Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up

Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification. Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol. Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.     

Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.     

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.     

Nimesulide affects antioxidant status during acute lung inflammation in rats

Antiradical activity of nimesulide, a commonly used COX-2 specific inhibitor, was estimated in vitro by 1, 1 diphenyl-2-picrylhydrazyl (DPPH) assay, nitroblue tetrazolium reduction assay and lipid peroxidation assay, respectively. The biochemistry of antioxidant functions of nimesulide was also investigated under control and inflammatory conditions, caused by intratracheal instillation of lipopolysaccharide (LPS). Pro-inflammatory conditions generally end up in oxidative insults, which have been suggested to be the cause of multiple organ failure in inflammation. A primary defense, constituted of antioxidant enzymes, against this oxidative damage has evolved in the body. In this study, male Wistar rats were orally administered with nimesulide (9 mg/kg/twice daily for 1 week), followed by intratracheal instillation with 2 microg of LPS and after 18 hr, antioxidant defense system and lipid peroxidation were measured in liver, lungs and kidneys. Nimesulide pretreatment was found to protect the tissue from enhanced levels of lipid peroxidation, and also stimulated the levels of glutathione-S-transferase (GST) in liver and glutathione reductase in kidneys. Surprisingly, nimesulide oral feeding also significantly suppressed superoxide dismutase (SOD) activity in all the three organs. Although, in our study, nimesulide proved to be an inducer of GST (a marker for chemoprevention) and a scavenger of superoxide anions at higher concentrations (> 250 microM), but the relevance of suppression of SOD enzyme activity, which may contribute to the drug's toxic effects cannot be ignored. The work suggests that further long term studies are needed to confirm nimesulide as a safe drug.     

Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.     

A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture

An efficient method has been developed for large-scale multiplication of Curculigo orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7 x 10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 microM), adenine (0.11 mM), indole butyric acid (1.0 microM) and polyvinyl pyrrolidone (250 mgL-1). About 95% of explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 microM) and gibberellic acid (3.5 microM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.     

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.     

Angiopoietin 2 expression in the retina: upregulation during physiologic and pathologic neovascularization

Vascular development in the embryo requires coordinated signaling through several endothelial cell-specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless beta-galactosidase (beta gal) gene behind the ang2 promoter, show constitutive beta gal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for beta gal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role.