Salil C. Datta

Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor in Leishmania donovani

A calcium dependent proteolytic enzyme was detected in the lysed promastigotes ofLeishmania donovani, the causative agent of kala-azar. The protease was able to hydrolyse and added substrate, azocasein and showed high affinity for calcium. Rate of azocasein digestion was primarily slow but boosted up after eight hours. It was not inactivated when heated at 55° C for 15 min at pH 7.4. Sulfhydryl reagents significantly reduced the enzymic activity but trypsin-like protease inhibitors hardly had any effect. The enzyme was not sensitive to calmodulin from a heterologous source but registered low activity when treated with chlorpromazine. The caseinolytic activity was stimulated when leishmanial cells were preincubated with ionophore A23187 in presence of 1 mM Ca2+. The enzyme is named caldonopain due to its similarity with a general class of calcium dependent protease calpain present in different tissues and cells. Caldonopain was found to be localized in cytosol along with its specific endogenous inhibitor caldonostatin. The ratio of caldonopain-caldonostatin unit was higher in the infected macrophage compared to the parasitic protozoa and Balb/c macrophage alone. It may be postulated that the amount of both calcium and its protein inhibitor may have a direct impact on the caldonopain-induced biological process to regulate cellular action of this pathogen.

Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection

A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca2+ affects proteolytic activity after 10 h. When host–parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.

Changes in Free Amino Acid Levels in Developing Human Foetal Brain Regions

The levels of free amino acids were determined in human foetal brain regions during prenatal development. Variation in the distribution of amino acids and their rate of change in five segments of the CNS at different stages of ontogeny was observed. Striking developmental changes were found in the levels of aspartic acid in medulla‐pons and spinal cord, glycine in the spinal cord, γ‐aminobutyric acid in the cerebral cortex, glutamic acid in the cerebral cortex, midbrain, and spinal cord, and taurine in the medulla‐pons and spinal cord. At a late gestational period, glutamic acid was found most abundantly over all the brain regions, whereas the level of taurine was highest at an early gestational stage but not in spinal cord.

Cyclic AMP mediates change in superoxide dismutase activity to monitor host-parasite interaction in Leishmania donovani

The present study is designed to understand the role of cyclic AMP (cAMP) in host-parasite interaction involving Leishmania donovani, the causative agent for Kala-azar. When Leishmania promastigotes or macrophages were pretreated with dibutyryl cAMP or theophylline and epinephrine, which are well defined initiators for cAMP release, a key enzyme of the oxygen defense system, superoxide dismutase (SOD), was inhibited. At the same time, parasite interaction was considerably reduced to the level of 54.5% and 46.2%, respectively, for pretreated promastigotes. Internalization of the organisms in phagolysosomes was similarly affected. Dibutyryl cAMP-treated promastigotes in the presence of SOD, on the other hand, restored in vitro infection to the normal level. At least 50% less cAMP entered into Leishmania promastigotes when SOD was added to the incubation system containing dibutyryl cAMP. Data reveal that cAMP perturbs the Leishmania-macrophage interaction through inhibition of SOD, pointing to the importance of a promastigote enzyme for the survival of this pathogen within phagolysosomes.

Reevaluation of Taurine Levels and Distribution of Cysteic Acid Decarboxylase in Developing Human Fetal Brain Regions

Abstract: The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HC1. Cysteic acid thus produced was separated from taurine by ion‐exchange chromatography using Biorad‐AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.

Repair of Impaired Host Peroxisomal Properties Cropped Up Due to Visceral Leishmaniasis May Lead to Overcome Peroxisome Related Genetic Disorder Which May Develop Later After Treatment

The leishmaniasis is a group of diseases caused by protozoan haemoflagelates of the genus Leishmania1,2. These parasites belong to the family of the Trypanosomatidae (order Kinetoplastida) and are closely related to the Trypanosomes3. Despite enormous efforts, it has proved difficult to predict the exact scale of the impact of leishmaniasis on public health, since many cases remain unreported or misdiagnosed4. It is estimated that approximately 12 million people are currently infected and a further 367 million are at risk of acquiring leishmaniasis in 88 countries, 72 of which are developing countries and 13 of them are among the least developed in the world1, 4. Hence we can link leishmaniasis to poverty, economic development and various environmental changes such as deforestation, urbanization, migration of people into endemic areas and building of damns etc5. The annual incidence rate is estimated to be 1 to 1.5 million cases of cutaneous leishmaniasis (CL) and 5,00,000 cases of visceral leishmaniasis (VL); these are the two major clinical types of leishmaniasis6. The only proven vector of the Leishmania parasite is the blood-sucking female sandfly1, 7 of the genus Phlebotomus in the old world and Lutzomyia in the new world8. The insects are 2-3 mm long (one-third the size of typical mosquitoes) and are found throughout the tropical and temperate parts of the world. The sandfly larvae require organic matter, heat and humidity for development and so are commonly found in house-hold rubbish, burrows of old trees and in cracks in house walls9. The sand flies usually feed at night while the host is asleep10. There are five most important Leishmania species namely L. tropica, L. major, L. donovani, L. braziliensis braszliensis, L. b. peruviensis and L. mexicana which cause the three forms of the disease dermal CL (oriental sore), VL and mucocutaneous leishmaniasis (Chicleros diseases and Espundi)11, 12, 13. Leishmania exhibits a dimorphic life cycle14 involving two life-cycle stages, the elongated promastigote with free flagellum present in the insect and the intracellular amastigote form15.