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.     

Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli

2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.     

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.     

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.     

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.     

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.     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Power and heat measurement by direct calorimetry of individual insect response to allelo- and toxic compounds

A microcalorimeter was constructed for the individual insect in order to measure the insect's power output as a function of time (the thermogram). The instrument has a figure of merit of 7 μW/μV. It includes a Peltier pumped thermoelectric control loop which protects from intruding ambient thermals, and holds baseline drift to less than 2 μV/day. The design is a conventional twin, or differential heat conduction calorimeter, with two insect-holding vessels of glass. The vessels are connected by a stopcock, to give the insect the option of crawling from the sample chamber to the reference chamber. Continuous, metered air flow is provided. A small pulse of compound may be born in, as vapor with the air flow, for the study of attractants, toxic compounds, anesthetics and allelochemicals. The insect's reaction to such compounds, appears in the thermogram. Cabbage looper larvae were examined in their irritative exothermic reaction to microgram amounts of benzene, and in their neutral behavior toward similar amounts of aliphatic hydrocarbons delivered as a single pulse of vapor. The male northern corn rootworm was monitored in his attraction to female rootworm extract (‘pheromone’) and his tendency to move upwind, or up airflow, toward the pheromone. The instrument enables discrimination of the transient metabolism of muscle use, from that of the resting state power which is probably the true basal metabolic rate power.