Mitotic Inhibition and Nucleic Acid Synthesis in Amoeba Nuclei

SEVERAL strains of large mononucleate amoebae contain specific proteins which inhibit mitosis and eventually kill amoebae of strains other than their own^1,2. We now describe experiments to show that this lethal antimitotic factor (AF) inhibits RNA synthesis when injected into susceptible cells.     

Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In Vivo

Abstract: d - and l -cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro. In vivo , l -cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l -cycloserine on a regimen of intraper-itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.     

Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.     

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

小鼠生殖细胞体外分化与发育机制

小鼠作为发育机制的模式动物,其生殖细胞分化与发育的研究一直是发育生物学研究的重点之一。主要综述了小鼠原始生殖细胞的起源、迁移与增殖的机制,以及原始生殖细胞向生殖细胞的分化,卵母细胞与精子的发生与发育机理,讨论了胚胎干细胞向生殖细胞体外诱导分化以及生殖细胞体外培养的应用前景。     

Studies on Nucleic Acid and Protein Synthesis and D evelopment of Male Gametnphyte of Clivia nobilis Cultured in Vitro

A simple method for culture male gametophyte (MG) of Clivia nobilis in vitro was established and the process of their development was observed. After research on dynamic of nucleic acid and protein synthesis of MG in various developmental stages by using of inhibitors and autoradiography authors found that DNA synthesis fro mrelease of tetrad to sperm only takes place in nucleus of interphase. There is no 3H-tymidine incorporation into vegetative nucleus (Vn), generative nucleus (Gn) or nucleus of sperm in 96 hours before dehiscence of anthers (BDA). The dynamic of protein synthesis is similar to the same of RNA’s. Both of them have three peaks and two intermissions. The 1st peak is in 12–9 days BDA. The 2nd is in 7–5 days BDA and intermitted from 48 hours BDA. The 3rd begins from the 1st hour after culture (AC) decleases at 6th hour AC and stops before 20th hour AC. The kind of inhibitor, the time and quanlity of treatment are affected the morphogenesis, showing the relationship to synthesis among DNA, RNA and protein and to the same between biomacromolecular and morphological development of MG.     

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.     

Nucleic Acid Profile of the Emt6 Cell Cycle In Vitro

Simultaneous RNA and DNA estimations were carried out during the cell cycle of EMT6/M/CC cells growing in vitro following synchronization by mitotic selection. the determinations were performed with a flow cytofluorimeter on individual cells stained with acridine orange. It was found that the RNA content increased during G₁ then remained virtually constant between early and mid S phase, but a second increase occurred during late S. the rate of uptake of tritiated uridine paralleled these changes in RNA levels, and it was also found that the rate of uptake in metaphase and anaphase was virtually zero, but a rapid increase occurred in telophase. the increase in DNA during S was approximately linear, and the intermitotic phase and cycle durations were very similar to previously reported results.     

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies—a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.     

False In Vitro and In Vivo Elevations of Uric Acid Levels in Mouse Blood

Uric acid (UA) levels in mouse blood have been reported to range widely from 0.1 μM to 760 μM. The aim of this study was to demonstrate false in vitro and in vivo elevations of UA levels in mouse blood. Male ICR mice were anesthetized with pentobarbital (breathing mice) or sacrificed with overdose ether (non-breathing mice). Collected blood was dispensed into MiniCollect® tubes and incubated in vitro for 0 or 30 min at room temperature. After separation of plasma or serum, the levels of UA and hypoxanthine were determined using HPLC. From the non-incubated plasma of breathing mice, the true value of UA level in vivo was 13.5 ± 1.4 μM. However, UA levels in mouse blood increased by a factor of 3.9 following incubation in vitro. This “false in vitro elevation” of UA levels in mouse blood after blood sampling was inhibited by allopurinol, a xanthine oxidase inhibitor. Xanthine oxidase was converted to UA in mouse serum from hypoxanthine which was released from blood cells during incubation. Plasma UA levels from non-breathing mice were 19 times higher than those from breathing mice. This “false in vivo elevation” of UA levels before blood sampling was inhibited by pre-treatment with phentolamine, an α-antagonist. Over-anesthesia with ether might induce α-vasoconstriction and ischemia and thus degrade intracellular ATP to UA. For the accurate measurement of UA levels in mouse blood, the false in vitro and in vivo elevations of UA level must be avoided by immediate separation of plasma after blood sampling from anesthetized breathing mice.     

蛋白核酸合成抑制剂和蛋白磷酸化对基因表达的调节

环己酰亚胺和放线菌素D明显降低小麦幼苗中BADH基因的表达,表明BADH基因表达在转录和转译水平上受到调控。氯霉素则有增加表达的效应。线粒体可能形成阻遏蛋白参与调节。H7和甘露糖降低BADH基因表达,相应地冈田酸(Okadaic acid)明显增加表达,说明蛋白磷酸化积极参与小麦幼苗中BADH基因表达的调节。     

Reversible Inhibition of Poliovirus RNA Synthesis In Vivo and In Vitro by Viral Products

In poliovirus-infected HeLa-S3 cells, the protease inhibitors tolylsulfonyl-phenylalanyl chloromethyl ketone and iodoacetamide cause an accumulation of large precursor proteins, and they block viral RNA synthesis most probably via these products. Viral RNA polymerase activity can, however, be extracted by detergent containing buffer (Tris/Nonidet P-40, deoxycholate) from the inhibited cells. Only cytoplasmic extracts from infected cells treated with tolylsulfonyl-phenylalanyl chloromethyl ketone or iodoacetamide contain a protein which inhibits the in vitro polymerase reaction.     

N- 糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗的 表达及免疫原性的影响

目的:研究N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗体外蛋白表达及小鼠体内体液免疫及细胞免疫应答的影响。方法:通过基因工程中定点突变技术,将乙型肝炎病毒表面抗原中蛋白(MHBs)中第4位氨基酸上连接的糖链去除,或将糖链依次移位至第5、6或7位氨基酸,来构建N-糖基化去除及移位的核酸疫苗,分别命名为Adr-dN4、Adr-N4-5、Adr-N4-6、Adr-N4-7。用上述核酸疫苗与野生型MHBs核酸疫苗(pSW3891/MHBs/Adr,简称Adr)及空载体质粒pSW3891分别用脂质体瞬时转染293T细胞,应用蛋白印迹法检测MHBs的表达。采用肌肉注射法,以各组疫苗分别对BALB/c小鼠于第0、2、4和6周进行免疫,用ELISA法检测小鼠血清中抗-HBs抗体、ELISPOT法检测小鼠表面抗原多肽特异性分泌IFN-γ的脾细胞数量。结果:蛋白印迹法结果显示Adr、Adr-dN4、Adr-N4-5、Adr-N4-6、Adr-N4-7体外转染293T细胞后,均可以在293T细胞内表达,且Adr、Adr-N4-5、Adr-N4-7可将表达产物分泌到细胞外。ELISA及EISPOT结果表明:Adr免疫组小鼠抗-HBs终点滴度及表面抗原特异性分泌IFN-γ的脾细胞数量,均略高于其他免疫组小鼠,但与Adr-N4-5、Adr-N4-7相比无统计学差异(P>0.05),与Adr-dN4和Adr-N4-6组相比有显著的统计学差异(P<0.05)。结论:在第5或7位氨基酸附加N-连接糖链,能修补或替代Asn4连接糖链引导MHBs分泌的功能。HBs表达蛋白分泌到细胞外对诱导机体产生特异性细胞和体液免疫是至关重要的。     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

In Vitro Protein Synthesis and Aging in Rhizoctonia solani

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.     

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear.     

N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗的表达及免疫原性的影响

目的：研究N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗体外蛋白表达及小鼠体内体液免疫及细胞免疫应答的影响。方法：通过基因工程中定点突变技术,将乙型肝炎病毒表面抗原中蛋白（MHBs）中第4位氨基酸上连接的糖链去除,或将糖链依次移位至第5、6或7位氨基酸,来构建N-糖基化去除及移位的核酸疫苗,分别命名为Adr—dN4、Adr-N4—5、Adr—N4．6、Adr—N4．7。用上述核酸疫苗与野生型MHBs核酸疫苗（pSW3891／MHBs／Adr,简称Adr）及空载体质粒pSW3891分别用脂质体瞬时转染293T细胞,应用蛋白印迹法检测MHBs的表达。采用肌肉注射法,以各组疫苗分别对BALB／c小鼠于第0、2、4和6周进行免疫．用ELISA法检测小鼠血清中抗．HBs抗体、ELISPOT法检测小鼠表面抗原多肽特异性分泌IFN-T的脾细胞数量。结果：蛋白印迹法结果显示Adr、Adr-dN4、Adr—N4．5、Adr—N4—6、Adr-N4—7体外转染293T细胞后,均可以在293T细胞内表达,且Adr、Adr-N4—5、Adr-N4—7可将表达产物分泌到细胞外。ELISA及EISPOT结果表明：Adr免疫组小鼠抗-HBs终点滴度及表面抗原特异性分泌IFN-γ的脾细胞数量,均略高于其他免疫组小鼠,但与Adr—N4—5、Adr．N4—7相比无统计学差异（P〉0．05）,与Adr-dN4和Adr—N4—6组相比有显著的统计学差异（P〈0．05）。结论：在第5或7位氨基酸附加N-连接糖链,能修补或替代Asn4连接糖链引导MHBs分泌的功能。HBs表达蛋白分泌到细胞外对诱导机体产生特异性细胞和体液免疫是至关重要的。     

Nucleic Acid Synthesis in Cytoplasm of Yaba Monkey Tumor Virus-Infected Cells

Yaba tumor virus progeny appeared in cynomolgus monkey kidney cells at 24 h postinfection and reached a plateau at 72 h in the first cycle of replication. Viral DNA synthesis was first detected at about 3 h and reached a peak in 18 h. Maximum coating of viral DNA in infected cells occurred at 4 days postinfection. Rapidly labeled RNA was synthesized in the cytoplasm of virus-infected cells. At 6 h postinfection 7 to 10S RNA was present; this species was present in greater amount at 12 h; at 24 h a truncated peak indicated the presence of 14 to 15S as well as 7 to 10S RNA. Hybridization data indicated that the largest peak of messenger RNA synthesis occurred at 11 to 13 h postinfection and a second, slightly smaller, peak occurred at 21 to 23 h.