Characterization of the DNA-binding domain and identification of the active site residue in the ‘Gyr A’ half of Leishmania donovani topoisomerase II

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. To investigate the biochemical properties of the individual domains of Leishmania donovani topoisomerase II, four truncation mutants were generated. Deletion of 178 aminoacids from the C-terminus (core and LdΔC1058) had no apparent effect on the DNA-binding or cleavage activities of the enzymes. However, when 429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (LdΔNΔC), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (LdΔNΔC, core and LdΔN429) render the mutant proteins incapable of performing ATP hydrolysis. The mutant proteins show cleavage activities at wide range of KCl concentrations (25–350 mM). In addition, the mutant proteins, excepting LdΔNΔC, can also act on kDNA and linearize the minicircles. Surprisingly, the mutant proteins fail to show the formation of the enhanced cleavable complex in the presence of etoposide. Our findings suggest that the conformation required for interaction with the drug is absent in the mutant proteins. Here, we have also identified Tyr(775) through direct sequencing of the DNA linked peptide as the catalytic residue implicated in DNA-breakage and rejoining. Taken together, our results demonstrate that topoisomerase II are functionally and mechanistically conserved enzymes and the variations in activity seem to reflect functional optimization for its physiological role during parasite genome replication

leishmania donovani DNA Topoisomerases i & ii : Molecular Biological Studies in Relation to Their Mode of Action

The term “leishmaniasis” covers a range of disease presentations, most commonly seen in the tropical areas of the New World and around the Mediterranean sea. Leishmaniasis is not a single entity but comprises of a variety of syndromes, primarily due to a variety of parasites affecting different populations and each related to a characteristic vector and animal reservoirs. It is caused by species of intracellular parasite belonging to the genus ‘Leishmania’. The problem is not only complex but also cosmopolitan. Until about 60 years ago leishmaniasis was thought to be important only in India, China, The Middle east, Southern Europe and parts of southern America. But now it is recognized in all continents except in Australia and Antarctica. The synonym “Kala azar” comes from Hindi, and means black poison or black fever, because of the darkening of skin pigmentation on the forehead and hands; other local names are given to different patterns of the disease Leishmaniasis has been called a “dynamic disease” gradually spreading, with new foci of infection arising in scattered areas of the globe. The WHO has estimated that there are 12 million people infected and 350 million at risk (1992) (Fig 1.1). Epidemiological data shows that leishmaniasis at present is the most fatal disease second only to malaria (WHO, 2000). Leishmaniasis infected areas also broadly overlap with areas in which human immunodeficiency virus (HIV) infections are increasing and about a third of the patients die during their first visceral leishmaniasis episode. Over the last 10 years, endemic regions have been spreading further afield and there has been a sharp increase in the number of recorded cases of the disease. Developing countries like Afghanistan, Bangladesh, Bolivia, Brazil, India, Iran, Peru, Saudi Arabia and Syria are worst affected. Unfortunately, as notification is obligatory in only 33 of the 88 countries affected, a substantial number of cases are never recorded. In fact, only 6,00,000 cases of the 2 million actual occurrences are officially recorded per year. In addition, deadly epidemics periodically flare up, as did in Sudan in early nineties (Mortality rate in an excess of 1,00,000) and in Kabul in late nineties (Estimated 2,00,000 cases). Moreover the lack of correlation between Leishmania taxa and the clinical disease produced makes the actual detection and treatment more difficult. Fig. 1.1. Areas showing world wide distribution of Leishmaniasi

Gelatinous Marrow Transformation: A Series of 11 Cases from a Tertiary Care Centre in South India

Gelatinous marrow transformation (GMT) or serous atrophy of bone marrow (BM) is a rare disease characterised by focal marrow hypoplasia, fat atrophy, and accumulation of extracellular mucopolysaccharides abundant in hyaluronic acid. This study reviews 11 cases of GMT from South India. Clinical and haematological parameters, BM aspirate, and biopsies of all patients diagnosed with GMT over a period of 7 years were studied. GMT was diagnosed in BM biopsy based on characteristic morphological appearance and was confirmed by alcian blue positive staining pattern at pH levels of 2.5 and 0.5. Eleven patients were diagnosed with GMT. All were males within the age range of 15 to 50 years. The underlying clinical diagnosis was human immunodeficiency virus positivity in 5 cases, 2 with coexistent disseminated tuberculosis, 1 with cryptococcal meningitis, and 1 with oral candidiasis; disseminated tuberculosis in 1 case; pyrexia of unknown origin in 2 cases; Hodgkin’s lymphoma in 1 case; acute lymphoblastic lymphoma with maintenance chemotherapy in 1 case; and alcoholic pancreatitis in 1 case. BM aspirates showed gelatinous metachromatic seromucinous material in 3 cases. BM biopsies were hypocellular in 7 and normocellular in 4 cases and showed focal GMT in 5 and diffuse GMT in 6 cases. Reactive changes were seen in 4 cases and haemophagocytosis in addition to GMT in 1 case. GMT is a relatively uncommon condition and an indicator of severe illness. It should be differentiated from myelonecrosis, amyloidosis, and marrow oedema. A high index of suspicion is required to diagnose this condition

Reactive Oxygen Species and Imbalance of Calcium Homeostasis Contributes to Curcumin Induced Programmed Cell Death in Leishmania donovani

Curcumin, a polyphenol compound, has been recognized as a promising anti-cancer drug. The purpose of the present study was to investigate the cytotoxicity of curcumin to Leishmania donovani, the causative agent for visceral leishmaniasis. Flow cytometric analysis revealed that curcumin induced cell cycle arrest at G2/M phase. Incubation of Leishmania promastigotes with curcumin caused exposure of phosphatidylserine to the outer leaflet of plasma membrane. This event is preceded by curcumininduced formation of reactive oxygen species (ROS) and elevation of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (DWm), release of Cytochrome c into the cytosol and concomitant nuclear alterations that included deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) and DNA fragmentation. Taken together, these data indicate that curcumin has promising antileishmanial activity that is mediated by programmed cell death and, accordingly, merits further investigation as a therapeutic option for the treatment of leishmaniasis

Characterization of the ATPase activity of topoisomerase II from Leishmania donovani and identification of residues conferring resistance to etoposide

We have cloned and expressed the 43 kDa N-terminal domain of Leishmania donovani topoisomerase II. This protein has an intrinsic ATPase activity and obeys Michaelis–Menten kinetics. Cross-linking studies indicate that the N-terminal domain exists as a dimer both in the presence and absence of nucleotides. Etoposide, an effective antitumour drug, traps eukaryotic DNA topoisomerase II in a covalent complex with DNA. In the present study, we report for the first time that etoposide inhibits the ATPase activity of the recombinant N-terminal domain of L. donovani topoisomerase II. We have modelled the structure of this 43 kDa protein and performed molecular docking analysis with the drug. Mutagenesis of critical amino acids in the vicinity of the ligand-binding pocket reveals less efficient inhibition of the ATPase activity of the enzyme by etoposide. Taken together, these results provide an insight for the development of newer therapeutic agents with specific selectivity

Primary extranodal non-Hodgkin lymphoma: A 3-year record-based descriptive study from a tertiary care center in Southern India

Introduction: Primary extranodal lymphoma (pENL) refers to group of disorders arising from tissues other than lymph nodes and even from sites, which normally do not contain lymphoid tissue. This study was undertaken to ascertain the prevalence, anatomical distribution, and histological subtypes of extranodal non-Hodgkin lymphoma (NHL) from a tertiary care institute in Southern India. Materials and Methods: This was a cross-sectional study conducted in the Department of Pathology over a period of 3-years. Detailed clinical history, routine complete blood count, microbiological status was obtained from the medical records. Hamatoxylin and eosin slides were reviewed and immunohistochemistry was performed using a panel of antibodies depending on the morphology. All cases were classified based upon morphologic and immunophenotypic criteria according to World Health Organization 2008 classification. Results: Primary extranodal NHL constituting 22.6% (68/300) of all NHL and the majority of patients were from higher age group with peak incidence seen in fourth to fifth decade of life. In the pediatric population, the most common site is ileo-caecal region (42.8%) and the most common morphology is lymphoblastic lymphoma (42.8%). Similarly in the adult population head and neck was the most common site constituting 36% and diffuse large B-cell lymphoma is the most common morphological subtype comprising 54% of all extranodal lymphomas. Conclusion: Although the reported incidence of pENL is low in India compared to other parts of the world, the possibility of pENL should always be kept in mind even though it arises in an extranodal site

A comparative study of dexamethasone ointment along with triamcinolone acetonide in treatment of recurrent aphthous stomatitis

Aim: To assess and compare the efficacy of dexamethasone ointment with triamcinolone acetonide, the gel form, in the treatment of minor recurrent aphthous stomatitis (RAS). Materials and Methods: This was a randomized clinical trial of 60 patients of either sex with clinically diagnosed RAS who were randomly divided into two groups—the dexamethasone ointment group (Group I) and the triamcinolone acetonide gel group (Group II). Patients were asked to apply the gel three times a day on each ulcer. Estimation of the efficacy of gel was done based on the time required for regression in pain, size, and number of ulcers. Results: The results showed there was a significant difference in size, pain, number, and duration of ulcers in Group I and Group II within 7 days. However, in both groups, no significant difference was noted in the treatment of RAS. Conclusion: Dexamethasone ointment was found to be efficient in this treatment and was safe as evaluated using clinical assessments

Clinical profile and in-hospital outcomes of COVID-19 patients: Findings from secondary data analysis

Background: Uttarakhand, a hilly state in north India, reported the first coronavirus disease (COVID) case on 15 March 2020. Since then, the case numbers rose multiple folds. As Uttarakhand has been on a 'war-footing' amidst the recent second wave and is gearing up to fight against the third wave, the present study aims to uncover baseline clinical profile and in-hospital outcomes of COVID patients in Dehradun district (Uttarakhand) during the first wave. Methods: A record-based descriptive analysis was carried out for 671 COVID patients admitted to a private dedicated COVID hospital in Dehradun district between August 2020 and February 2021. Data was collected from medical records on a standardized abstraction form. Data was entered and analyzed using Statistical Package for Social Sciences (SPSS) version 20. Results: The present study showed most admitted COVID patients were males, aged 40 years and above, moderately ill, had co-morbidities with about one-fourth lately succumbed to death. The proportions of deaths, moderate-to-severe and severe category of illness were invariably high for those with co-morbidities irrespective of the gender. Females, age <60 years, and absence of co-morbidities had overall high mean survival estimates from COVID. Conclusion: Females, younger age group, and absence of co-morbidities are more likely to survive from COVID than males, older age groups, and those with co-morbidities