Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples.

PURPOSE: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. METHODS: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65 kDa, 38 kDa and 85 B. RESULTS: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P < 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P> 0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. CONCLUSIONS: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

Protein antigen b (Pab) based PCR test in diagnosis of pulmonary and extra-pulmonary tuberculosis.

BACKGROUND AND OBJECTIVES: Diagnosis of tuberculosis (TB) is largely based on microscopy and culture examination which are either less sensitive, or time consuming. In the present study a PCR (polymerase chain reaction) test based on DNA sequence coding for a 38-kilodalton protein antigen b (Pab) ,specific for Mycobacterium tuberculosis was compared with Ziehl-Neelsen (ZN) stained AFB (acid fast bacilli) smear examination, culture based on conventional Lowenstein-Jensen (LJ) medium and radiometric BACTEC 460 system for the diagnosis of TB using clinical samples obtained from pulmonary and extra-pulmonary cases of TB. METHODS: Clinical samples obtained from 168 patients of suspected TB (pulmonary and extrapulmonary) were subjected to ZN smear examination, LJ culture, radiometric BACTEC culture and a PCR test by amplifying 419 bp sequence coding for Pab, a glycoprotein of molecular weight 38 kDa. RESULTS: A significant difference was seen in the sensitivity of different tests, the figures being 74.2 per cent for PCR test, 53.4 per cent for BACTEC culture, 47.1 per cent for LJ medium based culture and 35.2 per cent for ZN smear examination (P<0.05). However, there was no significant difference between different tests as far as specificity was concerned. PCR test sensitivity in pulmonary and extra-pulmonary clinical samples were 74.3 and 71.5 per cent respectively, being significantly higher (P<0.05) when compared with sensitivity of other tests. The mean detection time for M. tuberculosis was 24.0 days by LJ media culture, 12.8 days by BACTEC culture and less than 1 day by smear examination and PCR test. INTERPRETATION AND CONCLUSION: PCR test is more sensitive than ZN smear examination, LJ medium culture and BACTEC culture for diagnosing TB in pulmonary and extra-pulmonary clinical samples.

Amelioration of altered antioxidant status and membrane linked functions by vanadium and Trigonella in alloxan diabetic rat brains.

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on anti-oxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na+/K+ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P less than 0.001), decreased activities of SOD, catalase and Na+/K+ ATPase (P less than 0.01, P less than 0.001 and P less than 0.01), increased levels of GPx and MDA (P less than 0.01 and P less than 0.001) and decreased membrane fluidity (P less than 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose of Trigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain.

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.

Dietary protein deficiency induced changes in protein kinase C activity and phospholipid metabolism in rat hepatocytes.

Dietary protein deficiency is known to alter the protein kinase C activity in various tissues of rats. Protein kinase C activity is influenced by the metabolism of membrane phosphoinositides and phosphatidyl choline (PC). For metabolic studies, hepatocytes have been the cells of choice of various workers. Therefore, studies on protein kinase C and these phospholipids were conducted in hepatocytes of rats maintained on three different diets viz. casein (20% protein) deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) diet for 28 days. The protein deficiency in diet led to a decline in protein kinase C activity (P < 0.01) without effecting its translocation, an increase in phosphatidyl inositol 4,5-bisphosphate (P < 0.001) and a decrease in phosphatidyl inositol 4-monophosphate and phosphatidyl inositol (P < 0.01) but did not alter the PC contents, as compared to the casein group. Supplementation of deficient diet with L-lysine and DL-threonine could considerably reverse the effect of deficiency of protein in diet. The results suggest that quality of dietary protein is mainly relevant for maintaining phospholipid metabolism and physiology of hepatocytes and thus the signalling mechanism in these cells.

Effect of dietary protein manipulation on translocation of protein kinase C activity in various tissues of rats.

Ingestion of protein deficient diet is known to decrease the enzyme load, particularly drug metabolising enzymes in liver. It also leads to decrease in polyphosphoinositide pool in brain and kidney. Therefore, changes in protein kinase C activity and its translocation were speculated and studied in brain, lung, heart, spleen, liver and kidney of rats maintained on three different diets, viz. casein (20% protein) deficient (4% protein, rice flour as protein source) and supplemented (deficient diet supplemented with L-lysine and DL-threonine), for 28 days. A significant alteration in total protein kinase C activity and/or its translocation was observed in these tissues in the deficient group in comparison to casein group. Supplementation of diet with L-lysine and DL-threonine could partially reverse the affect. These changes in protein kinase C activity and its translocation indicate alteration in the mechanism of signalling system in dietary protein deficiency and hence an altered response of tissues to the external stimuli in dietary protein deficiency.