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Biomedical subjects

S Krishnan

Publications and source records attributed to S Krishnan.

At least 19 recordsLinked to original sources
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

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↗
PubMed1998

The ability of enteric diarrhoeal pathogens to ferment starch to short-chain fatty acids in vitro.

BACKGROUND: Short-chain fatty acids (SCFA), produced in the normal colon by bacterial fermentation, are decreased in acute diarrhoea. This may have deleterious effects on epithelial function in the colon. METHODS: The ability of several diarrhoeal pathogens to produce SCFA when incubated with starch in vitro was studied. Isolated pathogens were incubated for 24 h with either no added substrate, glucose, or starch under anaerobic conditions, and SCFA were quantitated by gas-liquid chromatography. RESULTS: Unlike the normal colonic flora, the pathogens produced acetate but not propionate or butyrate. D-Lactate was also produced by all the pathogens studied. When the pathogens were incubated in anaerobic medium containing starch, significantly greater amounts of acetate and significantly lesser amounts of lactate were produced. CONCLUSIONS: The inability of enteric pathogens to produce butyrate may impair epithelial cell function, whereas production of D-lactate may enhance mucosal damage in diarrhoeal disease. The presence of luminal starch may be helpful in shifting the fermentation profile to a more favourable pattern.

Butyrates↗
PubMed1998

Butyrate and glucose metabolism in isolated colonocytes in the developing rat colon.

BACKGROUND: The newborn colon is devoid of microflora, in that bacterial colonization is established after birth. Short chain fatty acids, products of bacterial fermentation, are the major energy source for colonocytes. Because it is not known whether colonocytes in the newborn can metabolize butyrate, this was examined in newborn and infant rat colon. METHODS: Isolated colonocytes from rats of different perinatal ages were incubated with 14C-labeled butyrate or glucose in vitro. Complete oxidation was estimated by the production of 14C-labeled carbon dioxide, whereas intermediate metabolites were measured enzymatically. RESULTS: Oxidation of butyrate (in micromoles per hour per milligram of protein) was highest in newborns (5.83+/-1.76), declining to 1.32+/-0.28 at day 10 and to 0.34+/-0.04 in adult rats. Glucose oxidation was also highest at birth (0.39+/-0.23), with a minor increase at approximately day 20 (weaning period) before decreasing to adult levels (0.05+/-0). Butyrate oxidation was substantially higher than was glucose oxidation in all age groups. Production of metabolic intermediates paralleled substrate oxidation. Acetoacetate production was 4.35+/-2.68, 2.07+/-1.29, and 0.27+/-0.09 nmol/hr per milligram of protein in newborns, at postnatal day 10, and in adults, respectively. The corresponding values for beta-hydroxybutyrate were 3.62+/-3.35, 0.2+/-0.07, and 0.09+/-0.03 nmol/hr per milligram of protein; and L-lactate production was 0.54+/-0.52, 0.06+/-0.04, and 0.02+/-0 micromol/hr per milligram of protein respectively. CONCLUSIONS: Neonatal rat colon epithelial cells resemble adult colonocytes in their preference for butyrate as a metabolic substrate, indicating a constitutive expression of this property.

3-Hydroxybutyric Acid↗
PubMed1998

Proficiency testing of creatine kinase and creatine kinase-2: the experience of the Ontario Laboratory Proficiency Testing Program.

The Ontario Laboratory Proficiency Testing Program has regularly monitored the analytical performance of total creatine kinase (CK) (approximately 230 participants) and CK isoenzyme-2 (CK-MB) (approximately 160 participants) throughout the entire province. Consistently, a wide dispersion of results has been observed not only between different analyzer systems but also among identical analyzers. Accordingly, the results of the last three proficiency surveys for these analytes were examined statistically to establish both the extent of these variations and the range of values reported for the male upper reference ranges. The results of many of the analyzer systems were significantly different from each other, as were many of the reference ranges. This unsatisfactory situation may only be remedied by the use of reference materials as shown by others. The consequences of these findings also effect the reliability of epidemiological surveys such as the WHO MONICA Project (Circulation 1994;90:583-612), which monitors deaths due to heart disease and includes cardiac enzyme results in its criteria.

Clinical Enzyme Tests↗
PubMed1998

Primary hyperparathyroidism presenting with pathological fracture.

Primary hyperparathyroidism (PHPT) is an intriguing condition. Routine automated biochemical screening has made the diagnosis commonplace in developed countries and the disease is diagnosed early in its course when it is often asymptomatic. In developing countries or in recent immigrants from these countries, PHPT is often seen in an advanced stage with bone involvement. Associated dietary deficiencies may alter the biochemical profile and cause a diagnostic dilemma. It is important to include it in the differential diagnosis of pathological fractures. We report three cases of PHPT presenting with pathological fractures and discuss their diagnosis and management.

Adult↗
PubMed1997

Larger increases in sensitivity to paracatalytic inactivation than in catalytic competence during experimental evolution of the second beta-galactosidase of Escherichia coli.

Second-order rate constants (M-1.s-1) at 25 degrees C and pH 7.5 for inactivation of first-generation (ebga and ebgb), second-generation (ebgab and ebgabcd) and third-generation (ebgabcde) experimental evolvants of the title enzyme by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside are 0.042, 0.30, 10, 24 and 57 respectively. Only partial inactivation is observed, except for ebgabcde. At a single high inactivator concentration, inactivation of the wild-type ebgo is also seen. The changes in sensitivity to the paracatalytic inactivator (over a range of 10(3.3)) are larger than changes in kcat/Km for lactose (over a range of 10(2.7)) or nitrophenyl galactosides (over a range of only 10(1.3)), or changes in degalactosylation rate (over a range of 10(1.7)). These data raise the possibility that evolution in the reverse sense, towards insensitivity to a paracatalytic inactivator with a proportionally lower effect on transformation of substrate, may become a mechanism for the development of bacterial resistance to antibiotics that act by paracatalytic enzyme inactivation.

Binding Sites↗
PubMed1997

Adaptive filtering, modelling and classification of knee joint vibroarthrographic signals for non-invasive diagnosis of articular cartilage pathology.

Interpretation of vibrations or sound signals emitted from the patellofemoral joint during movement of the knee, also known as vibroarthrography (VAG), could lead to a safe, objective, and non-invasive clinical tool for early detection, localisation, and quantification of articular cartilage disorders. In this study with a reasonably large database of VAG signals of 90 human knee joints (51 normal and 39 abnormal), a new technique for adaptive segmentation based on the recursive least squares lattice (RLSL) algorithm was developed to segment the non-stationary VAG signals into locally-stationary components; the stationary components were then modelled autoregressively, using the Burg-Lattice method. Logistic classification of the primary VAG signals into normal and abnormal signals (with no restriction on the type of cartilage pathology) using only the AR coefficients as discriminant features provided an accuracy of 68.9% with the leave-one-out method. When the abnormal signals were restricted to chondromalacia patella only, the classification accuracy rate increased to 84.5%. The effects of muscle contraction interference (MCI) on VAG signals were analysed using signals from 53 subjects (32 normal and 21 abnormal), and it was found that adaptive filtering of the MCI from the primary VAG signals did not improve the classification accuracy rate. The results indicate that VAG is a potential diagnostic tool for screening for chondromalacia patella.

Adult↗
PubMed1997

A histopathological study of liver and kidney in male Wistar rats treated with subtoxic doses of t-butyl alcohol and trichloroacetic acid.

Tertiary butyl alcohol and trichloroacetic acid are known to be contaminants in drinking water. In order to evaluate the interactive toxicity of t-butyl alcohol with trichloroacetic acid, young male Wistar rats were dosed through water at a dose level of t-butyl alcohol (TBA)-0.5% (v/v), trichloroacetic acid (TCA)-25 ppm and a combined dose of TBA + TCA (0.5% v/v TBA-25 ppm TCA) for a period of 10 weeks ad libitum and were maintained on normal diet. The control animals received plain water and normal diet. The liver and kidney histology was undertaken to see whether subtoxic administration of TBA and TCA individually as well as combined administration for a period of 10 weeks would bring about any histological alterations. It was observed that TBA, TCA and TBA + TCA caused histological alterations in the liver such as centrilobular necrosis, vacuolation in hepatocytes and loss of hepatic architecture. TBA and TBA + TCA caused periportal proliferation and lymphocytic infiltration. Hypertrophy of hepatocytes in the periportal area was a characteristic feature in the liver of TCA treated rats. Moreover, in the histology of the kidney, in the three treated groups, degeneration of renal tubules, with syncitial arrangements of the nucleus of renal tubular epithelial cells was evident. In addition to this, degeneration of the basement membrane of the Bowmans capsule, diffused glomeruli and vacuolation of glomeruli was also evident in the three treated rat kidneys. Renal tubular proliferation in certain areas was also evident in certain areas of the kidney in TCA treated rats. The results indicate that, TBA and TCA do bring about alterations in histology of liver and kidney, but on combined administration, do not show enhanced toxicity in the form of increased hepatic and renal injury.

Animals↗
PubMed1997

Parametric representation and screening of knee joint vibroarthrographic signals.

We have been investigating analysis of knee joint vibration or vibroarthrographic (VAG) signals as a potential tool for noninvasive diagnosis and monitoring of cartilage pathology. In this paper, we present a comprehensive comparative study of different parametric representations of VAG signals. Dominant poles and cepstral coefficients were derived from autoregressive models of adaptively segmented VAG signals. Signal features and a few clinical features were used as feature vectors in pattern classification experiments based on logistic regression analysis and the leave-one-out method. The results using 51 normal and 39 abnormal signals indicated the superior performance of cepstral coefficients in VAG signal classification with an accuracy rate of 75.6%. With 51 normal and 20 abnormal signals limited to chondromalacia patella, cepstral coefficients again gave the highest accuracy rate of 85.9%.

Algorithms↗