Behaviour of the germ cell specific lamin through mammalian spermatogenesis as probed with monoclonal antibodies.

We had earlier identified a 60 kDa nuclear lamin protein (lamin(g)) unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin(g) antigen. While all the eight Mabs reacted with lamin(g) antigen in an immunoblot analysis, only three Mabs (A(11)C(7), A(11)D(4), C1F7) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A(11)C(7) and A(11)D(4) showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin(g) is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin(g) as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin(g) antibodies. The selective retention of lamin(g) in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

Peanut agglutinin from callus and cell suspension cultures of Arachis hypogaea L

Synthesis of peanut agglutinin was induced in callus and cell suspension cultures of cotyledons of peanut (Arachis hypogaea L.). The lectin was synthesised in cultures through several passages. Biosynthesis of peanut agglutinin was regulated by the type and concentration of exogenous growth regulators and was positively correlated to the growth of the cultures,indicating that the agglutinin may have a role to play during cell growth. Movement of agglutinin from the cells into the medium not only facilitated easy isolation of the lectin but also provided a clue that it may probably serve as a defence molecule. The synthesized lectin purified from culture, was found to be biologically active, and was found to be comparable with the lectin from seeds, in terms of its electrophoretic mobility.

Design and development of a self-supported polymer electrolyte fuel cell system with anodic dead-end operation

Design and operational details for a self-supported polymer electrolyte fuel cell (PEFC) system with anodic dead-end fuel supply and internally humidified cathodic oxidant flow are described. During the PEFC operation, nitrogen and water back diffuse across the Nafion membrane from the cathode to the anode and accumulate in the anode flow channels affecting stack performance. The accumulated inert species are flushed from the stack by purging the fuel cell stack with a timer-activated purge valve to address the aforesaid problem. To minimize the system complexity, stack is designed in such a way that all the inert species accumulate in only one cell called the purge cell. A pulsed purge sequence comprises opening the valve for purge duration followed by purge-valve closing for the hold period and repeating the sequence in cycles. Since self-humidification is inadequate to keep the membrane wet, the anodic dead-end-operated PEFC stack with composite membrane comprising perflourosulphonic acid (Nafion) and silica is employed for keeping the membrane humidified even while operating the stack with dry hydrogen and internally humidified air.

Ultrasonic spray pyrolysis of CZTS solar cell absorber layers and characterization studies

CZTS (Copper Zinc Tin Sulphide) is a wide band gap quartnery chalcopyrite which has a band gap of about 1.45 eV and an absorption coefficient of 10(4) cm(-1); thus making it an ideal material to be used as an absorber layer in solar cells. Ultrasonic Spray Pyrolysis is a deposition technique, where the solution is atomized ultrasonically, thereby giving a fine mist having a narrow size distribution which can be used for uniform coatings on substrates. An Ultrasonic Spray Pyrolysis equipment was developed and CZTS absorber layers were successfully grown with this technique on soda lime glass substrates using aqueous solutions. Substrate temperatures ranging from 523 K to 723 K were used to deposit the CZTS layers and these films were characterized using SEM, EDAX and XRD. It was observed that the film crystallized in the kesterite structure and the best crystallites were obtained at 613 K. It was observed that the grain size progressively increased with temperature. The optical band gap of the material was obtained as 1.54 eV.

The level of sequestered calcium in vegetative amoebae of Dictyostelium discoideum can predict post-aggregative cell fate

When freshly starved amoebae of Dictyostelium discoideum are stained with chlortetracycline (CTC), a cell type-specific fluorescent probe for membrane-associated calcium (Ca2+) the resulting fluorescence distribution falls into two functional classes. Fluorescence-activated cell sorting shows that highly fluorescing amoebae tend to enter the prestalk pathway while those with low fluorescence tend to become prespores. In the light of previous findings, these results indicate that in addition to cell cycle phase at starvation, phenotypic variation in the level of sequestered calcium is an early correlate of cell fate.

Azidothymidine induces apoptosis in mouse myeloma cell line Sp2/0

Azidothymidine (AZT), which has been extensively used as an antiviral agent in the treatment of AIDS, showed strong inhibition of growth of Sp2/0 cells in vitro. AZT-treated cells showed a decrease in viability in a dose-dependent manner. AZT specifically induced typical apoptotic cell death with DNA double-strand cleavage and subsequent formation of apoptotic bodies. The induction of DNA double-strand cleavage into the oligonucleosomal ladder by AZT was protected in the presence of thymidine or uridine. An increase in endonuclease activity from nuclear extract of AZT-treated cells was observed. The enzyme activity was found to be Ca2+- and Mg2+-dependent and was inhibited by zinc acetate. A marked enhancement of PARP activity was observed in AZT-treated cells. These observations show that AZT can trigger both morphological and biochemical changes typical of apoptosis in the mouse myeloma cell line Sp2/0.

Cell versus message level performances in ATM networks

The goal of this paper is to provide some insight into the relations that exist between cell level and message level performance guarantees in the context of ATM networks. Cell level guarantees are typically what the network is capable of providing, while message level guarantees are the ones of interest to users. It is, therefore, important to understand how the two are related, and which factors influence this relation. There are many different performance measures that are of importance, and in this paper we try to touch on the (three) most relevant ones. This includes comparing cell and message loss probabilities, average cell and message delays, and cell and message jitter. Specifically, we show that cell and message loss probabilities can exhibit significant differences, which strongly depend on traffic characteristics such as peak rate and burst size, i.e., for a fixed cell loss probability, the message loss probability can greatly vary when peak rate and burst size change. One reason for this sensitivity, is that message loss depends on what happen to all the cells in a message. For delay and jitter, we also find that peak rate and burst size play a role in determining the relation between cell and message performance. However, this sensitivity is not as acute as with losses since message delay and jitter are typically determined by the performance seen by only one cell, the last cell in a message. In the paper, we provide quantitative examples that illustrate the range of behaviors and identify the impact of different parameters.

Effect of TiO2 electrode thickness on photovoltaic properties of dye sensitized solar cell based on randomly oriented Titania nanotubes

Anatase titania nanotubes (TNTs) have been synthesized from P25 TiO2 powder by alkali hydrothermal method followed by post annealing. The microstructure analysis by X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed the formation of anatase nanotubes with a diameter of 9-10 nm. These NTs are used to make photo anode in dye-sensitized solar cells (DSSCs). Layer by layer deposition with curing of each layer at 350 C is employed to realize films of desired thickness. The performance of these cells is studied using photovoltaic measurements. Electrochemical impedance spectroscopy (EIS) is used to quantitatively analyze the effect of thickness on the performance of these cells. These studies revealed that the thickness of TiO2 has a pronounced impact on the cell performance and the optimum thickness lies in the range of 10-14 mu m. In comparison to dye solar cells made of P25, TNTs based cells exhibit an improved open circuit voltage and fill factor (FF) due to an increased electron lifetime, as revealed by EIS analysis. (C) 2011 Elsevier B.V. All rights reserved.

Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata)

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type-the Sertoli cells-to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction (>40%; P < 0.05) in [H-3] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition (>50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block (<90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

Determination of the Gibbs energy of diamond using a solid state cell

he chemical potential of carbon in diamond, relative to its value in graphite, has been directly determined using a solid state electrochemical cell incorporating single crystal CaF2 as the solid electrolyte. The cell can be represented as Pt, C(graphite) + CaC2 + CaF2double vertical barCaF2double vertical barCaF2 + CaC2 + C(diamond), Pt The reversible emf of this cell is directly related by the Nernst equation to the Gibbs free energy change for the conversion of diamond to graphite. The difference in the chemical potential of carbon in the two crystal structures varies linearly with temperature in the range 940 to 1260 K ?C(diamond) ? ?C(graphite) = 1100 + 4.64T (±50) J mol?1 On the average, the values given by the equation are 320 J mol?1 less positive than the currently accepted ones based on calorimetric studies. The difference is primarily in the enthalpy term.

What makes a cell tick? The A, B and C of the matter

Nuclear lamina in an eukaryotic cell is primarily composed of the lamins A, B and C. The A type lamins are found only in differentiated cell types while the B type lamins are present both in differentiated and undifferentiated cells. Lamin B interacts with the inner nuclear membrane, In recent years there have been extensive studies on the relationship between the dynamic state of lamin B and the nuclear envelope integrity with respect to the fate of a particular cell, In this article, we have analysed the recent developments and have considered the sequence of events that might be contributing to the fate of a cell either to undergo normal cell division or uncontrolled cellular proliferation or apoptosis.

A novel zinc bis(thiosemicarbazone) complex for live cell imaging

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), Zn (GTSC)](3), complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, Zn(GTSC)](3) had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that Zn(GTSC)](3) had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that Zn(GTSC)](3) appeared to have a nuclear localization within 30 mm of addition to MCF7 cells. Moreover, Zn(GTSC)](3) showed minimal cytotoxicity compared with Zn(ATSM), suggesting that Zn(GTSC)](3) may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel Zn(GTSC)](3) complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.