Template-directed oligomerization catalyzed by a polynucleotide analog

A pyrophosphate-linked analog of polycytidylic acid has been synthesized and shown to catalyze the oligomerization of the complementary monomer 2'-deoxyguanosine 3',5'-bisphosphoimidazolide. Analogs of polynucleotides are of interest in studies of the origins of life as possible precursors of the first RNA molecules. These results demonstrate that such molecules are capable of serving as templates for further synthesis.     

Deprotection of Acetyl Group on Amino Group with Thionyl Chloride and Pyridine

[Objective] This study aimed to analyze the deprotection of acetyl group on amino group.[Method] A simple,convenient one-pot amino protection group of amide removed by thionyl chloride and pyridine via efficient chlorination and hydrolysis with 1,2-dichloroethane as solvent at ambient temperature has been developed.[Result] Pyridine is crucial to the reaction;the best solvent is 1,2-dichloroethane,and the most suitable reaction temperature is the ambient temperature;in addition,the yield is the highest as the molar ratio of pyridine to N-(4-bromophenyl) acetamide is 1:1.[Conclusion] The significant features of this protocol include short reaction time,cleaner reaction profiles,under mild reaction conditions and easy purification,and simple workup that precludes the use of toxic solvents.     

Recombinant origin of the retrovirus XMRV

The retrovirus XMRV (xenotropic murine leukemia virus-related virus) has been detected in human prostate tumors and in blood samples from patients with chronic fatigue syndrome, but these findings have not been replicated. We hypothesized that an understanding of when and how XMRV first arose might help explain the discrepant results. We studied human prostate cancer cell lines CWR22Rv1 and CWR-R1, which produce XMRV virtually identical to the viruses recently found in patient samples, as well as their progenitor human prostate tumor xenograft (CWR22) that had been passaged in mice. We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages. In particular, we found that the host mice contained two proviruses, PreXMRV-1 and PreXMRV-2, which share 99.92% identity with XMRV over >3.2-kilobase stretches of their genomes. We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10(-12)); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event.     

"Dip-Pen" nanolithography

A direct-write "dip-pen" nanolithography (DPN) has been developed to deliver collections of molecules in a positive printing mode. An atomic force microscope (AFM) tip is used to write alkanethiols with 30-nanometer linewidth resolution on a gold thin film in a manner analogous to that of a dip pen. Molecules are delivered from the AFM tip to a solid substrate of interest via capillary transport, making DPN a potentially useful tool for creating and functionalizing nanoscale devices.     

Glycine inhibition of asparaginase

Administration of either Escherichia coli asparaginase or guinea pig serum to C3H/HE mice with the 6C3HED lymphosarcoma is followed by depression of glycine in the tumor. This decrease in cellular glycine concentration does not occur in a tumor resistant to asparaginase. The inhibition of the lymphosarcoma by asparaginase can be reversed by intraperitoneal injection of asparagine or glycine. This reversal appears to be specific because lysine, threonine, serine, and aspartic acid were ineffective. Loss of cellular glycine may be more important than loss of asparagine because of the requirement for glycine in purine synthesis.     

ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis

Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.     

Murine lymphoma-induced immunosuppression: requirement for direct tumour cell contact

The FBL-3 lymphoma cell line caused impaired antibody formation in vivo when injected into mice intraperitoneally, and in vitro when added to normal syngeneic spleen cells immunized in vitro with sheep erythrocytes. Immunosuppression occurred only when intact viable tumor cells were cocultivated with the normal spleen cells. As few as 10(5) FBL-3 cells, when added to 5 X 10(6) normal cells, impaired antibody formation. However, cell-free extracts of filtrates from even much larger numbers of tumor cells did not affect antibody formation, either in vitro or in vivo. Heating the tumor cells at 56 degrees C or irradiation with as little as 1000 rads completely abolished immunosuppressive activity, both in vitro and in vivo. Separation of viable tumor cells from target antibody-forming cells by cell-impermeable membranes prevented immunosuppression, showing that direct cell-to-cell contact is required for immunosuppression.     

Induction of tumors in mice by genomic hypomethylation

Genome-wide DNA hypomethylation occurs in many human cancers, but whether this epigenetic change is a cause or consequence of tumorigenesis has been unclear. To explore this phenomenon, we generated mice carrying a hypomorphic DNA methyltransferase 1 (Dnmt1) allele, which reduces Dnmt1 expression to 10% of wild-type levels and results in substantial genome-wide hypomethylation in all tissues. The mutant mice were runted at birth, and at 4 to 8 months of age they developed aggressive T cell lymphomas that displayed a high frequency of chromosome 15 trisomy. These results indicate that DNA hypomethylation plays a causal role in tumor formation, possibly by promoting chromosomal instability.     

Nebulin and N-WASP cooperate to cause IGF-1-induced sarcomeric actin filament formation

Insulin-like growth factor 1 (IGF-1) induces skeletal muscle maturation and enlargement (hypertrophy). These responses require protein synthesis and myofibril formation (myofibrillogenesis). However, the signaling mechanisms of myofibrillogenesis remain obscure. We found that IGF-1-induced phosphatidylinositol 3-kinase-Akt signaling formed a complex of nebulin and N-WASP at the Z bands of myofibrils by interfering with glycogen synthase kinase-3β in mice. Although N-WASP is known to be an activator of the Arp2/3 complex to form branched actin filaments, the nebulin-N-WASP complex caused actin nucleation for unbranched actin filament formation from the Z bands without the Arp2/3 complex. Furthermore, N-WASP was required for IGF-1-induced muscle hypertrophy. These findings present the mechanisms of IGF-1-induced actin filament formation in myofibrillogenesis required for muscle maturation and hypertrophy and a mechanism of actin nucleation.     

Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA

A rat dopamine (DA) transporter complementary DNA has been isolated with combined complementary DNA homology and expression approaches. The DA transporter is a 619-amino acid protein with 12 hydrophobic putative membrane-spanning domains and homology to the norepinephrine and gamma-aminobutyric acid transporters. The expressed complementary DNA confers transport of [3H]DA in Xenopus oocytes and in COS cells. Binding of the cocaine analog [3H]CFT ([3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane) to transfected COS cell membranes yields a pharmacological profile similar to that in striatal membranes.     

High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease

An analog of staphylococcal nuclease has been prepared in which all amino acids, except the six following, are fully deuterated: tryptophan; methionine; tyrosine, in ring positions 2 and 6; histidine, in ring position 2; aspartic acid and asparagine, beta-methylene; and glutamic acid and glutamine, gamma-methylene. The analog has a much simpler high-resolution nuclear magnetic resonance spectrum than the fully protonated enzyme. The effects of calcium ion and of the inhibitor 3', 5'-thymidine diphosphate on the spectrum of the analog were readily detected.     

Tumor formation dependent on proteoglycan biosynthesis

The role proteoglycans play in tumor formation was examined by measuring the tumorigenicity of proteoglycan-deficient Chinese hamster ovary cell mutants in nude mice. When 10(7) cells were injected subcutaneously, mutants with less than about 15% of the wild-type level of proteoglycan synthesis did not produce tumors. Mutants defective in the synthesis of heparan sulfate proteoglycans also did not form tumors, whereas mutants with altered chondroitin sulfate proteoglycans were tumorigenic. Tumors arose from mixtures of wild-type and nontumorigenic mutant cells and contained both cell types, suggesting that wild-type cell proteoglycans enabled mutant cells to survive. The failure of heparan sulfate-deficient mutants to form tumors depended on the ability of the host to mount a B cell-mediated immune reaction.     

In vivo determination of the pyridine nucleotide reduction charge by carbon-13 nuclear magnetic resonance spectroscopy

An intracellular coenzyme has been observed by carbon-13 nuclear magnetic resonance spectroscopy. The pyridine nucleotides in Escherichia coli were specifically labeled with carbon-13 from the biosynthetic precursor, nicotinic acid. The intracellular redox status and metabolic transformations of the pyridine nucleotides were examined under a variety of conditions. A highly reduced nicotinamide adenine dinucleotide pool was observed under anaerobic conditions only in cells that were cultured aerobically on glycerol.     

基于微生物16SrRNA测序技术对ICR小鼠传代后肠道菌群变化的分析

为探讨不同代次雄性ICR小鼠生长发育与其肠道微生物的关系,本研究通过对16SrRNA基因V3~V4区测序,分析F0到F3代雄性ICR小鼠肠道菌群多样性和菌种组成。结果显示:1)从F0到F3代,组间微生物的Alpha多样性中的Chao1指数,Observed-species指数和香侬指数(Shannon)指数随代次积累显著降低(P<0.05),Simpson指数无显著差异(P>0.05)。2)从F0到F3代的优势菌门均为厚壁菌门(Firmicutes)(F0、F1、F2和F3丰度分别为83.3%、79.0%、81.8%和90.3%)和拟杆菌门(Bacteroides)(F0、F1、F2和F3丰度分别为6.0%、5.1%、4.0%和3.2%)。3)F0,F1和F2代三组的优势菌属均为乳酸菌属(Lactobacillus)(F0、F1和F2丰度分别为83.1%、62.8%和38.2%),但F3代的一些未确认分类的菌属占比较高,丰度约为45.3%;从F0到F3代在属水平上微生物组成上存在显著差异(P<0.05),各组的乳酸菌属丰度随代次的积累显著降低(P<0.05)。4)对差异菌属进行了KEGG差异代谢通路预测,发现乙醛酸盐代谢、胰岛素信号通路、苯丙氨酸,色氨酸和酪氨酸生物合成途径,萜类化合物生物合成途径,肽聚糖生物合成和糖酵解糖异生作用在F3代中显著降低,表明F3代氨基酸合成、糖类代谢等多条基础代谢通路受损。综上,本研究基于微生物16SrRNA测序技术对ICR小鼠传代后肠道菌群变化进行了一系列分析和功能预测,发现微生物多样性发生改变,F0至F2代优势菌门未发生明显变化,但在F3代出现了较多的为确认分类菌属,并且在属水平上各组的乳酸菌属丰度随代次的积累显著降低,F3代氨基酸合成、糖类代谢等多条基础代谢通路受到影响。本研究有助于从肠道微生物与宿主间的相互作用角度解释ICR雄性小鼠传代后出现的体重偏低等现象产生的原因。     

Steroidogenesis-activator polypeptide isolated from a rat Leydig cell tumor

A cycloheximide-sensitive protein responsive to adenosine 3',5'-monophosphate has been postulated to participate in the regulation of cholesterol side-chain cleavage activity in steroidogenic tissues. Such a steroidogenesis activator polypeptide (SAP) had been isolated from rat adrenocortical tissue and partially characterized. Now a polypeptide with comparable chromatographic behavior and biological activity has been purified from the rat H-540 Leydig cell tumor in quantities sufficient for amino acid sequencing. The activator contains 30 amino acid residues and has a molecular weight of 3215. The synthetic construct based on this sequence is virtually equipotent with native H-540 tumor SAP in an adrenal mitochondrial cholesterol side-chain cleavage assay. Hormonal regulation of the intracellular concentration of this activator may control the rate of cholesterol metabolism in steroidogenic organs.     

THE PRODUCTION OF FOLIC ACID BY RAT LIVER IN VITRO

The occurrence in urine and in acid-autoclaved grass and liver extracts of a substance which appears to participate in the synthesis of folic acid by rat liver in vitro is described. A similar effect is produced by synthetic xanthopterin. The effect of these materials might be accomplished by (1) catalysis of the enzymatic synthesis of folic acid; (2) the release of folic acid not liberated by takadiastase from tissue complexes; or (3) their serving as substrate material for the enzymatic synthesis of folic acid. The data presented favor the last hypothesis and suggest that xanthopterin, or a substance derived from it, may constitute a portion of the folic acid molecule. The probable involvement of compounds related to ranthopterin in the formation of hemocytopoietic substances in several animal species is discussed.     

BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity

Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.     

Autoradiographic investigations of incorporation of H3-thymidine into cells of the rat and mouse

The application of H(3)-thymidine results in labeling of those nuclei of cells in which deoxyribonucleic acid (DNA) is synthesized during the interval between application and the sacrifice of tne animal (1-3). This paper reports autoradiographic investigation with H(3)-thymidine of rats and mice. This method permits a more exact statement of the number of dividing cells than does the microscopic estimate of mitosis. The latter method is practically impossible in tissues with small fusiform cells. Moreover, it is possible to obtain information about the relative time of DNA synthesis in different cells.     

饲料中添加烟酸对夏季奶牛产奶性能的影响

选择9头泌乳早期处于热应激的荷斯坦奶牛,采用3×3拉丁方设计,分别饲喂5、10 g/d烟酸,研究其对奶牛生产性能的影响.结果表明:添加5、10 g/d烟酸可使产奶量分别提高13.20%、9.39%;而4%标准乳量无显著差异(P＞0.05);添加烟酸不影响乳蛋白含量和乳脂率.