Biomedical subjects
P Sundaram
Publications and source records attributed to P Sundaram.
Viral hepatitis B with pleural complication.
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A simplified 'coupled' probing approach for the detection of proteins on dot and western blots.
Western blotting is one of the most commonly and widely used methods for identifying the presence of an antigen in biochemical studies. The present study describes a novel 'coupled blotting' approach that allows antigen on dot and Western blots to be simultaneously probed with primary and secondary probing reagents. The study utilized the highly sensitive enhanced chemiluminescence (ECL) detection system, purified E7 protein of cottontail rabbit papillomavirus (CRPV) and E7-specific antibodies. Comparison of the abilities of sequential primary antibody followed by secondary reagent and coupled treatments to detect E7 protein on blots showed no reduction in signal strength after coupled probing. The new 'coupled' probing has the advantage of saving hands-on time and buffer solutions over the standard procedure. Overall, the results suggest that the 'coupled blotting' is useful for the rapid detection of proteins without compromising quality.
Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen).
Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta-galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants.
Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus.
Cottontail rabbit papillomavirus (CRPV) E7 protein is one of the :high risk' papillomavirus E7 oncoproteins that are partially insoluble in aqueous solution. An Escherichia coli expression system was used for purification of CRPV E7 protein in quantities sufficient for immunologic studies and structural analysis. A glutathione S-transferase (GST)-CRPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione Sepharose beads. The CRPV E7 portion was cleaved from the column with thrombin at a thrombin cleavage site between the fused partners. Thrombin was removed subsequently by adsorption to benzamidine. This method is rapid, requiring just one week, and efficient, yielding 3 mg of pure CRPV E7 protein per liter of bacterial culture. It produced a protein product that was about 95% pure. High-titered polyclonal antisera generated to the product recognized CRPV E7 but not GST. Purified CRPV E7 protein exhibited the ability to bind pRB, making it the first unfused, non-denatured CRPV E7 product reported to do so. This attribute could facilitate structure-function studies of CRPV E7-pRB interactions.
Bochdalek hernia presentation in an adult.
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Adenocarcinoma of the lung--pleomorphic radiographic appearances.
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Chiladiti's syndrome in an adult with chronic obstructive airway disease.
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Lipid peroxidation, antioxidant defences and red-cell membrane changes in relation to coronary risk index and symptomatic coronary heart disease.
OBJECTIVE: To establish the normal lipoprotein profile in the population and identify the early warning signs of coronary heart disease (CHD). DESIGN: Random blood sampling of healthy adults and patients with symptomatic CHD including that complicated with acute myocardial infarction. METHODS: Plasma lipids, lipoproteins, lipid peroxidation, antioxidant enzymes and scavengers, red-cell membrane lipids and glycoproteins were assayed. RESULTS AND CONCLUSION: The normal levels of plasma lipids and lipoproteins were established. Levels of plasma free fatty acids, fibrinogen, white blood cell counts, echinocytes, red-cell membrane lipids and protein-bound carbohydrate components are significantly higher in healthy subjects with coronary risk index above 4.5 than they are in normal individuals. Antioxidant defences appear to be the distinguishing factor, remaining higher in normal individuals and thus keeping lipid peroxidation under control. In symptomatic CHD, antioxidant defences are significantly lowered.
Inhibition of tyrosylprotein sulfotransferase by sphingosine and its reversal by acidic phospholipids.
Although tyrosylprotein sulfation has been implicated in the processing of several secretory proteins, nothing is known about the regulation of the enzyme responsible for this event. When poly(Glu6, Ala3, Tyr1) (EAY; M(r) 47,000) was employed as sulfate acceptor, the tyrosylprotein sulfotransferase (TPST) from Golgi membranes of submandibular salivary gland was used to study the effect of various lipids on the expression of its activity. The TPST activity in the Golgi membrane was 38 pmol (mg of protein)-1 (30 min)-1. Approximately 90% of the total activity present in Golgi membranes was extracted by NaCl and Triton X-100 treatment. The Km values of solubilized TPST for EAY and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were 0.04 and 0.25 microM, respectively. Among the various lipids tested, sphingosine showed maximum inhibition of TPST activity followed by sphingomyelin and phosphatidylcholine (PC). Of the two sphingosine analogs tested, threosphinganine was as effective as sphingosine in TPST inhibition, while erythrosphinganine had little effect. In contrast, the acidic phospholipids phosphatidylinositol (PI) and phosphatidylserine (PS) and oleic acid showed slight stimulation. Half-maximal inhibition of TPST was obtained at 150 microM sphingosine (6 mol % when expressed as mole percent of sphingosine to total phospholipids plus Triton X-100). The inhibition was competitive with respect to EAY and uncompetitive with respect to PAPS. The inhibition caused by sphingosine could be reversed by PI, PS, and oleic acid but not by PC and sphingomyelin. Sphingosine inhibition of TPST activity was also observed in the enzyme isolated from several other tissues such as liver, lung, heart, and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium transport and calcium activated ATPase activity in microsomal vesicles of rat gastric mucosa.
1. Microsomal and plasma membrane vesicles, isolated from rat gastric mucosa, were found to exhibit Ca(2+)-dependent ATPase activities of 14.1 +/- 1.4 and 7.8 +/- 1.1 mumol/mg/hr, respectively. The optimum conditions for the microsomal Ca(2+)-ATPase was pH 6-7, and required Mg2+, while divalent cation such as Cu2+, Zn2+, Fe2+, Ba2+ and Cd2+ had no significant effect. 2. As in the case of Ca2+, Mg(2+)-ATPase, the Ca2+ uptake activity of the microsomal membrane required Mg2+. Both processes were stimulated by submicro molar concentrations of Ca2+ and the apparent Km for Ca2+, Mg2+ ATPase and Ca2+ uptake activities were 0.06 microM and 0.02 microM, respectively. 3. Divalent cations Ba2+ and Fe2+, inhibited both microsomal activities, while Zn2+ and Cd2+ showed no effect on them. However, the monovalent cation K+ did not stimulate Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities. 4. The Ca2+ pumping ATPase of rat gastric mucosal microsome cross-reacted with a monoclonal antibody (mAb-5F10) against the human erythrocyte Ca2+ pump. The apparent molecular weight of mucosal Ca2+ pump was 98 kDa. 5. Close relationship between the kinetic parameters of Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities, and the cross reaction of 98 kDa protein of mucosal microsome with erythrocyte Ca2+ pump antibody, strongly suggest the expression of Ca2+ pump in rat gastric mucosa.
Glycosulfatase activity of Helicobacter pylori toward gastric mucin.
A glycosulfatase activity toward sulfated gastric mucus glycoprotein was identified in the extracellular material elaborated by H. pylori, a bacteria implicated in the etiology of gastric disease. Upon acetone precipitation, an active enzyme fraction at 64% acetone was obtained which on SDS-PAGE gave a major 30kDa protein band. The H. pylori glycosulfatase exhibited maximum activity (314.8 pmol/mg protein/h) at pH 5.7 in the presence of Triton X-100 and CaCl2, and was capable of removal of the sulfate ester groups situated at C-6 of N-acetylglucosamine, galactose and glucose. However, the enzyme was ineffective toward galactosylceramide and lactosylceramide sulfates which contain the sulfate ester group on C-3 of galactose. The results suggest that H. pylori is capable of overcoming the interference by sulfated mucus glycoprotein with its colonization of gastric mucosa.
Tyrosylprotein sulfotransferase in rat submandibular salivary glands.
1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu6, Ala3, Tyr1) (EAY; Mr 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2 mM 5'AMP, 20 mM MnCl2 and 50 mM NaF at pH 6.2. 2. The apparent Km values for EAY and PAPS were 1.6 x 10(-6) and 1.9 x 10(-6) M, respectively. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.
Diagnostic significance of circulating immune complexes in patients with pulmonary tuberculosis.
A polyethylene glycol (PEG) precipitation method was used to examine sera of patients with active pulmonary tuberculosis (PT), leprosy and non-tuberculous pulmonary diseases and of healthy control subjects for immune complexes (ICs). Mycobacterium tuberculosis antigen 5 was detected in the ICs in 80% of patients with PT by the indirect (sandwich) enzyme-linked immunosorbent assay (ELISA). Detection of mycobacterial antigen in ICs has diagnostic potential as an adjunct in the laboratory diagnosis of PT, particularly when repeated bacteriological investigations for M. tuberculosis in clinical specimens are negative. Levels of ICs tend to decrease with the duration of anti-tuberculosis chemotherapy and their detection can also be used to assess the clinical response to therapy in patients with PT.
Identification of tyrosylprotein sulfotransferase in rat gastric mucosa.
An enzyme activity which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-Glu6,Ala3,Tyr1 (EAY; M(r) 47,000) has been demonstrated in the antral and body mucosa of the rat stomach. The distribution of this tyrosylprotein sulfotransferase was similar to that of the Golgi marker enzyme, glycoprotein sulfotransferase, and its activity from body mucosa was 23% higher than that from the antrum. The optimum for tyrosylprotein sulfotransferase activity was obtained at pH 6.8, in the presence of 0.5% Triton X-100, 20 mmol/l MnCl2, 50 mmol/l NaF, 2 mmol/l 5'-AMP, and 1 mmol/l DTT, whereas Ca2+, Mg2+, Cu2+, Zn2+, EDTA, NEM, NaCl and Na2SO4 were inhibitory. The apparent Km of the sulfotransferase for EAY was 1.5 x 10(-6) mol/l and for PAPS 0.75 x 10(-6) mol/l. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition as compared to that required for phenol sulfotransferase inhibition. The tyrosine sulfation by the tyrosylprotein sulfotransferase was independent of the sulfation of carbohydrate residues in mucous glycoproteins and glycolipids, thus indicating that the identified sulfotransferase is specific for sulfation of the tyrosyl residues in the peptide core.
Tyrosylprotein sulfotransferase activity is increased in human gastric mucosa of alcoholics.
Sulfation plays a major role in the processing of secretory proteins. We report here on tyrosylprotein sulfotransferase activity in human gastric mucosa of normal and alcoholics. The tyrosylprotein sulfotransferase was identified in the Golgi-enriched fraction. In alcoholics, the activity of sulfotransferase was 2- to 3-fold higher than in normals. However, no change in the activity of UDP-glucose-ceramide glucosyltransferase, a marker for Golgi, between alcoholics and normals was observed. The tyrosylprotein sulfotransferase enzyme required Triton X-100, MnCl2 and 5'-AMP, and obtained optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 20 mM MnCl2, 50 mM NaF, and 2 mM 5'-AMP. The apparent Km for poly-Glu6, Ala3, Tyr1 (EAY; 47,000) was 1.9 x 10(-6) M and for 3'-phosphoadenosine 5'-phosphosulfate (PAPS), 1.4 x 10(-6) M. The results suggest that alcohol abuse causes enhancement in the expression of gastric mucosal tyrosylprotein sulfotransferase activity.
Double-blind cross-over study of modified Anna Pavala Sindhooram in patients with hyperlipidemia or ischemic heart disease.
The plasma lipid lowering effect of modified Anna Pavala Sindhooram (APSm) was tested in 30 patients with plasma lipid abnormalities and with symptomatic ischemic heart disease (IHD) in a double-blind cross-over design. HDL2 cholesterol and total HDL cholesterol were increased during APSm therapy leading to a reduction in the coronary risk index. Plasma cholesterol, LDL and VLDL cholesterol, triglycerides, phospholipids and free fatty acids were significantly reduced during APSm therapy while withdrawal of the drug led to a slow increase in these lipids. No adverse side effects were noticed. Some patients were able to reduce their daily doses of antihypertensive and vasodilator drugs during APSm therapy.