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Biomedical subjects

D K Ghosh

Publications and source records attributed to D K Ghosh.

At least 55 records · Page 3Linked to original sources
PubMed1991

Biochemical and immunological characterization of exometabolites from an Indian strain of Leishmania donovani promastigotes grown in a chemically defined medium.

Exometabolites (EXOM) of an Indian strain of Leishmania donovani promastigotes isolated from a chemically defined medium by ultrafiltration consisted of proteins, glycoproteins, lipid and lipophosphopolysaccharide (LPPS). LPPS of Mr 40-28 kDa in SDS-PAGE could be labelled metabolically with [32P]-phosphate and recovered in the aqueous phase of hot-phenol-water extraction of EXOM (PE-Aq) along with a glycoprotein of Mr 150-130 kDa (GP150-130). These two molecules could be eluted from DE-52 column with 200 mM NaCl (D2). The 300 mM NaCl (D3) and 400 mM NaCl (D4) eluates from DE-52 column contained one unsaturated polar lipid component. The LPPS had Rf value of 0.65-0.75 in Thin Layer Chromatography (TLC) using saturated phenol water solvent system. EXOM revealed 15 bands in SDS-PAGE of which proteins of Mr 84, 66, 56, 50 and 29 kDa were prominent. When EXOM were fractionated through Con A-Sepharose column, the fraction eluted with alpha-methyl-D-mannoside (Con A-E) had seven bands as revealed by SDS-PAGE of which 25, 16, 13 and 12 kDa glycoproteins were prominent. The antigens present in EXOM can be classified as slower anodic migrating and faster anodic migrating antigens as revealed by immunoelectrophoresis (IEP). The slower anodic migrating antigens, LPPS and GP150-130 recovered in PE-Aq and D2 did not cross-react with kala-azar patients' sera but cross-reacted with homologous anti-promastigote sera. Two faster anodic migrating antigens which could be recovered in organic phase of hot phenol extraction of EXOM (PE-O) and eluted in D3 and D4 and Con A-E, cross-reacted with kala-azar patients' sera. The antigens of both the classes were sensitive to periodic acid oxidation.

Animals↗
PubMed1991

Role of Ca2+ ion on Leishmania-macrophage attachment.

Leishmania donovani, the etiological agent for the disease visceral leishmaniasis, attach themselves to the macrophages for initiation of the disease. The attachment process has been found to be regulated by Ca2+ ions. Verapamil, a Ca(2+)-channel blocker inhibits Leishmania-macrophage attachment. The inhibitory effect is increased with time. Nifedipine, another Ca(2+)-channel blocker exhibits the same effect. The attachment process is stimulated by Ca(2+)-ionophore alone. The inhibitory effects of the calcium channel blockers are reversed by the ionophore.

Animals↗
PubMed1991

Direct enzyme-linked immunosorbent assay: a simple immunoassay using Leishmania donovani promastigote for diagnosis of kala-azar.

For immunodiagnosis of kala-azar enzyme-linked immunosorbent assay (ELISA) and immunofluorescence testing (IFAT) are commonly used. In IFAT, whole parasite antigen and in ELISA the soluble antigen have been used. Preparation of ELISA antigen has certain inherent difficulties. We have developed a simple, specific, and quantitative immunoassay, "direct ELISA" for diagnosis of kala-azar. Intact formalinized promastigote suspension has been used to combine with the antibodies of the patient sera. The colour developed in the supernatant by the enzyme conjugate combined on the parasite surface was measured with a spectrophotometer. The test was able to detect kala-azar-specific antibodies at very high serum dilution and could discriminate between kala-azar and the common diseases prevalent in Asia. The optical densities of the sera of different control groups were significantly low. The method has potential for use as a diagnostic tool in less well equipped laboratories.

Animals↗
PubMed1991

Evaluation of direct agglutination test (DAT) and ELISA for serodiagnosis of visceral leishmaniasis in India.

The direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) were used for serodiagnosis of parasitologically confirmed Indian visceral leishmaniasis (VL) cases. All the sera of VL cases were positive by both the methods. DAT titres of VL cases were greater than or equal to 1:3,200, and ELISA values were greater than or equal to 0.55 1:400 dilution. In the control group, sera of widely prevalent diseases of India, such as leprosy, tuberculosis, malaria, and liver cirrhosis, were included. Both tests could discriminate between VL and other patients of the control group. The sera of post-kala-azar dermal leishmaniasis (PKDL) patients gave OD values of greater than 0:55 and had DAT titres of 1:1,600. Both tests are sensitive and specific for the diagnosis of VL cases. DAT, being simpler and more economical, will be suitable for diagnosis and epidemiological studies for VL under rural conditions of India.

Agglutination Tests↗
PubMed1991

A cloned kinetoplast DNA mini-circle fragment from a Leishmania spp. specific for post-kala-azar dermal leishmaniasis strains.

DNA-DNA hybridization techniques have been found to be very useful in the classification and identification of Leishmania parasites. We report here the cloning of a mini-circle from Leishmania strain UR6 in a plasmid and subcloning of the mini-circle fragments in M13 mp9. Clone MLURk32, containing a 560 bp fragment of mini-circle, has been found to have unique specificity. Application of this specific probe in identifying different Leishmania isolates reveals that the probe reacted only with strains of post-kala-azar dermal leishmaniasis but not with strains or isolates of visceral leishmaniasis.

Animals↗
PubMed1991

Plasma membrane of Leishmania donovani.

Plasma membrane of Leishmania donovani promastigotes was isolated by disrupting the cells in Dounce homogenizer and found to be having two fractions M1 and M2. Chemical analysis of the two membrane fractions revealed that M1 had less RNA content and high sterol-phospholipid molar ratio than M2. M1 was also rich in membrane marker enzymes, e.g., 5' nucleotidase and acid phosphatase. Glucose-6-phosphatase, the marker enzyme of endoplasmic reticulum was higher in M2 fraction. The electron micrograph also revealed the presence of plasma membrane vesicles in M1 fraction.

Animals↗
PubMed1991

Identification of immune complex antigens in sera of Indian kala-azar patients.

Level of circulating immune complex (IC) in visceral leishmaniasis is much higher than that in control sera. In immunoblot experiment, treatment of kala-azar IC with patient sera showed at least 6 bands of which the band around 55 kDa region was most prominent. The band at 55 kDa is primarily due to the presence of an antigen recognized by its corresponding antibody present in the patient sera. This was confirmed by using radiolabelled antibody from kala-azar patient serum and antipromastigote serum. The heavy chain of IgG originating from IC is also present in the same region which was detected by its recognition with antihuman IgG. The IC gave a band at 55 kDa region with sea-urchin antitubulin. Kala-azar sera also reacted with purified rat brain tubulin. The present results suggest that a tubulin like protein is present at 55 kDa region along with the heavy chain of IgG.

Antigen-Antibody Complex↗
PubMed1991

Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles.

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.

Acid Phosphatase↗
PubMed1990

An approach for immunodiagnosis of clinical cases of filariasis.

In this communication an immunodiagnostic approach has been adopted for detection of antigen and antibody in amicrofilaeamic Mf(-) patients by countercurrent immuno electrophoresis (CCIE) and immunodiffusion (ID). Using Setaria cervi and Immune Complex (IC) antigens, out of fifteen clinical cases the number of positive patients in CCIE were twelve and ten respectively. Sixty percent of the Mf(-) cases were positive in antigen detection against both the homologous and heterologous antibody. In ID nine Mf(-) cases gave precipitin bands against S. cervi antigen while with IC antigens ten patients were positive. In similar experiments, it was found that out of fifteen Mf(-) cases nine and eleven patients were positive in antigen detection against microfilaraemic Mf(+) sera and S. cervi antibody respectively. All the Mf(+) cases were positive in both antibody and antigen detection. From the standpoint of immunodiagnosis the data were analysed by two-way analysis of variance study and a newly developed system using Binomial distribution. The sera from the control group were negative in all the immunodiagnostic tests.

Adolescent↗
PubMed1990

Characterization of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India.

1. It is widely accepted that foul or polluted environments are the principal sources of potentially pathogenic species of free-living amoebae. The present paper is the first report of occurrence of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India. 2. We describe the occurrence, isolation, specific identification and comparative mouse pathogenicity test of two pathogenic amoebae, viz., Naegleria fowleri (N. aerobia) Carter, 1970, causing human meningoencephalitis and Acanthamoeba castellanii Douglas, 1930, causing granulomatous amoebic encephalitis, and one non-pathogenic amoeba, viz., A. astronyxis Ray and Hayes, 1959, in sewage samples of Calcutta, India. 3. The existence of both pathogenic and non-pathogenic amoebae living side by side is of considerable epidemiological relevance.

Acanthamoeba↗
PubMed1990

Kinetoplastid flagellates: surface-reactive carbohydrates detected by fluorescein-conjugated lectins.

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.

Animals↗
PubMed1990

Effect of ureastibamine on Leishmania donovani amastigote.

Ureastibamine, a pentavalent antimonial, reduced the parasitic load in the 60-day model of infection of L. donovani in hamsters. It also inhibited the in vivo multiplication of I donovani amastigotes in hamster peritoneal macrophages. No inhibition in either promastigote multiplication or amastigotes transformation was noted with filtrate obtained after incubation of the drugs for 72 h in the macrophage culture. Incubation of macrophages with ureastibamine revealed an impairment in the uptake of deoxyglucose. The effect of ureastibamine was compared with that of another pentavalent antimonial, sodium stibogluconate. It is suggested that impairment of macrophage membrane may contribute towards the adverse effect of these drugs against the intracellular parasite.

Animals↗
PubMed1989

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins.

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.

Acanthamoeba↗
PubMed1989

Evaluation of antileishmanial activity of trans-aconitic acid.

TAA, an inhibitor of the enzyme aconitase, inhibits the growth of L. donovani promastigotes. Morphogenic transformation of the amastigote to the promastigote (table; see text) form in vitro was also inhibited by 2 mM TAA. TAA also reduced multiplication of the parasite in macrophage culture. In the hamster model of leishmania, TAA significantly reduced the parasitic burden of liver. In acute toxicity tests with BALB/c mice no deaths were recorded even at a dose level of 2 g/kg body wt/day.

Aconitic Acid↗
PubMed1988

Estrogen increases precursor for pregnenolone synthesis with temperature-sensitive occupancy of P-450scc in mitochondria of rabbit corpus luteum.

To examine the mechanism of estrogen's direct stimulation of steroidogenesis in the rabbit corpus luteum, we tested the hypothesis that the effect of estrogen on progestin production occurs at the site of processing of the precursor for pregnenolone (i.e. cholesterol) in the mitochondrion. For this purpose, we manipulated a model of estrogen stimulation by 1) removing sc estradiol-filled polydimethylsiloxane capsules from superovulated rabbits on day 9 of pseudopregnancy or 2) leaving the capsules in place to preserve a chronic estrogen stimulus. In the estrogen-deprived rabbits, the serum progesterone level fell precipitously in vivo within 24 h, but in rabbits with chronic estrogen stimulation, serum progesterone levels remained high. Our results show that the loss in progestin production caused by estrogen deprivation could not be attributed to loss of the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc), a common rate-limiting step in progestin synthesis in many steroidogenic tissues. In addition, we confirmed that there was no loss in the catalytic activity of this enzyme. Treatment with aminoglutethimide in vivo followed by electron paramagnetic resonance spectroscopic analysis of mitochondria (prepared in aminoglutethimide-free buffers) showed that incubation of isolated mitochondria at 37 C and pH 6.2 caused an increased high spin state (g = 8.2 signal) and a concomitant decreased low spin state. This shift from low to high spin states, which is indicative of cholesterol-P-450scc complex formation, occurred in the luteal mitochondria from both estrogen-deprived and estrogen-stimulated rabbits. In further studies to localize estrogen's regulatory point, we determined that the initial (first minute) rate of production of pregnenolone (per mg protein or per U P-450scc) from endogenous precursor proceeded equally fast in mitochondria from estrogen-deprived and those from estrogen-stimulated rabbits. However, the rapid pregnenolone production in the estrogen-deprived group lasted for a shorter time and, after 30 min, yielded less pregnenolone per mg protein or per U P-450scc than did mitochondria from estrogen-stimulated rabbits. Addition of 25-hydroxycholesterol did not increase the initial rate of pregnenolone formation, indicating that precursor availability is not limiting during the initial period. In aggregate, these observations suggest that the effect of estrogen on progestin production in the rabbit corpus luteum is not regulation of the movement of cholesterol to the catalytic site on the inner mitochondrial membrane, even though this is a step in the regulation of protein hormone-stimulated steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoglutethimide↗
PubMed1988

Modulation of progesterone synthesis and cytochrome P450 levels in rat luteal cells during human chorionic gonadotropin-induced desensitized state.

Down-regulation of hCG receptors and a steroidogenic lesion in progesterone production were observed in isolated luteal cells after 24-h treatment of pseudopregnant rats with 50 IU hCG. The luteal cells from hCG-desensitized rats exhibited a complete loss of steroidogenic response to lipoproteins in both the absence and presence of hCG. This loss of response was not overcome by the addition of 25-hydroxycholesterol. To examine if the loss of steroidogenic response is due to a transient loss of steroidogenic enzymes or inadequate delivery of substrate cholesterol, cholesterol side-chain cleavage enzyme (P450scc) activity was measured in the isolated mitochondria. No differences appeared in enzyme activity between control and hCG-treated groups. Furthermore, electron paramagnetic resonance spectra of mitochondrial cytochrome P450scc from control and hCG-treated groups exhibited no appreciable differences in low spin electron paramagnetic resonance signal (g = 1.9, 2.2, and 2.4) or high spin EPR signal (g = 8.2), suggesting that the binding of the substrate to P450scc was not impaired. Additionally, the iron sulfur protein signal (g = 1.95 and 2.03) of the reduced mitochondria remained unchanged in the desensitized group. These data were supported by immunoblotting analysis which revealed no difference in the relative amounts of iron sulfur protein or cytochrome P450scc between the two groups. Moreover, the inner mitochondrial membranes, the locus of cytochrome P450scc, showed an identical cholesterol content in control and desensitized mitochondria, indicating that the availability of cholesterol substrate is not impaired during desensitization. These results suggest that the intramitochondrial transfer of cholesterol and cholesterol side-chain cleavage enzyme activity are unaffected in the desensitized state, but an inhibitory substance might be responsible for the lack of steroidogenic response.

Animals↗