Specific immunoneutralization of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat

Although requirement for follicle stimulating hormone (FSH) in the initiation of spermatogenesis is well documented, its role in adult spermatogenesis is still debated. In the present communication, we have investigated the effect of specific immunoneutralization of FSH on apoptotic cell death in the testicular germ cells both in immature and adult rats. The germ cells of control animals showed predominantly high molecular weight DNA while the antiserum (a/s) treated group showed DNA fragmentation characteristic of apoptosis. The pattern could be detected within 24 hours of a/s treatment, and became more pronounced after 48 hours. The germ cells were purified from FSH a/s treated rats by centrifugal elutriation and vulnerability of each cell type to undergo apoptosis on FSH neutralization was investigated. The pachytene spermatocytes were found to be most sensitive to absence of FSH, even in the adult animals suggesting the involvement of FSH in spermatogenesis. The in situ analysis of DNA strand breakage following FSH a/s treatment showed fragmentation of the DNA of the pachytene spermatocytes confirming this observation. The in situ analysis also showed that the spermatogonia undergo apoptosis in addition to the pachytene spermatocytes. These data clearly demonstrate the role of FSH in the adult rat spermatogenesis.

Voltage gated Na+ channels contribute to membrane voltage fluctuation in \alpha T3-1 pituitary gonadotroph cells

\alpha T3-1 cells showed a slope resistance of 1.8 G\omega. The cell membrane surface was not smooth and a scanning electron micrograph showed a complex structure with blebs and microvilli like projections. The cells showed spontaneous fluctuations at zero current resting membrane potential and hyperpolarization increased the amplitude of membrane potential fluctuations. The amplitude of membrane potential fluctuations at hyperpolarized membrane potential was attenuated on application of TTX to the bath solution. The potential at which half steady state inactivation of isolated sodium current occurred, was at a very hyperpolarized potential (-95.4 mV). The study presented in this paper shows that the voltage gated sodium channels contribute to the increase in the amplitude of electrical noise with hyperpolarization in \alpha T3-1 cells.

Poly (vinyl alcohol) hydrogel membrane as electrolyte for direct borohydride fuel cells

A direct borohydride fuel cell (DBFC) employing a poly (vinyl alcohol)hydrogel membrane electrolyte (PHME) is reported. The DBFC employs an AB(5) Misch metal alloy as anode and a goldplated stainless steel mesh as cathode in conjunction with aqueous alkaline solution of sodium borohydride as fuel and aqueous acidified solution of hydrogen peroxide as oxidant. Room temperature performances of the PHME-based DBFC in respect of peak power outputs; ex-situ cross-over of oxidant, fuel,anolyte and catholyte across the membrane electrolytes; utilization efficiencies of fuel and oxidant, as also cell performance durability are compared with a similar DBFC employing a NafionA (R)-117 membrane electrolyte (NME). Peak power densities of similar to 30 and similar to 40 mW cm(-2) are observed for the DBFCs with PHME and NME, respectively. The crossover of NaBH4 across both the membranes has been found to be very low. The utilization efficiencies of NaBH4 and H2O2 are found to be similar to 24 and similar to 59%, respectively for the PHME-based DBFC; similar to 18 and similar to 62%, respectively for the NME-based DBFC. The PHME and NME-based DBFCs exhibit operational cell potentials of similar to 1 center dot 2 and similar to 1 center dot 4 V, respectively at a load current density of 10 mA cm(-2) for similar to 100 h.

Division of the Salmonella-Containing Vacuole and Depletion of Acidic Lysosomes in Salmonella-Infected Host Cells Are Novel Strategies of Salmonella enterica To Avoid Lysosomes

Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

DNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4',5':4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4+-grown Azospirillum lipoferum cells

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed.

Growth inhibition of human promonocytic leukaemic U937 cells by interferon gamma is irreversible and not cell cycle phase-specific.

Growth of human promonocytic leukaemic U937 cells was found arrested within 24 h upon exposure to interferon gamma (IFN-gamma). Removal of the interferon did not result in the resumption of growth, as is evident from the absence of doubling of viable cell count and(3)H-thymidine incorporation. 5-Bromo-2'-deoxyuridine-based flow cytometric analysis of the growth-arrested cells, 24 h subsequent to the removal of IFN-gamma, showed absence of DNA synthesis, confirming the irreversible nature of the growth inhibition. Propidium iodide-based flow cytometric analysis of the growth-arrested cells showed a distribution which is typical of a growth inhibition without resulting in the accumulation of cells in any specific phase of the cell cycle. These results indicated that IFN-gamma arrested growth of U937 cells in an irreversible and cell cycle phase-independent manner. These observations were in contrast to our earlier report on the reversible and cell cycle phase-specific growth inhibition of human amniotic (fetal epithelial) WISH cells by the interferon. Copyright 1999 Academic Press.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Processing of Mycobacterium tuberculosis bacilli by human monocytes for CD4+ alphabeta and gammadelta T cells: role of particulate antigen.

Mycobacterium tuberculosis readily activates both CD4+ and Vdelta2+ gammadelta T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and gammadelta T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vdelta2+ gammadelta T cells. For gammadelta T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vdelta2+ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4+ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vdelta2+ gammadelta T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for gammadelta T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4+ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4+ and gammadelta T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4+ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4+ and gammadelta T cells.

Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

A Methanol-Tolerant Carbon-Supported Pt-Au Alloy Cathode Catalyst for Direct Methanol Fuel Cells and Its Evaluation by DFT

A Pt-Au alloy catalyst of varying compositions is prepared by codeposition of Pt and Au nanoparticles onto a carbon support to evaluate its electrocatalytic activity toward an oxygen reduction reaction (ORR) with methanol tolerance in direct methanol fuel cells. The optimum atomic weight ratio of Pt to Au in the carbon-supported Pt-Au alloy (Pt-Au/C) as established by cell polarization, linear-sweep voltammetry (LSV), and cyclic voltammetry (CV) studies is determined to be 2:1. A direct methanol fuel cell (DMFC) comprising a carbon-supported Pt-Au (2:1) alloy as the cathode catalyst delivers a peak power density of 120 mW/cm2 at 70 °C in contrast to the peak power density value of 80 mW/cm2 delivered by the DMFC with carbon-supported Pt catalyst operating under identical conditions. Density functional theory (DFT) calculations on a small model cluster reflect electron transfer from Pt to Au within the alloy to be responsible for the synergistic promotion of the oxygen-reduction reaction on a Pt-Au electrode.

Biotransformation of cholesterol to diosgenin by freely suspended and immobilised cells of Dioscorea bulbifera L.

The cell suspension cultures, established from the friable callus which was risen from the nodal segments of Dioscorea bulbifera L. in Murashige-Skoog (MS) medium supplemented with indole-3-butryic acid (20 mg L- 1), was examined for cell growth in MS medium fed with cholesterol (50 mg L- 1 and 100 mg L- 1) after 8, 10, 12, 14, 16, and 18 days of culture. The growth index of the cell suspension culture on the 8th day was 1.2 and gradually inclined to 1.9 on the 16th day and remained the same at the 18th day. There is no marked difference in the cell growth of cholesterol-treated and control cell suspension culture. The maximum accumulation of diosgenin was noticed on the 14th day in control and cholesterol-treated cell suspension culture and immobilised cell cultures. The highest concentration of diosgenin, 2.94% and 2.14% dry weight, was obtained in immobilised cell culture and cell suspension culture treated with 100 mg L- 1 cholesterol, respectively.

Nafion and modified-Nafion membranes for polymer electrolyte fuel cells: An overview

Polymer electrolyte fuel cells (PEFCs) employ membrane electrolytes for proton transport during the cell reaction. The membrane forms a key component of the PEFC and its performance is controlled by several physical parameters, viz. water up-take, ion-exchange capacity, proton conductivity and humidity. The article presents an overview on Nafion membranes highlighting their merits and demerits with efforts on modified-Nafion membranes.