Culture of granulosa cells in collagen gels: the influence of cell shape on steroidogenesis

The gonadotropic regulation of granulosa cells steroidogenesis in vitro has been shown to be accompanied by cellular rounding. In this study, the possible relationship between cell shape, microtubules, and granulosa cell steroidogenesis in vitro was further explored by culturing (24 h) granulosa cells obtained from antral follicles of pregnant mare's serum gonadotropin-treated rats in either Eagles's Minimum Essential Medium alone (MEM-cells) or in collagen gels (GEL-cells) in the absence or presence of colchicine, a microtubule-depolymerizing agent previously shown to inhibit cell-spreading in vitro. Cellular morphology was assessed by electron microscopy and compared with that seen in vivo. In addition, the influence of the various culture conditions on progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) secretion was determined by specific radioimmunoassays. Whereas the majority of granulosa cells in sections of antral follicles appeared rounded in shape, cells cultured in MEM underwent considerable spreading and assumed a variety of shapes at the end of 24 h of culture. GEL-cells, on the other hand, remained rounded and had cellular diameters only slightly larger than those observed in vivo. They also secreted more progesterone (almost 3-fold) and less 20 alpha-OH-progesterone (0.6-fold) than MEM-cells. Colchicine increased the secretion of progesterone (1.6-fold) and 20 alpha-OH-progesterone (1.8-fold) comparably in MEM-cells but had no influence on the secretion of either progestin by GEL-cells. Hence, although colchicine-stimulated progestin secretion by granulosa cell monolayers appeared to reflect increased metabolism of substrate-possibly due to a closer association between lipid droplets and mitochondria, the elevated secretion of progesterone by GEL-cells may have been largely due to a shift in the equilibrium between progesterone and its inactive 20 alpha-reduced metabolite. The high ratio of 20 alpha-OH-progesterone to progesterone secretion seen in MEM-cultured cells may be an adaptation of granulosa cell metabolism to culture as monolayers on plastic or glass surfaces. The morphology of GEL-rather than MEM-cells resembled closely that seen in vivo. This culture method may represent a more physiologic approach to the maintenance of granulosa and other steroidogenic cells in vitro and provide a more appropriate means of assessing cytoskeletal function in the regulation of steroid hormone production.     

Identification of the Dimerization Domain of Dehalogenase IVa of Burkholderia cepacia MBA4

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.     

Visual detection of β-fructofuranosidase (inulase) —Regulatory mutants ofKluyveromyces fragilis

Summary Inulase constitutive mutant cells of the yeastKluyveromyces fragilis were enumerated in continuous culture cell populations. After cloning and growth on glycerol agar plates, mutant colonies stained red when exposed to a mixture of sucrose and a chromogenic reagent for glucose.Mutants with improved inulase production on glucose were isolated from opaque agar plates containing undissolved inulin. Mutant colonies were surrounded with clearing zones. Attempts to isolate similar mutants by selection for 2-deoxyglucose resistance proved unsuccessful withK. fragilis.     

Reduced immunogenicity of pancreatic progenitor cells derived from first-trimester human fetal pancreas

The relatively low immunogenic and tumorigenic nature of fetal stem cells makes them attractive candidates for transplantation. Pancreatic progenitor cells (PPCs) derived from human fetal pancreas that are amenable to growth and differentiation into transplantable insulin-producing islet-like cell clusters (ICCs) have been reported recently; however, the immunological nature of these cells has yet to be characterized. We thus investigated and compared the immunogenicity of pancreatic progenitor cells and islet-like cell clusters from first- and second-trimester human fetal pancreas. Polymerase chain reaction demonstrated that pancreatic progenitor cells and islet-like cell clusters express immune-related genes of major histocompatibility complex, MHC-I and MHC-II, complement component 3 (C3), chemokine ligand (CCL19), and tumor necrosis factor super family (TNFSF10), but no expression of the co-stimulatory genes, CD80 and CD86. Interestingly, pancreatic progenitor cells showed a differential expression of MHC-I and MHC-II with advancing gestational age with a greater expression in pancreatic progenitor cells from the second trimester. Pre-incubation of the second-trimester cells with interferon-γ (IFN-γ) increased MHC molecule expression. Functional alloreactivity of pancreatic progenitor cells was investigated via mixed lymphocyte reactions (MLRs). Relative to first-trimester pancreatic progenitor cells, second-trimester pancreatic progenitor cells induced a greater extent of proliferation of peripheral blood mononuclear cells (PBMCs) and resulted in more IFN-γ production in phytohaemagllutinin-stimulated peripheral blood mononuclear cells following co-culture. Results of the study indicated that first-trimester pancreatic progenitor cells and islet-like cell clusters have a distinctively lower immunogenicity relative to second-trimester pancreatic progenitor cells, even after a pro-inflammatory cytokine challenge.     

Effects of calcium channel activators BAY-K8644 and CGP-28392 on steroidogenesis in granulosa cells of the domestic hen

The effects of calcium agonists BAY-K8644 and CGP-28392 on steroidogenesis was examined in chicken granulosa cells in short term incubation. BAY-K8644 (5-500 nM) and low doses of CGP-28392 (1-10 microM) failed to appreciably affect basal and LH-stimulated progesterone production whether tested in calcium free, low (0.05 mM) or high (3 mM) calcium containing medium. However, higher concentrations of CGP-28392 (50-250 microM) inhibited significantly (P less than 0.01) both basal and LH-stimulated steroidogenesis in a dose-related manner independently of extracellular calcium availability. The suppressive effect of CGP-28392 was manifest with submaximally and maximally stimulating LH doses. In additional experiments with non-hormonal agonists such as forskolin, dibutyryl cyclic AMP and kaurenol, BAY-K8644 and low CGP-28392 concentrations were again without effect on steroidogenesis. By comparison, higher CGP-28392 doses suppressed the stimulatory effects of all three agonists dose-dependently. These results demonstrate that, the calcium channel agonists are incapable of inducing a steroidogenic response in chicken granulosa cells. Since BAY-K8644 and CGP-28392 (low dose, 1-10 microM) failed to influence steroidogenesis in the dose range that induced maximal physiologic responses and calcium influx in a variety of cells, it is concluded that chicken granulosa cells lack the type(s) of channels specific for them. Hence the usefulness of BAY-K8644 and CGP-28392 as Ca2+ probes may be tissue-specific. The inhibitory effects of CGP-28392 appear to be non-specific.     

Expression of an altered cAMP binding protein by rapid-developing strains of Dictyostelium discoideum

Immunoblotting with a monoclonal antibody raised against a novel cAMP binding protein termed CABP1 revealed that the molecular weights of the two CABP1 subunits are altered in certain strains of Dictyostelium discoideum. Cell-free translation followed by immunoprecipitation showed that the altered CABP1 polypeptides are derived from primary translation products. In addition, the affinity of the altered CABP1 for cAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABP1. These results indicate a good correlation between an altered CABP1 and rapid development.     

Genetics and development

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in genetics and development.     

Effect of polymyxin B on outer membranes of Serratia marcescens: morphological alterations of the outer membranes and their lipopolysaccharide components.

The in vitro or the in vivo treatment of outer membranes and their lipopolysaccharide (LPS) components from Serratia marcescens with the antibiotic polymyxin B appeared to alter their normal morphology in a sequential manner. The normal spherodial morphology was destablized into a flattened structure after the in vitro treatment of either the resistant strain 08 or the sensitive strain Bizio. The more severe in vivo treatment of the outer membranes from the resistant strain converted the flattened forms further into spheres with undefined periphery and diminished sizes. On the other hand, the same treatment of the outer membranes from the sensitive strain resulted in numerous incomplete spheres and short rods, which were similar to the various morphological forms of the LPS components after polymyxin B treatment. The difference in the morphological changes of the outer membranes and their LPS components of the resistant and sensitive strains after polymyxin B treatment may be explained by the variation of the susceptiblity of the membrane components to the degradative effects of the antibiotic.     

Bis(7)-tacrine attenuates beta amyloid-induced neuronal apoptosis by regulating L-type calcium channels

Beta amyloid protein (Abeta) and acetylcholinesterase (AChE) have been shown to be closely implicated in the pathogenesis of Alzheimer's disease. In the current study, we investigated the effects of bis(7)-tacrine, a novel dimeric AChE inhibitor, on Abeta-induced neurotoxicity in primary cortical neurons. Bis(7)-tacrine, but not other AChE inhibitors, elicited a marked reduction of both fibrillar and soluble oligomeric forms of Abeta-induced apoptosis as evidenced by chromatin condensation and DNA specific fragmentation. Both nicotinic and muscarinic receptor antagonists failed to block the effects of bis(7)-tacrine. Instead, nimodipine, a blocker of L-type voltage-dependent Ca2+ channels (VDCCs), attenuated Abeta neurotoxicity, whereas N-, P/Q- or R-type VDCCs blockers and ionotropic glutamate receptor antagonists did not. Fluorescence Ca2+ imaging assay revealed that, similar to nimodipine, bis(7)-tacrine reversed Abeta-triggered intracellular Ca2+ increase, which was mainly contributed by the extracellular Ca2+ instead of endoplasmic reticulum and mitochondria Ca2+. Concurrently, using whole cell patch-clamping technique, it was found that bis(7)-tacrine significantly reduced the augmentation of high voltage-activated inward calcium currents induced by Abeta. These results suggest that bis(7)-tacrine attenuates Abeta-induced neuronal apoptosis by regulating L-type VDCCs, offers a novel modality as to how the agent exerts neuroprotective effects.     

生物破乳菌形态、表面性质和物质特征研究方法与进展

生物破乳菌在石油开采与加工行业的研究已经引起各界的广泛关注,然而由于生物破乳菌菌体形态、表面性质和表面物质的复杂性,使菌体的破乳活性特征尚未被揭示。本文介绍了生物破乳剂的来源、合成及破乳机制;归纳了影响生物破乳菌破乳活性的菌体形态、表面性质和表面物质三方面因素的研究进展,特别是总结了相关研究的方法;最后在此基础上对今后研究方向提出展望。