Glucose utilization by enzymatically-formed trophoblastic vesicles and Day-14 porcine blastocysts

The total glucose metabolism of 48-h spherical trophoblastic vesicles, Day-60 trophoblastic vesicles sections and Day-14 porcine blastocyst sections was measured by the method of O'Fallon and Wright (1). Trophoblastic vesicles were formed by enzyme dispersal in Day-14 porcine blastocysts. Glucose was based on DNA content of the tissue measured by diamino benzoic acid reaction with DNA (2). Slope of the lines (PMoles glucose utilized/4 h x DNA content) was different between Day-14 blastocyst sections and 48 h trophoblastic vesicles (P /= 0.05). Slopes of the lines were identical between 48-h trophoblastic vesicles and Day-60 trophoblastic vesicles sections (P >/= 0.87). Average glucose utilization on a per ng DNA basis was calculated. Day-14 blastocyst sections utilized 0.67 Pmoles glucose/4 h per ng DNA, Day-60 trophoblastic vesicles sections; 0.57; and 48-h sperical trophoblastic vesicles used 0.29. It is hypothesized that the change in glucose utilization between the Day-14 porcine blastocyst and enzymatically formed trophoblastic vesicles may be due to a decrease in metabolism as a consequence of in vitro culture. Further, it is theorized that Day-60 trophoblastic vesicles sections used higher quantities of glucose than 48-h sperical trophoblastic vesicles on a per ng DNA basis due to the increased availability of glucose to the cells of the inner layers, caused by the sectioning of the tissue. The results of this study identify changes in glucose metabolism of enzymatically formed porcine trophoblastic vesicles during culture. It is proposed that enzymatically-formed trophoblastic vesicles be used as a model system for the study of embyro metabolism.     

Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

Summary The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a)N-nitrosomethyl-urea (NMU)-induced and (b) 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGF₂, PGE₁, and PGF_2α (6.7, 4.7, and 1.7 ng/10⁶ cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF_1α and TXB₂, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE₂ (1 and 4 ng/10⁶ cells per 48 h, respectively). PGE₂ production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1. 14. 99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE₂ levels incresed at low concentration of ibuprofen and then decreased at higher concentration. At 100 μg/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respecively. There was no obvinous dse-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiogically relevant amounts of PGs. This work was supported by Grant CA 29602 from the National Cancer Institute, Bethesda, MD, and Grant PDT-208 from the American Cancer Society.     

Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a) N-nitrosomethylurea (NMU)-induced and (b) 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGE2, PGE1, and PGF2 alpha (6.7, 4.7, and 1.7 ng/10(6) cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF1 alpha and TXB2, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE2 (1 and 4 ng/10(6) cells per 48 h, respectively). PGE2 production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1.14.99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE2 levels increased at low concentrations of ibuprofen and then decreased at higher concentrations. At 100 micrograms/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respectively. There was no obvious dose-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiologically relevant amounts of PGS.     

Protein production by auto-induction in high density shaking cultures

Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).     

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.     

Proteins secreted by day-16 to -18 bovine conceptuses extend corpus luteum function in cows

Corpus luteum function, interoestrous interval and spontaneous uterine PGF-2 alpha (PGF) production were evaluated in 9 cyclic Holstein cows (3/group) after intrauterine injections of pooled conceptus secretory proteins, 5 beta-pregnan-3 alpha-ol-20-one, or homologous serum proteins on Days 15.5 through 21 after oestrus. A significant extension of corpus luteum lifespan and interoestrous interval were detected in cows treated with conceptus secretory proteins compared to the other 2 groups. CL lifespan and interoestrous interval were not different (P greater than 0.25) between 5 beta-pregnan-3 alpha-ol-20-one and control groups. Evaluation of spontaneous PGF responses suggested that proteins synthesized and secreted by the bovine conceptus accommodate luteal maintenance during early gestation via an attenuation of endometrial PGF production.     

Oestrogens and androgens in blastocoelic fluid and cultures of cells from equine conceptuses of 10-22 days gestation.

Six samples of blastocoele fluid recovered between 10 and 22 days gestation were tested in human clinical radioimmunoassay systems measuring total oestrogens and total androgens. The results were erratic but in 5 cases measurements for oestrogen equivalent to between 1000 and 70,000 pg/ml and for androgen between 1000 and 85,000 pg/ml were recorded. Cells from two blastocysts were cultured in medium 199 with and without horse serum. When the used media were assayed, values equivalent to at least 8000 pg oestrogen/ml were obtained on 7 of 11 occasions. In 9 of 11 samples the androgen concentration exceeded 700 pg/ml. In control media the oestrogen and androgen concentrations did not exceed 120 and 360 pg/ml respectively.     

14-3-3蛋白和神经退行性疾病

14-3-3蛋白以二聚体形式存在,识别磷酸丝氨酸／苏氨酸连接的信号分子,通过与其配体蛋白质相互作用,参与细胞信号转导、细胞周期调控和细胞问运输。14-3-3蛋自在脑组织中含量丰富,所有神经元、星形胶质细胞、少突肢质细胞、小胶质细胞的胞液与胞核中都有表述。14-3-3蛋白与神经退行性疾病阿尔茨海默病和帕金森病的发生过程关系密切。     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如：新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

Protein production by sheep embryos during the period of maternal recognition of pregnancy

An embryo must be present in the uterus 12-13 days after estrus to prevent regression of the ovine corpus luteum. The present experiments were designed to determine if embryo-specific secretory proteins could be detected in the maternal blood at the time of maternal recognition of pregnancy. In two experiments, 92 embryos were flushed from 47 ewes at 14-15 days after estrus. Embryos were incubated in vitro for 24 h and the proteins in the media were harvested. Antisera to proteins in both flushing and incubation medium were produced in rabbits. In experiment 1, crude fractions were used for antibody production and radioimmunoassays were established for protein peaks separated on a 1.1 X 75 cm G-100 Sephadex column. Two low molecular weight fractions (EPiv and EPv) appeared to be embryo specific but were not detectable in jugular vein sera of 14- to 15-day pregnant animals. In experiment 2, proteins derived from uterine flushes and from embryo incubations were chromatographed on a 2.5 X 85 cm column of G-100 Sephadex. The protein peaks were measured, pooled, lyophilized, and used for immunization of rabbits. As in experiment 1, antisera were generated, some of which seemed to be directed against embryo-specific proteins. However, we could not detect these fractions in the uterine vein blood of pregnant animals. Thus, embryo-specific proteins are either confined to the uterus or they appear in the blood in quantities that are undetectable with our assay system.     

土生隐球酵母14-3-3蛋白耐铝能力的研究

14-3-3蛋白家族是由多个高度保守的成员构成的调节性蛋白质家族,它们主要以磷酸化的形式与伴侣蛋白相互作用,并能够以多种方式来影响靶蛋白。通过构建14-3-3蛋白原核表达载体,纯化重组蛋白获得14-3-3蛋白抗体。为了验证14-3-3蛋白基因在耐铝中的作用,构建14-3-3酵母表达载体,得到14-3-3过表达酵母菌株。在5mmol/L铝浓度下,转基因酵母比对照酵母长势好,这表明14-3-3蛋白通过促进生长赋予酵母对铝胁迫的耐受性。     

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.     

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Abstract The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO₃⁻ or O₂ as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha . Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.     

Gene targeting in primary fetal fibroblasts from sheep and pig

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.     

Formation of ketone bodies from [14C]palmitate and [14C]glycerol by tissues from ketotic sheep

Labelled ketone bodies were produced readily from [U-(14)C]palmitate, [2-(14)C]palmitate and [1-(14)C]glycerol by sheep rumen-epithelial and liver tissues in vitro. On a tissue-nitrogen basis, both tissues had similar capacities for ketogenesis. Palmitate was a ketogenic substrate in both rumen-epithelial tissue and liver, and more of its (14)C appeared in ketone bodies than in the (14)CO(2) liberated. Glycerol was actively metabolized to ketone bodies, but more readily underwent complete oxidation to carbon dioxide; this complete oxidation was most pronounced in rumen-epithelial tissue from ketotic ewes. These experiments with labelled compounds confirm earlier observations that rumen-epithelial tissue, like liver, actively forms ketone bodies from long-chain fatty acids and show further that normal rumen-epithelial tissue can convert palmitate into ketone bodies as readily as into carbon dioxide. Free glycerol, which is metabolized only by liver tissue in non-ruminants, is also metabolized by rumen epithelium. The rumen epithelium thus has unique metabolic capacity among extrahepatic tissues.