Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype

Proofs for Pearson's HSAB principle

1-3<span style="font-size:12.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-in;mso-fareast-language:en-in;mso-bidi-language:ar-sa"="" lang="EN-IN">Two previously published rules are used in an attempt to provide a qualitative proof for Pearson's HSAB principle and another attempt is made to prove the principle quantitatively by using a heterolytic dissociative version of Pauling's bond energy equation.</span

Use of barbituric acid as a "padlock" to generate azamacrocyclic complexes of Ni(II) containing fused aromatic rings

985-9901,5-Di(4,5-disubstituted-2-aminophenyl) penta- 1,5-diaza- 1,3-dienato nickel(II) chlorides are reacted with formaldehyde and barbituric acid in the presence of catalytic amount of  perchloric acid to synthesize two members of a new type of aza macrocyclic complex of Ni(II). The macrocyclic complexes, which are diamagnetic, have been characterised by conductivity, electronic spectra, thermal analysis and NMR (1H and 13C) spectroscopy. The structure of the cation in one of the macrocyclic complexes has been examined by using molecular mechanics.</span

Evaluation of s.c. route of immunization by homologous radio attenuated live vaccine in experimental murine model of visceral leishmaniasis

Our previous studies in BALB/c mice showed substantial protection against the experimental murine visceral leishmaniasis (MVL) when the animals were immunized with γ-irradiated live Leishmania donovani parasites through intra peritoneal (i.p.) and intra muscular (i.m.) routes respectively. The observations encouraged us to check the prophylactic efficacy of subcutaneous (s.c.) route as it is better alternative for human trial. The mice immunized with two subsequent doses of the radio attenuated homologous vaccine were challenged with virulent L. donovani parasites. Seventy-five days post infection, the animals were sacrificed. The extent of protection against the disease was evaluated by assessing the reduction of parasite burden in spleen and liver, the generation of free radicals (NO &amp; ROS) and release of the cytokines from T-lymphocyte helper 1 (Th 1) and T-lymphocyte helper 2 (Th 2) along with the measurement of the serum immunoglobulins. The reductions in parasitic burden were observed up to 21 and 24 % in spleen and liver of the immunized groups with NO and ROS productions 27 and 34 % respectively. Whereas the increase in IFN gamma releases was between 19 and 34 %, the decrease in IL-10 release was not more than 22 %. This indicates the failure of the establishment of pronounced Th1 ambience which was further corroborated by the observed IgG2a and IgG1 ratio. The present study when compared with our previous observations with i.m. and i.p. routes revealed that s.c. route may not be a good choice for the use of radio attenuated vaccine

<span style="font-size:14.0pt;font-family: "Times New Roman","serif";mso-fareast-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA" lang="EN-US">Structure and metal binding of a 1<i style="mso-bidi-font-style:normal">H</i>-1,5-benzodiazepine</span>

553-557The X-ray crystal structure of 2,3-dihydro-2,2,4-trimethyl-1H-1,5-benzodiazepine (L), which has medicinal effect on the central nervous system, has been determined. The solid state conformation of the seven-membered heterocyclic ring in L is found to be somewhat like an armchair. Its modes of binding to three metal ions – Ni2+, Cu2+ and Zn2+ – are identified. In cases of Ni2+ and Cu2+ ,the seven-membered heterocyclic ring in L opens up following a hydrolysis of the imino bond promoted by these two metal ions. Zn2+ cannot effect such ring opening

New copper complexes of diacetyl hydrazone oxime and its acetone azine

571-578Using diacetyl hydrazone oxime (LH), we have synthesised a neutral copper(I) complex of the type Cu(L)(LH). Such stabilisation of copper(I) has not been achieved using the acetone azine (L'H) of LH. Both LH and L'H yield hexanuclear copper(II) complexes - CU6L6(O)(OH)(ClO4)3.3CH3COOH (1a) and CU6L'6(O)(OH)(ClO4)3 (1b), respectively. X-ray crystal structure of 1b (space group PbcZ1; Z = 4) indicates that both 1a and 1b contain an unprecedented Cu3O···H···OCu3 core where the copper atoms are staggered. In 1b, two of the three perchlorate anions are coordinated to the metals. The cationic part in 1b comprises two [(ClO4)Cu3O(L')3] units held together by a proton. The O··:H···O bond (2.428Å) is one of the shortest H-bonds known. Each copper(II) center in 1b is square pyramidal with a perchlorato oxygen occupying the apical position. The average bond distances around each copper(II) center are: Cu-perchlorato O, 2.54; Cu-central O, 1.91; Cu-oximato O, 1.92; Cu-oximato N, 1.95; Cu-azine N, 1.98 A. In a [(ClO4)Cu3O(L')3] unit, the three copper atoms are linked to each other via a centrally bridging oxygen atom and two adjacent copper atoms are further linked by a peripheral oximato bridge. It is indicated that the hexanuclear copper(II) moiety in 1a and 1b represents a case of two unpaired spins interacting through an H-bond

Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis

After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman–Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further

RFLPs of ITS, ITS1 and hsp70 amplicons and sequencing of ITS1 of recent clinical isolates of Kala-azar from India and Bangladesh confirms the association of L. tropica with the disease

Visceral Leishmaniasis or Kala-azar (KA) is a serious health concern in India. In the present study, Restriction Fragment Length Polymorphism (RFLP) of three genetic markers viz., Internal Transcribed Spacer (ITS), ITS1 and heat shock protein 70 (hsp70) have been employed for typing the clinical isolates [n = 15] of KA and post Kala-azar Dermal Leishmaniosis (PKDL) collected from India and Bangladesh in the period of 2006–2010. Experimentally, ITS, ITS1 and hsp70 regions of genomes of all the clinical isolates were separately amplified by PCR and then digested with restriction enzymes: ITS with Alu1, EcoR1 and Msp1, ITS1 with Hae III and Rsa1 and hsp70 with Hae III. The resultant fragments were analyzed by agarose gel electrophoresis and the RFLP profiles of the clinical isolates were compared with that of the WHO reference strains for Leishmania donovani (DD8) and Leishmania tropica (K27), respectively. Also, the ITS1 regions of all the clinical isolates along with the two WHO reference strains were sequenced and a phylogram was constructed to ascertain the extent of similarity or dissimilarity. Interestingly, the RFLP profiles of one of the isolates showed a significant homology with K27 and the phylogram revealed its closeness with the same putting credence to our earlier typing of isolates by RAPD method. This observation also supported an earlier report claiming that both the species are responsible for KA in India and thus, emphasizes urgent need for thorough systematic characterization of the clinical isolates of Indian KA as appropriate treatment regime relies primarily on proper diagnosis