A and R522K showed no sulfurylase activity, and H506A had normal sulfurylase activity but produced an effect on kinase activity (<10% residual activity). The single aspartate, D523A, which is part of the highly conserved GRD sequence of the PP-loop, affected both sulfurylase and kinase activity. This mutational analysis indicates that the HXXH motif plays a role only in the sulfurylase activity, whereas the PP-loop is involved in both sulfurylase and kinase activities. Residues specific for sulfurylase activity have also been distinguished from those involved in kinase activity.

Amino Acid Sequence↗
PubMed1999

Isolation of subgenus B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in Sibu, Sarawak.

BACKGROUND: In mid-1997, several children died in Sarawak, Malaysia, during an epidemic of enterovirus-71 (EV71) hand, foot, and mouth disease. The children who died had a febrile illness that rapidly progressed to cardiopulmonary failure and the cause was not satisfactorily resolved. We describe the isolation and identification of a subgenus B adenovirus from the children who died. METHODS: We studied two groups of children presenting to Sibu Hospital from April 14 to Sept 30, 1997. For children who died, the inclusion criterion was death after febrile illness, and for those who did not die it was acute flaccid paralysis (AFP). Serum and cerebrospinal fluid samples were tested for IgM antibodies to Japanese encephalitis and dengue viruses. Viruses isolated were identified by immunofluorescence, reverse-transcriptase PCR, or PCR and DNA sequencing. FINDINGS: Enterovirus was isolated in three (19%) of 16 children who died and in none of the eight surviving children with AFP. However, an agent that was initially difficult to identify was found in ten (63%) children who died and five (63%) surviving children who had AFP. The agents isolated from ten (66.7%) of these 15 children were eventually identified as adenoviruses and were isolated mainly from clinically important sterile sites or tissues. All the enterovirus-positive children who died had this second agent. INTERPRETATION: Our data raises doubts that EV71 was the only aetiological agent in these deaths.

Adenoviridae↗
PubMed1999

Variation in volatile organic compounds in the breath of normal humans.

We studied the variation in volatile organic compounds (VOCs) in the breath of 50 normal humans, using gas chromatography and mass spectroscopy. An average breath sample contained 204.2 VOCs (SD=19.8, range 157-241). The alveolar gradient of each VOC (abundance in breath minus abundance in air) varied with rate of synthesis minus rate of clearance. A total of 3481 different VOCs were observed: 1753 with positive alveolar gradients, 1728 with negative alveolar gradients. Twenty-seven VOCs were observed in all fifty subjects. This study confirmed previous reports of wide inter-individual variations. Two new findings were the comparatively small variation in total number of breath VOCs, and the presence of a 'common core' of breath VOCs in all subjects.

Breath Tests↗
PubMed1999

Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase.

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.

Amino Acid Sequence↗
PubMed1999

Intradermal DNA immunization by using jet-injectors in mice and monkeys.

We have used spring powered jet injectors to deliver a solution of a naked DNA vaccine encoding the influenza hemagglutinin HA into the skin of mice and monkeys. We compared the immune responses induced by this needleless injection technique into the skin to the responses induced by a classical i.m. immunization. Both routes of immunization induced significant ELISA antibody titers and hemagglutination inhibition (HI) titers that were above the usual threshold values predictive of protection against influenza in mice and monkeys. In mice, both ways of immunization were equally efficient in inducing HA-specific CTL responses. Regarding antibody isotypes, the IgG1/IgG2a ratio was in favour of the IgG2a isotype for i.m. immunization and more balanced for i.d. immunization. The ability of the two injection techniques to induce immunity in mice did not correlate with transgene expression in the site of administration. In fact, local gene expression was 10-100 fold more important in the injected muscle as compared to the jet-injected skin when assessed by using the luciferase reporter system.

Animals↗
PubMed1999

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.

Apoptosis↗
PubMed1999

An overview of quality control practices in Ontario with particular reference to cholesterol analysis.

BACKGROUND: The Laboratory Proficiency Testing Program (LPTP) assesses the analytical performance of all licensed laboratories in Ontario. The LPTP Enzymes, Cardiac Markers, and Lipids Committee conducted a "Patterns of Practice" survey to assess the in-house quality control (QC) practices of laboratories in Ontario using cholesterol as the QC paradigm. DESIGN AND METHODS: The survey was questionnaire-based seeking information on statistical calculations, software rules, review process and data retention, and so on. Copies of the in-house cholesterol QC graphs were requested. A total of 120 of 210 laboratories were randomly chosen to receive the questionnaires during 1995 and 1996; 115 laboratories responded, although some did not answer all questions. RESULTS: The majority calculate means and standard deviations (SD) every month, using anywhere from 4 to >100 data points. 65% use a fixed mean and SD, while 17% use means calculated from the previous month. A few use a floating or cumulative mean. Some laboratories that do not use fixed means use a fixed SD. About 90% use some form of statistical quality control rules. The most common rules used to detect random error are 1(3s)/R4s while 2(2s)/4(1s)/10x are used for systematic errors. About 20% did not assay any QC at levels >5.5 mmol/L. CONCLUSIONS: Quality control data are reviewed daily (technologists), weekly and monthly (supervisors/directors). Most laboratories retain their QC records for up to 3 years on paper and magnetic media. On some QC graphs the mean and SD, QC product lot number, or reference to action logs are not apparent. Quality control practices in Ontario are, therefore, disappointing. Improvement is required in the use of clinically appropriate concentrations of QC material and documentation on QC graphs.

Chemistry, Clinical↗
PubMed1999

Annexin II: a mediator of the plasmin/plasminogen activator system.

The annexins constitute a family of calcium-dependent membrane binding proteins. Recently, annexin II has been shown to accelerate the activation of the clot-dissolving protease plasmin by complexing with the plasmin precursor plasminogen and with tissue plasminogen activator. Binding of plasminogen to annexin II is inhibited by the atherogenic lipoprotein, lipoprotein(a), while binding of tissue plasminogen activator to annexin II is blocked by the thiol amino acid homocysteine. Formation of the plasminogen/tissue plasminogen activator/annexin II complex may represent a key regulatory mechanism in fibrinolytic surveillance.

Amino Acid Sequence↗
PubMed1999

Stimulation of sodium chloride absorption from secreting rat colon by short-chain fatty acids.

Inhibition of electroneutral NaCl absorption by cyclic adenosine monophosphate (cAMP) results in fluid malabsorption in cholera. Short-chain fatty acids (SCFA) stimulate electroneutral NaCl absorption from the colon. The present study investigated effects of elevated cAMP on SCFA-stimulated NaCl absorption in rat distal colon. The effect of SCFA on fluxes of 22Na and 36Cl was studied under voltage-clamp conditions, in the presence and absence of secretagogues inducing mucosal cAMP elevation [ie, theophylline, cholera toxin (CT) and forskolin]. The effect of butyrate concentration on Na absorption in CT- and theophylline-treated mucosa was compared with control normal mucosa. cAMP was measured in isolated colonocytes in the presence of secretagogues with and without SCFA using a radioassay method. All secretagogues were noted to inhibit net Na absorption and to induce net Cl secretion. In the presence of SCFA, net Na absorption was normal, and net Cl secretion was partly reversed. The flux data indicated that NaCl absorption from secreting colon was stimulated by SCFA and that Cl secretion was partially inhibited. The effects of SCFA on NaCl absorption were similar regardless of the secretagogue used. The kinetics of butyrate-stimulated Na absorption were altered by theophylline and CT, which decreased Km (6.87 and 7.17, respectively, compared to 10.75 mM for control) and increased Vmax (4.55 and 8.33 compared to 3.45 mM/eq/cm2/hr for control). cAMP production by colonocytes in response to secretory stimuli was significantly reduced (34.4%) by butyrate but not by acetate or propionate. In conclusion, SCFA-stimulated Na absorption is up-regulated by cAMP and may be an absorptive pathway that can be utilized in the therapy of cholera. Effects of butyrate on cAMP generation are also likely to be useful in secretory diarrhea.

Animals↗
PubMed1999

Effect of shiga toxin on NaCl transport in rat distal colon.

BACKGROUND: Shiga toxin causes net fluid secretion in rabbit jejunum by selectively targeting, and inhibiting protein synthesis in, absorptive villous cells. The effect of Shiga toxin on the colon, where it is presumably produced, is not known. This study was undertaken to investigate the effect of Shiga toxin on the rat distal colon. METHODS: Net absorption of water and Na was determined by in vivo perfusion of closed loops of rat colon pre-exposed to Shiga toxin or saline. Unidirectional and net fluxes of 22Na and 36Cl were measured in vitro under voltage-clamp conditions across rat distal colon mucosa pre-exposed to Shiga toxin. Shiga toxin binding to sections of rat distal colon was localized by immunohistochemistry. Protein synthesis was measured in surface and crypt colonocytes with 3H-leucine incorporation. RESULTS: In the in vivo perfusion studies net absorption of Na and water was increased in Shiga toxin-treated colon compared with controls (P < 0.01). In the studies carried out in vitro, J(net)Na and J9net)Cl across Shiga toxin-treated mucosa were found to be significantly higher than in control tissue (P < 0.001 and P < 0.01, respectively). Net absorption of Na or Cl did not increase further in the presence of 25 mM butyrate, indicating the absence of short-chain fatty acids (SCFA)-linked NaCl absorption in Shiga toxin-treated colon. Moreover, Shiga toxin-treated colon failed to respond to theophylline, which induced secretion in the normal colon. Immunohistochemistry showed Shiga toxin binding to crypt cells but not to surface cells in the distal colon. Shiga toxin inhibited protein synthesis (by 27.3%) in crypt cells but not in surface cells (P < 0.05). CONCLUSIONS: An unexpected increase in water and NaCl absorption was noted in Shiga toxin-treated rat distal colon, which appears to result from selective effects of the toxin on secretory crypt cells.

Animals↗
PubMed1999

Linear motion correction in three dimensions applied to dynamic gadolinium enhanced breast imaging.

Quantitative analysis of dynamic gadolinium-DTPA (diethylenetriamine pentaacetic acid) enhanced magnetic resonance imaging (MRI) is emerging as a highly sensitive tool for detecting malignant breast tissue. Three-dimensional rapid imaging techniques, such as keyhole MRI, yield high temporal sampling rates to accurately track contrast enhancement and washout in lesions over the course of multiple volume acquisitions. Patient motion during the dynamic acquisitions is a limiting factor that degrades the image quality, particularly of subsequent subtraction images used to identify and quantitatively evaluate regions suggestive of malignancy. Keyhole imaging is particularly sensitive to motion since datasets acquired over an extended period are combined in k-space. In this study, motion is modeled as set of translations in each of the three orthogonal dimensions. The specific objective of the study is to develop and implement an algorithm to correct the consequent phase shifts in k-space data prior to offline keyhole reconstruction three-dimensional (3D) volume breast MR acquisitions.

Algorithms↗
PubMed1999

Mortality associated with concurrent strongyloidosis and cytomegalovirus infection in a patient on steroid therapy.

Disseminated strongyloidosis has been recognized with increasing frequency, often in patients who are immunocompromised or have received steroid therapy. In addition, disease due to cytomegalovirus (CMV) is noted in immunodeficient hosts. We report on a 55-year-old Puerto Rican man who received steroid treatment for orpharyngeal pemphigus vulgaris and developed abdominal symptoms with alternating constipation and diarrhea. The clinical work-up did not reveal specific abnormalities, but the patient died of cardiopulmonary failure. At the postmortem examination, the patient had evidence of strongyloidosis and CMV disease. This report reviews both this case and the literature, and discusses the overlapping infections of strongyloidosis and CMV disease in this patient who had received steroid therapy.

Anti-Inflammatory Agents↗
PubMed1998

A member of a family of sulfate-activating enzymes causes murine brachymorphism.

Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member, SK2, colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the SK2 coding sequence of bm mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of SK genes are responsible for sulfate activation in mammals, that a mutation in SK2 causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.

Amino Acid Sequence↗
PubMed1998

Deletion and site-directed mutagenesis of the ATP-binding motif (P-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5'-phosphosulfate kinase enzyme.

The P-loop is a common motif found in ATP- and GTP-binding proteins. The recently cloned murine ATP-sulfurylase/adenosine 5'-phosphosulfate (APS) kinase contains a P-loop (residues 59-66) in the APS kinase portion of the bifunctional protein. A series of enzymatic assays covering the multiplicity of functions of this unique protein (reverse ATP-sulfurylase, APS kinase, and an overall assay) were used to determine the effect of deleting or altering specific residues constituting this motif. In addition to the full-length cDNA construct (1MSK), two deletion mutants that progressively shortened the N terminus by 34 amino acids (2MSK) and 70 amino acids (3MSK) were designed to examine the effects of translation initiation before (2MSK) and after (3MSK) the P-loop. The 2MSK protein possessed sulfurylase and kinase activity equivalent to the full-length construct, but 3MSK exhibited no kinase activity and reduced sulfurylase activity. In light of the evident importance of this motif, a number of site-directed mutants were designed to investigate the contribution of key residues. Mutation of a highly conserved lysine in the P-loop to alanine (K65A) or arginine (K65R) or the following threonine (T66A) to alanine ablated APS kinase activity while leaving ATP-sulfurylase activity intact. Three mutations (G59A, G62A, and G64A) addressed the role of the conserved glycines as follows: G64A showed diminished APS kinase activity only, whereas G62A had no effect on either activity. G59A caused a significant decrease in ATP-sulfurylase activity without effect on APS kinase activity. A series of highly conserved flanking cysteines (Cys-53, Cys-77, and Cys-83) were mutated to alanine, but none of these mutations showed any effect on either enzyme activity.

Adenosine Triphosphate↗
PubMed1998

Usefulness of ST-segment elevation in lead III exceeding that of lead II for identifying the location of the totally occluded coronary artery in inferior wall myocardial infarction.

The presence of ST-segment elevation in lead III exceeding that of lead II, particularly if combined with ST elevation in lead V1, proved to be a powerful marker for occlusion of the proximal or midportion of the right coronary artery. These findings helped to determine the extent of myocardium at risk in inferior wall myocardial infarction and may further guide the decision to administer thrombolytics.

Algorithms↗