Biomedical subjects

P Ramaprasad

Publications and source records attributed to P Ramaprasad.

11 recordsLinked to original sources

PubMed2005

Identification and characterization of tyrosylprotein sulfotransferase from human saliva.

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl(2) for its activity. Maximum TPST activity was observed at 20 mM MnCl(2). The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.

Chlorides↗

PubMed1998

Isolation of tyrosylprotein sulfotransferase from rat liver.

1. Tyrosylprotein sulfotransferase (TPST) is involved in the posttranslational modification of proteins and plays a critical role in the biological activity and secretion of proteins. A simple method has been developed to isolate the TPST (28% yield) from rat liver, using polyclonal anti-TPST antibodies. 2. The protein fractions eluted from antibody affinity column showed TPST activity and revealed a 50-54 kDa protein band in the silver stained SDS-polyacrylamide gels. 3. The enzyme exhibited optimum activity at pH 5.5 with 20 mM MnCl2. Unlike the TPST activity of the Golgi membrane, the activity of the purified enzyme was not stimulated by NaF, 5'-AMP, and Triton X-100. 4. The antibody was also used to study the TPST protein turnover in rat liver of animals that were given [35S]methionine. The TPST protein synthesis assessed by measuring initial rates of incorporation of [35S]methionine into TPST protein showed enzyme synthesis for up to 60 min. 35S-labeled TPST protein of rat liver was degraded with a half-life of 30 hr. 5. The immunoaffinity purification method using rat liver as an enzyme source appeared to be very simple, rapid, and easy to perform with significant enzyme recovery. Further, the antibody was also found to be useful in the study involving TPST protein metabolism.

Animals↗

PubMed1998

Stimulation of tyrosylprotein sulfotransferase activity by ethanol: role of increased enzyme level.

Tyrosylprotein sulfotransferase (TPST), an enzyme involved in the posttranslational modification of proteins, plays important role in the biological activity and secretion of proteins. Previously we have shown an increased activity of this enzyme in gastric mucosa of alcoholics. In the present study, effect of ethanol on TPST was examined in rat liver and gastric mucosa utilizing enzyme assays and Western blot analyses for TPST levels. Male Sprague-Dawley rats were pair-fed Leiber-DeCarli liquid diets for 10 days and controls received a liquid diet in which dextrose was isocalorically substituted for ethanol. After ethanol feeding, rats were sacrificed and liver and gastric mucosa were processed for Golgi membrane preparation. The TPST activity was measured using poly(Glu6, Ala3, Tyr1) as the sulfate acceptor and PAPS as sulfate donor. There was a threefold increase in TPST activity of gastric mucosa of animals subjected to chronic alcohol feeding. In the liver, the increase in tyrosine sulfating activity was also around threefold. The kinetic studies performed to understand the mechanism involved in ethanol stimulation of TPST activity showed no change in the Km values of the enzyme by ethanol. In control and ethanol-treated animals, the Km for EAY was 0.41-0.53 and 0.43-0.53 microM, and the Km for PAPS was 10-12.5 and 9-17 microM, respectively. The Vmax in ethanol-fed animals was increased by 1.5- to 2-fold. The increase in TPST activity in experimental rats was further tested by analyzing the Western blots by Imaging Densitometer for TPST levels. Analysis of TPST levels also showed over threefold increase in the stomach and liver of ethanol-fed rats. Our results indicate that stimulation of TPST by ethanol involves increased TPST level rather than change in affinity for its substrates.

Animals↗

PubMed1998

In vivo induction of tyrosylprotein sulfotransferase by ethanol: role of increased enzyme synthesis.

Tyrosine sulfation is a posttranslational modification involved in the synthesis, secretion, and biological activity of proteins and peptides. Our previous studies have demonstrated that the enzyme activity was induced by ethanol. In the present work, the induction was studied in detail. Initial experiments were conducted to examine the time course of tyrosylprotein sulfotransferase (TPST) induction in rats pair-fed liquid diets containing either ethanol or carbohydrate substitute (controls). Marked elevation of TPST activity (3-fold) was measured on day 10 in the liver and gastric mucosa of ethanol-fed rats. Ethanol-mediated enhancement was also noticed by Western-blot analysis with anti-TPST antibody in both the liver and gastric mucosa on days 5 and 10. We then determined the steady-state TPST protein turnover in ethanol-fed and control animals that were given 35S-methionine after 10 days of pair-feeding with liquid diet. The rates of TPST synthesis assessed by measuring initial rates of incorporation of 35S-methionine into TPST was increased in the liver and gastric mucosa of animals fed with ethanol. Monophasic exponential decay curves showed that TPST protein half-lives for liver (control: 34 hr, ethanol: 32 hr) and gastric mucosa (control: 52 hr, ethanol: 48 hr) did not differ between control and ethanol groups. Our overall results indicate that the in vivo induction of TPST by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation.

Animals↗

PubMed1997

Purification of tyrosylprotein sulfotransferase from rat submandibular salivary glands.

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands. In the present study, this enzyme was purified from the Golgi membranes of rat submandibular salivary glands using a Cibacron blue F3GA affinity column chromatography. Antibodies raised in rabbit against TPST detected the purified enzyme (50-54 kDa) and proteins consisting of molecular mass 50-54 kDa in the Golgi membranes of liver, submandibular salivary glands, stomach, cerebellum, thalamus, and pituitary. The protein levels in liver and salivary glands were higher compared to those found in the stomach, cerebellum, thalamus, and pituitary. The levels of immunoreactivity in cytosol and endoplasmic reticulum fractions of salivary glands were either undetectable or very low. The antibody was also used to immunoprecipitate the TPST activity and to isolate protein by immunoaffinity column. MnCl2 was required for the purified TPST. The enzyme exhibited optimum activity between pH 6.2 and 6.8 at 20 mM MnCl2. The apparent K(m) values of the purified enzyme for poly-(Glu6, Ala3, Tyr1) (EAY: M(r) 47,000) and 3'-phosphoadenosine 5'-phosphosulfate were 3 and 20 microM, respectively. The results presented here collectively demonstrate the purification of TPST and, for the first time, development of polyclonal antibody that recognizes this enzyme.

Animals↗

PubMed1997

Transmission and protection in leprosy: indications of the role of mucosal immunity.

Recent advances in treatment have achieved a large drop in the prevalence of active leprosy cases, but the incidence is at best decreasing slowly. Most people within leprosy-endemic populations have been exposed to Mycobacterium leprae, but few develop disease and it seems likely that the majority of the population develops protective immunity. If the site of initial infection is in the nose, dissemination of bacilli around the body to skin and nerve implies that the initial infection is bacilliferous and it has been shown that nasal M. leprae are detectable by polymerase chain reaction (PCR) of nasal swabs. Since salivary anti-M. leprae IgA (sMLIgA) levels are correlated with protection, we have surveyed groups of leprosy patients, contacts and the general population for both their sMLIgA and nasal PCR positivity. A total of 304 subjects were enrolled in the study: PCR and mucosal challenge tests were performed in 204 of these individuals. sMLIgA was present in 66% of treated patients, 76% of leprosy workers and 72% of healthy contacts. However, only 33% of indigenous subjects were sMLIgA+, in contrast to the earlier studies showing 74% positivity. PCR for M. leprae was present in both household contacts (2%) and indigenous controls (5%). In a subsequent follow-up study, nasal swabs were taken from 97 of those studied in the first series: three PCR+ individuals followed up after one year became negative, while of the remaining 94 PCR- individuals retested, 2 became positive. Of 112 subjects retested with the mucosal challenge test for sMLIgA: 22 converted from positive to negative and 12 from negative to positive. These results suggest that there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response, despite the fact that few individuals will develop clinical disease. This may explain the current lack of effect of multidrug therapy (MDT) control programmes on incidence, although the reduction in general population immunity is consistent with some effect of MDT on transmission.

Adolescent↗

PubMed1995

Development of a mucosal challenge test for leprosy using leprosin A.

There is little information about the mucosal immune response in leprosy. We have developed a nasal provocation test with leprosin A which will be used to investigate mucosal immunity to Mycobacterium leprae. Initial studies were performed with increasing doses of leprosin A (1.0 pg/ml-10 micrograms/ml) to determine the optimal safe dose of leprosin A. Anti-M. leprae IgA antibody and normal IgA concentrations were measured in the saliva of leprosy contacts and controls before and after instillation of leprosin A. Nasal leprosin A was well tolerated up to a concentration of 10 micrograms/ml without side effects. None of the six subjects who had not been exposed to leprosy had salivary IgA against whole M. leprae, whereas IgA was detected from 64 h to 140 h following instillation of leprosin A in all of the leprosy hospital workers and in 15 out of 18 healthy household contacts tested. There was no correlation between serum and salivary anti-M. leprae IgA levels before and after testing. Salivary IgA anti-lipoarabinomannan responses were seen in 12 out of 20 household contacts. Normal salivary IgA concentrations varied from 8 to 240 mg/l. The leprosin A nasal provocation test appears to be a safe method for the investigation of the role of mucosal immunity in the pathogenesis of leprosy.

Adolescent↗

PubMed1995

Use of fractionated urinary filarial antigen in the diagnosis of human filariasis.

Fractionated urinary filarial antigen UFA C2 has shown high antigenic activity after absorption of urinary albumin present in the fraction. As little as 500 ag (10(-18) g) of albumin absorbed UFA C2, labelled as UFA C2-A, was found to be sufficient to detect filarial antibody. Stick enzyme immunoassay to assess the immunodiagnostic potential of UFA C2-A indicated filarial IgG antibody in 89% of microfilaraemic (mf) cases, 84% of clinical filariasis and 7% of endemic normals. UFA C2-A was found to be present in circulation in active as well as clinical infections as observed by inhibition assay using UFA C2-A penicillinase conjugate. Eighty-six per cent of mf, 50% of clinical cases and 6% of endemic normal subjects revealed parasite antigen to UFA C2-A on further serological analysis. None of the non-endemic normal sera showed the presence of filarial antibody/antigen to UFA C2-A. Furthermore, the test to determine phosphorylcholine (PC) bearing epitopes in UFA C2-A indicated no immunological reaction with anti-PC monoclonal antibody by avidin-biotin enzyme linked immunosorbent assay (ELISA). The highly sensitive and more easily obtainable non-PC urinary filarial antigen, UFA C2-A, is of great immunodiagnostic interest for lymphatic filariasis.

Animals↗

PubMed1990

Efficacy of two different DEC regimens in the treatment of human filarial infection.

Two regimens of diethyl carbamazine (DEC) viz., 14 day and 5 day, were compared for microfilaricidal effect and side effects, in the treatment of bancroftian filariasis. The rate of successful treatment, cure rate and decrease in mf count were found to be significantly high with the 14 days regimen when assessed immediately after treatment. About 40 per cent of subjects on the 14 days regimen and 66 per cent of patients on 5 days regimen experienced side reactions. The severity of side reactions was more in patients on 5 days regimen. When the effect of DEC was assessed one year after treatment with the 14 days regimen and compared with the results immediately after treatment, the rate of successful treatment, cure rate and decrease in mf count were reduced significantly. The 14 days DEC regimen with initial low dose of DEC along with antipyretics may be better accepted in the control programmes of filariasis.

Adolescent↗

PubMed1988

Microfilaraemia, filarial antibody, antigen and immune complex levels in human filariasis before, during and after DEC therapy. A two-year follow-up.

A study on the effect of DEC therapy on microfilaraemia and the immune status in 27 patients with W. bancrofti infection was carried out for two years. Persistence of microfilaraemia was observed in 4 out of 27 cases after one course of DEC therapy and were treated again for one week. On further follow-up, none was microfilaraemic upto Day 60. The mean filarial antibody titres of IgM and IgG showed a gradual decrease as assessed by enzyme linked immunosorbent assay (ELISA). The mean titres of circulating microfilarial excretory-secretory (ES) antigens and immune complexes (ICs) showed an initial increase during therapy, followed by a gradual fall upto Day 60. Filarial antigen was detected in urine of all the carriers during therapy. Excretion pattern of antigen in urine showed correlation with DEC dose. Reappearance of microfilariae (mf) in circulation in 12 patients after a year showed that DEC had temporary attenuating effect on adult worms or no effect on developing larvae, suggesting further treatment and follow-up of patients. Parasitological and immunoscreening at the end of 2 years showed that the presence of mf ES antigen in blood correlated with the appearance of microfilariae in blood.

Animals↗

PubMedDate not supplied

Fractionation, characterization and diagnostic potential of filarial antigens isolated from hydrocoele fluid in bancroftian filariasis.

Filarial antigen was isolated from hydrocoele fluid and fractionated on Ultrogel AcA 44. Six protein fractions (HFA C1 to HFA C6) were chromatographically separated from the column. Of the 4 antigenic fractions (HFA C1, HFA C2, HFA C3 and HFA C5), HFA C3 was a major reactive fraction with filarial serum immunoglobulin G. Analysis of HFA C3 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded 18 bands of Mr 18,000-160,000. Two fractions, HFA C3-7 and HFA C3-9 of Mr 48,000-55,000 and 32,000-39,000 obtained by gel elution, were antigenic in an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE immunoblotting confirmed the ELISA by identifying 3 immunoreactive bands of Mr 51,000, 38,000 and 35,000. The diagnostic potential of HFA C3-7 and HFA C3-9 was compared in serodiagnosis of active infections and diseased states. HFA C3-9 showed greater sensitivity in detection of filarial specific IgM antibody in cases with microfilaraemia. Physicochemical characterization indicated the protein nature of HFA C3-9.

Animals↗