Synchronization of mammalian cells with tritiated thymidine

Short exposures of mammalian cells to tritiated thymidine of high specific activity destroys the proliferative capacity of mammalian cells. Since the killing is limited to cells that have synthesized DNA in the presence of the labeled compound, an exposure duration of less than one generation can yield a synchronized population.     

Beta-hydroxybutyrate dehydrogenase: lack in ruminant liver mitochondria

The enizyme D(-)-beta-hydroxybutyrate dehydrogenase has been reported to occur in all mitochondria isolated from mammalian tissues and is especially high in liver. By contrast only very low concentration of the enzyme is detectable in mitachondria from bovine and sheep liver. The amount of the enzyme in mitochondria from other ruminant tissues such as kidney and heart are comparable with those from mammalian sources. The verv low activity of beta-hydroxybutyrate dehydrogenase is not referable to the iniability of substrates to penetrate milochondria or to decreased electron transfer activity but rather to a lack of the enzyne in ruminant liver mitochondria.     

Measurement of suppressor transfer RNA activity

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.     

Mammalian and yeast ras gene products: biological function in their heterologous systems

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.     

鸡肉钙激活酶与其它畜禽肉中钙激活酶 对钙离子浓度敏感性的比较

 【目的】研究鸡肉中钙激活酶的种类和性质,并与其它畜禽肉以及鱼肉中钙激活酶对钙离子浓度敏感性进行比较。【方法】提取宰后0 d鸡胸肉中的钙激活酶,通过硫酸铵沉淀和透析的方法对鸡肉钙激活酶进行纯化,采用活性电泳的方法检测钙激活酶种类和活性。通过设置0、0.01、0.03、0.05、0.10、0.50和5.00 mmol·L-1的钙离子浓度,比较鸡肉、鱼肉、鸭肉、兔肉、猪肉、牛肉在宰后0 d肉样中不同种类的钙激活酶对钙离子的敏感性。【结果】禽肉鸡肉、鸭肉中μ-钙激活酶的电泳迁移率小于哺乳类兔肉、猪肉、牛肉,当Ca2+浓度从0.01 mmol·L-1升到0.03 mmol·L-1时,兔肉、猪肉和牛肉μ-钙激活酶活性增加,但鸡肉和鸭肉钙激活酶活性没有改变（P>0.05）,说明禽类μ-钙激活酶对钙离子的敏感程度高于哺乳类;在禽类鸡肉、鸭肉中存在另外一种钙激活酶,其对钙离子的敏感性介于哺乳动物中的m-钙激活酶和μ-钙激活酶之间,并且它在禽肉中的活性分别占67.4%和88.4%。【结论】鸡肉中的钙激活酶对钙离子浓度敏感性高于哺乳类。     

Spontaneous impulse activity of rat retinal ganglion cells in prenatal life

The existence of spontaneous neural activity in mammalian retinal ganglion cells during prenatal life has long been suspected. This activity could play a key role in the refinement of retinal projections during development. Recordings in vivo from the retinas of rat fetuses between embryonic day 17 and 21 found action potentials in spontaneously active ganglion cells at all the ages studied.     

Synaptic connections in vitro: modulation of number and efficacy by electrical activity

The functional architecture of synaptic circuits is determined to a crucial degree by the patterns of electrical activity that occur during development. Studies with an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons slow that electrical activity induces competitive processes that regulate synaptic efficacy so as to favor activated pathways over inactive convergent pathways. At the same time, electrical activity initiates noncompetitive processes that increase the number of axonal connections between these sensory and spinal cord neurons.     

Diphtheria toxin subunit active in vitro

Exposure of diphtheria toxin to dithiothreitol (and similar thiols) resulted in a subunit which was active in catalyzing the adenosine diphosphateribosylation of mammalian aminoacyl-transferase II in the presence of nicotinamide adenine dinucleotide. At the same time there was a marked increase in total ADP-ribosylation activity. A molecule which was apparently identical to the derived subunit in size and activity was detected in partially purified preparations of toxin.     

Prolactin: evidence that it is separate from growth hormone in human blood

A highly sensitive bioassay has been developed for prolactin, which uses a mammalian end organ and which is capable of measuring the hormone in unextracted human plasma. High prolactin activity, largely neutralizable with antiserum to human growth hormone, is stimulated along with high immunoassayable growth hormone by insulin-induced hypoglycemia. High prolactin activity, not neutralizable with antiserum to growth hormone, exists with low concentrations of plasma growth hormone in postpartum patients and patients with galactorrhea.     

Electrophoretic heterogeneity of mammalian galactose dehydrogenase

Electrophoretically distinct forms of galactose dehydrogenase were demonstrated in various tissues of the rat. Phylogenetic comparisons revealed considerable variation with species in mammalian liver zymograms; virtually no activity was demonstrable in fish, pigeon, and frog liver. Ontogenetic studies of the rat revealed sequential appearance of liver isoenzymes.     

SIRT6 promotes DNA repair under stress by activating PARP1

Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.     

SEPARATION OF TRANSDUCER AND IMPULSE-GENERATING PROCESSES IN SENSORY RECEPTORS

New evidence is presented that spike and transducer processes in sensory receptors are independent events; impulse activity in tile crustacean stretch receptor neuron and the mammalian pacinian corpuscle was selectively blocked by a compound (tetrodotoxin) without affecting any of the parameters of the generator potential.     

人H1启动子转录shRNA的细胞种属特异性研究

利用siDirect软件预测绿色荧光蛋白(GFP)基因特异性小干扰RNA(siRNA),将人工合成的相应shRNA插入含人H1启动子的pSuper载体,获得表达载体pSuper-shRNA,再将H1-shRNA插入表达GFP基因的peGFP-N1载体,获得表达载体peGFP-H1-shRNA。分别以pSuper-shRNA peGFP-N1和peGFP-H1-shRNA转染COS-1、293-T、鸡胚肝(CEL)和鸡胚成纤维(CEF)细胞,根据相同条件下GFP阳性细胞数及荧光强度变化判断产生的siRNA对GFP基因表达的沉默作用,比较人H1启动子在哺乳动物和禽源细胞中的转录活性。结果表明:人H1启动子在2种哺乳动物细胞中能有效转录shRNA,但在2种禽源细胞中的转录活性很弱,提示在禽源细胞中表达siRNA和进行基因沉默研究应选用禽源启动子。     

Coherin: a new peptide of the bovine neurohypophysis with activity on gastrointestinal motility

A factor with potent activity in the regulation of mammalian gastrointestinal motor function has been isolated from the bovine posterior pituitary gland by a process allowing minimal dissociation of neurophysin-bound complexes and the separation of free unbound peptides. This substance alters the frequency, amplitude, rhythm, and duration of peristaltic contraction.     

Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation

The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. However, by metabolic labeling of mammalian cells with 35S, we show that the sulfinic form of peroxiredoxin I, produced during the exposure of cells to H2O2, is rapidly reduced to the catalytically active thiol form. The mammalian cells' ability to reduce protein sulfinic acid might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated.     

捻转血矛线虫H11蛋白提取及其氨肽酶活性分析

试验对体外培养的捻转血矛线虫L4期幼虫和自然感染的成虫的H11天然蛋白分别进行了提取和纯化,并进一步测定了其氨基肽酶活性及酶抑制剂敏感性,为利用捻转血矛线虫天然抗原进行疫苗研制进行了初步探索。结果表明,H11蛋白在L4期幼虫和自然感染成虫体内均有表达,且具有氨肽酶M(ApM)活性和氨肽酶A(ApA)活性,其酶活性能被EDTA及邻二氮杂菲等哺乳动物氨肽酶抑制剂所抑制。H11蛋白是寄生阶段虫体的一种酶类,在对H11蛋白进行操作时,应按照相应的酶处理方法进行,以防变性,影响抗原免疫效果。     

PB2蛋白E627V突变可增强H7N9病毒对小鼠的致病力

【背景】低致病性H7N9病毒自2013年在我国首次出现以来,研究发现其对家禽均呈现低致病力,对哺乳动物模型小鼠也不表现任何的致病力。但是,2015年在湖南分离的1株低致病性H7N9病毒(简称HuN/S40726),却对哺乳动物小鼠表现为高致病力。分析、推测导致该病毒对哺乳动物致病力改变的原因可能在于该病毒PB2蛋白E627V的改变。【目的】为了揭示该病毒致病力的变化原因以及对哺乳动物致病性增强的机制,提供人类H7N9病毒感染、危害增强风险预警,展开该研究。【方法】选取2013年低致病性H7N9病毒代表株(简称SH/S1053)和上述湖南HuN/S40726病毒,开展了哺乳动物致病力对比试验以及可能导致病毒发生致病力变化的相关基因位点对比分析。然后以HuN/S40726病毒为模式毒株,利用反向遗传学技术,成功建立了病毒的反向遗传操作系统;利用基因点突变技术定点突变了HuN/S40726病毒PB2蛋白的627位氨基酸,救获了重组病毒r HuN/S40726、r HuN/S40726-PB2/627E和r HuN/S40726-PB2/627K。通过小鼠感染模型评估了以上3株重组突变病毒对哺乳动物的致病力,分析了PB2蛋白627位氨基酸的突变对小鼠致病力的差异。然后通过构建HuN/S40726病毒及其突变病毒的聚合酶复合表达质粒系统,以SH/S1053病毒为背景毒株构建聚合酶复合表达质粒系统作为对照,使用双荧光素酶法检测了PB2蛋白627位氨基酸的不同突变体在293T细胞中的聚合酶活性,进一步分析PB2蛋白627位氨基酸影响病毒毒力的内在机制。【结果】通过哺乳动物致病力对比试验以及可能导致病毒发生致病力变化的相关基因位点对比分析,推测导致HuN/S40726病毒对哺乳动物致病力改变的原因可能在于该病毒PB2蛋白E627V的改变。重组救获病毒和突变株对小鼠致病性试验结果显示,PB2蛋白E627V的改变显著的增强了HuN/S40726病毒对小鼠的致病力,使得病毒MLD50由≥6.5 log10EID50变化为3.5 log10EID50,病毒毒力增强了至少1 000倍以上。聚合酶活性试验结果表明,无论是在33℃还是37℃下,PB2蛋白E627V的改变,显著提高了HuN/S40726病毒在哺乳动物细胞中的聚合酶活性,与病毒对小鼠的致病性增强呈正相关性。【结论】PB2蛋白627位氨基酸(V)决定了HuN/S40726病毒对小鼠的高致病力。PB2蛋白E627V的改变能够显著增强HuN/S40726病毒在哺乳动物细胞中的聚合酶活性,是引起HuN/S40726病毒对哺乳动物致病力的重要因素。