Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H 2 O 2 . The present study shows that upon activation of death response by H 2 O 2 , a dose-and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca 2؉ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na ؉ and mitochondrial Ca 2؉ . Partial inhibition of cytosolic Ca 2؉ increase achieved by chelating extracellular or intracellular Ca 2؉ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca 2؉ is an additive result of release of Ca 2؉ from intracellular stores as well as by influx of extracellular Ca 2؉ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like Nacetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.Mitochondria are pivotal in controlling cell life and death (1). Maintenance of proper mitochondrial transmembrane potential (⌬ m ) 1 is essential for the survival of the cell as it drives the synthesis of ATP and maintains oxidative phosphorylation (2). Recently, the study of mitochondrial potential has become a focus of apoptosis regulation as many investigations demonstrate a major functional impact of mitochondrial alterations on apoptosis (2). Apoptosis is a process of cell death in which the cells undergo nuclear and cytoplasmic shrinkage; the chromatin is condensed and partitioned into multiple fragments, and finally the cells are broken into multiple membrane-bound bodies. In a number of experimental systems, disruption of ⌬ m constitutes a constant early event of the apoptotic process that precedes nuclear disintegration (3-5). For example, in thymocytes or tumor necrosis factor-stimulated U937 cells (3, 6), thymocytes or imexon-treated myeloma cells (5, 7), and PC-12 cells (8), a loss of ⌬ m occur as an early change associated with apoptosis. Lymphocytes with low ⌬ m show irreversible commitment to apoptosis in comparison to cells with high ⌬ m that do...

Leishmania donovani promastigotes introduced into the bloodstream by sandfly vectors, are exposed to reactive oxygen species like H2O2 during phagocytosis by the host macrophages. H2O2 can induce promastigote death, but the mechanism of induction of this death is not known. Studies presented in this paper demonstrate that exposure to 4 mM H2O2 results in a pattern of promastigote death that shares many features with metazoan apoptosis. Motility and cell survival in these parasites show a gradual decline with increasing doses of H2O2. Features common to metazoan apoptosis, such as nuclear condensation, DNA fragmentation with accompanying DNA ladder formation and loss of cell volume, are observed after exposure to 4 mM H2O2. Within 30 minutes of the exposure, there is a significant increase in the ability of the cell lysates to cleave the fluorogenic tetrapeptide acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin, which is a substrate for the CED-3/CPP32 group of proteases. Pretreatment of cells with a specific inhibitor of CED-3/CPP32 group of proteases, Z-DEVD-FMK, reduces the number of cells showing apoptosis-like features, prevents DNA breakage and inhibits cleavage of a PARP-like protein. Activation of the caspase-like proteases is followed at 2 hours by the cleavage of a poly(ADP)ribose-polymerase-like protein and a reduction in intracellular glutathione concentration. DNA breakdown as detected by TdT labelling of cells and agarose gel electrophoresis is visible at 6 hours. Taken together, the above data show for the first time that there is a distinct pathway for apoptosis-like death in L. donovani.

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of 'Sertoli cell only' animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of <9.8x10(-7) M for testosterone and 9x10(-6) M for oestradiol respectively.

A comparative study was made regarding the stability and catalytic activity of two related enzymes (Alcohol dehydrogenase from Tbermoanaerobacter brockii TBADH and the mesophilic enzyme from Yeast YADH)in the medium of various organic solvents of different degrees of hydrophobicity. It was clearly found that both enzymes remains stable at high temperatures after prolonged incubation in hydrophobic solvents than in thier hydrophilic counterparts. However, in the presence of pyridine the catalytic activity of TBADH reduced considerably at high temperatures whereas YADH is inactivated completly even at room temperature. The effect of water-miscible solvents on the activity of both enzymes have also been investigated. Interestingly, the thermophilic enzyme was more active at high concentrations of dioxane, but low amount of dimethylformamide (DMF) inhibit its catalytic activity, while these solvents exert adverse effect on mesophilic counterpart. These observations indicate that (i) TBADH is more resistant than YADH toward extreme organic meliue as well as high temperatures and (ii) it is possible to provide thermophilic characteristics to a mesophilic enzyme by using a proper microenvironment.The semineferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis primarily through the secretions of the seminiferous tubule (ST). Previous studies from this laboratory have shown the presence of GSTs in adult rats. In the present study we have shown the isoform composition, testicular source and possible function of rat GST. The major GST isoforms present in rat STF-GST share extensive N terminal similarity with rat GST-MI, M2, M3 and Alpha subunits. Molecular mass of the individual peaks have been done both in STF-GST and GST from liver. Rat STF GST M1 was higher by 325 Da.Peptide digest analysis data of the selected peaks were done from liver and STF revealed considerable simi1arity.The above studies showed consideraable stuctural similarity between liver and STF-GST. Further studies with radiolabelled GST showed that Seroli cells were the prime secretors of GST.Functionally, STF GSTs have the ability to bind to oestradiol and testosterone, two important hormones in the ST that are essential for spermatogenesis. Rat GST-MI carries out this function. 01Hyperthemostable AONS: the first committed enzyme in E. coli biotin biosynthesis has a tropical relative An essential cofactor for fatty acid metabolism in all organisms, biotin is produced in small quantities only in bacteria, fungi and plants. The catalyst for the first committed step of this process in E.coli, 8-amino-7-oxononanoate synthase ( A O N S EC 2.3.1.47), is the product of the bioF gene and catalyses the decarboxylative condensation reaction between L-alanine and Pimeloyl CoA to produce 8-amino-7-oxononanoate. The structure of this protein (1) confirms that AONS functions as a homodimer, each monomer of which has a mass of 41.7 kDa and is composed of three distinct domains. The active site is found in a deep cleft within the central,...