Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.     

Environmental variables influencing in vitro development of hamster 2-cell embryos to the blastocyst stage

This study is a systematic analysis of environmental variables influencing development of 2-cell hamster embryos to the blastocyst stage in vitro. Experiments were done using a chemically defined (protein-free) culture medium (HECM-2). Physicochemical variables examined were temperature, the concentrations of CO2, HCO3-, Ca2+, Mg2+, K+, and O2, the presence of a silicone oil overlay, and osmotic pressure. The optimal temperature for development in vitro was 37.5 degrees C; lower temperatures were inhibitory. There was no significant effect on blastocyst development of alterations in the concentrations of NaHCO3, CaCl2, MgCl2, and KCl, or in the ratios of Ca2+:Mg2+ and Na+:K+, over the ranges tested. Development to the blastocyst stage was significantly stimulated by increased CO2 concentrations (7.5% and 10%), by reduced O2 concentrations (10% and 5%), and by the presence of silicone oil overlying the culture medium. Reduction of blastocyst development in the absence of an oil overlay was not caused by increased osmotic pressure. Cleavage stage embryos were not affected by osmolalities ranging from 250 to 350 mOsmols, but blastocyst development was inhibited at greater than or equal to 325 mOsmols. Under optimized conditions (37.5 degrees C, 10% CO2, 25 mM HCO3-, 2.0 mM Ca2+, 0.5 mM Mg2+, 3.0 mM K+, 10% O2, 250-300 mOsmols, with silicone oil overlay), 51-57% of 2-cell hamster embryos developed to the blastocyst stage. This represents a significant improvement over previous "standard" culture conditions, which supported development of 26% blastocysts from 2-cell hamster embryos. The culture system described here provides an adequate baseline response to support detailed investigations into the regulation of embryo development in the hamster.     

鼠李糖乳杆菌LV108及其发酵乳免疫调节作用的比较

目的探讨鼠李糖乳杆菌LV108及其发酵乳对免疫抑制小鼠免疫功能的调节作用。方法将BALB/c小鼠随机分为5组,每组10只,即空白组(正常小鼠)、模型组(免疫抑制小鼠)、药物组(免疫抑制小鼠食物中添加左旋咪唑)、LV108菌悬液组(免疫抑制小鼠食物中添加LV108菌悬液)和LV108发酵乳组(免疫抑制小鼠食物中添加LV108发酵乳),除空白组外其余组构建免疫抑制小鼠模型。干预4周后,分别测定各组小鼠体质量和脏器指数,血清中白细胞介素2(IL2)、肿瘤坏死因子α(TNFα)和免疫球蛋白G(IgG)含量,血清溶血素含量、耳肿胀度和肝、脾巨噬细胞吞噬能力。结果相比模型组,LV108菌悬液组和LV108发酵乳组小鼠体质量增长速度、脏器指数、血清IL2与IgG水平、血清溶血值、耳肿胀度和巨噬细胞吞噬能力显著升高(均P<0.05);在脾脏指数、血清IL2与TNFα水平、血清溶血素含量和耳肿胀度免疫指标上,LV108菌悬液组与LV108发酵乳组之间比较差异具有统计学意义(均P<0.05)。结论LV108菌体及发酵乳对免疫抑制小鼠具备较全面的免疫调节作用,均可提高小鼠的自身免疫力;LV108发酵乳对小鼠的免疫调节作用强于LV108菌体。     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.     

11beta-Hydroxysteroid Dehydrogenase Type 1 Regulation by Intracellular Glucose 6-Phosphate Provides Evidence for a Novel Link between Glucose Metabolism and Hypothalamo-Pituitary-Adrenal Axis Function

Microsomal glucose-6-phosphatase-alpha (G6Pase-alpha) and glucose 6-phosphate transporter (G6PT) work together to increase blood glucose concentrations by performing the terminal step in both glycogenolysis and gluconeogenesis. Deficiency of the G6PT in liver gives rise to glycogen storage disease type 1b (GSD1b), whereas deficiency of G6Pase-alpha leads to GSD1a. G6Pase-alpha shares its substrate (glucose 6-phosphate; G6P) with hexose-6-phosphate-dehydrogenase (H6PDH), a microsomal enzyme that regenerates NADPH within the endoplasmic reticulum lumen, thereby conferring reductase activity upon 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 11beta-HSD1 interconverts hormonally active C11beta-hydroxy steroids (cortisol in humans and corticosterone in rodents) to inactive C11-oxo steroids (cortisone and 11-dehydrocorticosterone, respectively). In vivo reductase activity predominates, generating active glucocorticoid. We hypothesized that substrate (G6P) availability to H6PDH in patients with GSD1b and GSD1a will decrease or increase 11beta-HSD1 reductase activity, respectively. We investigated 11beta-HSD1 activity in GSD1b and GSD1a mice and in two patients with GSD1b and five patients diagnosed with GSD1a. We confirmed our hypothesis by assessing 11beta-HSD1 in vivo and in vitro, revealing a significant decrease in reductase activity in GSD1b animals and patients, whereas GSD1a patients showed a marked increase in activity. The cellular trafficking of G6P therefore directly regulates 11beta-HSD1 reductase activity and provides a novel link between glucose metabolism and function of the hypothalamo-pituitary-adrenal axis.