Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.     

Kinetic Changes of Nucleic Acids and Protein During Embryogenesis of Wheat (Triticun vulgaris L.)

Per embryonic total nucleic acid, RNA content and per cell RNA content increased during embryogenesis, reached maximun at 21 day after anthersis. The per embryo and per cell protein content also increased concomitantly. But the protein content continued to increase up to 24 days after anthersis. On the basis of dry weight, RNA content decreased in the early stage of embryogenesis, but then increased over the period of later developmental stage. The protein content on the basis of dry weight also changed in similar way. It was likely the protein and RNA content changes concomitantly during the developmental process of wheat embryo. As to per embryo DNA content, it increased in early developmental stage, but then remained in a similar level during the later stage. The relationship between the changes of RNA content and protein synthesis, embryonie develope is also discussed in present paper.     

非洲紫罗兰叶片脱分化过程中核酸蛋白质和滨粉含量动态的研究

本文测定了非洲紫罗兰（Ｓａｉｎｔｐａｕｌｔａｉｏｎａｎｔｈａ）叶片脱分化过程中核酸、蛋白质和波粉的含量。结果表明：发生脱分化的叶片中的蛋白质和淀粉含量均低于对照,而ＲＮＡ含量则高于对照,ＤＮＡ含量无明显差异。叶片培养的第一天内,发生脱分化的叶片中的蛋白质含量明显下降,对照中的蛋白质含量上升。脱分化过程中,淀粉含量有一个上升、下降、再上升的变化过程,对照中淀粉含量一直上升。     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

ULTRASTRUCTURAL CYTOCHEMISTRY : Enzyme and Acid Hydrolysis of Nucleic Acids and Protein

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 µ sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3°C are more rapidly digested by proteinases than those fixed for the same length of time at 20°C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 µ sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Effect of Fluoride on Nucleic Acids and Growth in Germinating Corn Seedling Roots

Corn seeds were treated with 0.01 M sodium fluoride for various time periods. The treated seeds were germinated and grown until the seedling roots reached a standard size of 12±3 mm. Analyses were made for RNA and DNA contents of 3-mm seedling root tips. Determinations also were made for growth rate, rate of cell elongation, cell multiplication, and tissue maturity of 12-mm roots. RNA contents of 3-mm root tips were found to be directly proportional to the growth rates of the entire seedling root of corn seeds treated with sodium fluoride for various periods of time. The RNA content was reduced on a cell basis and was independent of the root tip cell number. The amount of DNA was not related to the growth rate of the intact seedling roots. Since fluoride reduced the number of mitotic figures, it was likely that fluoride inhibited DNA synthesis during the interphase of the mitotic cycle. Growth by cell multiplication was inhibited more than that by cell elongation in the sample treated with fluoride for a shorter period. The two types of growth, however, showed a similar level of growth reduction in the sample treated with fluoride for a longer period. Fluoride seemed to reduce the rates of cellular elongation and multiplication not more than about 40 per cent of the control value in these tissues under present experimental conditions. Fluoride also induced maturity in the seedling roots in proportion to the periods of fluoride treatment.     

Labeling During Cleavage of Nucleic Acids for Their Detection on DNA Chips

Abstract

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.     

在继代培养中玉米花粉愈伤组织无性系的核酸和蛋白质变化

本实验室筛选出具有不同分化能力的单倍体玉米无性系组织,挑选其大小相同、新鲜的、具有分化能力的无性系No.1和完全丧失分化能力的无性系No.250组织,转接到分化和继代两种培养基上,分析其DNA和RNA以及蛋白质含量的差异,以了解组织在分化过程中DNA、RNA和蛋白质动态。试验结果表明,在分化和继代两种培养基上,No.1的DNA、RNA和蛋白质含量都高于No.250;No.1和No.250在分化培养基上DNA和RNA含量增加的速度比在继代培养基上要快;在组织分化过程中,No.1会出现一些新的小分子量蛋白质分子,而No.250中却没有,这些特殊的蛋白质分子可能与组织分化有关。     

Effect of UV-Irradiation on Total Protein and Nucleic Acids in Alternaria alternata and Fusarium solani

UV-A^* irradiation caused increases in total protein in Fusariumsolani, while its effect on Alternaria alternata was variable,and not as clear-cut as in F. solani. On the other hand, UV-Birradiation stimulated protein production in both fungi. UV-Airradiation showed an inhibitory effect on total DNA in bothfungi, while the effect on RNA was stimulatory in F. solanibut had no effect on A. alternata. Short fluences of UV-B inhibitedDNA production to some extent in both fungi, however longerfluences increased DNA content especially in F. solani. Theeffect of UV-B on RNA production was inhibitory in F. solanibut not in A. alternata. A. alternata is much more resistantto UV-irradiation than is F. solani, and increases in proteinin the former after UV-irradiation suggests that protein mayplay a part in protection against the harmful effect of UV-irradiation. UV-A, UV-B, fluence, protein, nucleic acids, Alternaria alternata, Fusarium solani     

Enzyme and Nucleic Acid Formation During Synchronous Growth of Rhodopseudomonas spheroides

The synthesis of various cell components was examined during the anaerobic photosynthetic growth of synchronous populations of Rhodopseudomonas spheroides. Net deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein increased continuously as did the rate of incorporation of radioactive precursors into protein. The rates of incorporation of radioactive precursors into RNA and DNA were marked by abrupt discontinuities. It is not clear whether these discontinuities represent changes in rates of synthesis or fluctuations in precursor pools. Although the synthesis of bacteriochlorophyll occurred in a continuous manner, those enzymes examined which are involved in the synthesis of tetrapyrroles, i.e., succinyl CoA thiokinase, delta-aminolevulinic acid synthetase, and delta-aminolevulinic acid dehydrase, increased discontinuously. Two other enzymes not involved in tetrapyrrole biosynthesis were examined. Alkaline phosphatase increased in a stepwise manner during the division cycle, whereas the synthesis of ornithine transcarbamylase increased rapidly before leveling off for a period of time until synthesis began again. In each instance of discontinuous enzyme synthesis, increases occurred at regular and characteristic times during the division cycle. Ammonium sulfate precipitation was employed to remove low molecular weight end product inhibitors from enzyme preparations. These studies suggested that the stepwise increases in enzyme activity observed in the present investigation were not affected by periodic end product inhibition. A temporal map of enzyme synthesis during the division cycle was constructed. Both delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydrase appeared early in the division cycle, whereas alkaline phosphatase and succinyl CoA thiokinase appeared later on.     

Keto Acids in Pollen and Pollen Tubes of Sesbania aegyptica

Pollen and pollen tubes of Sesbania aegyptica Pers. contain α-ketoglutaric acid, oxaloacetic acid and pyruvic acid. Changes in the keto acids have been correlated with their corresponding amino acids during different phases of germination. It is suggested that keto acids were readily turned over during the elongation of pollen tubes.     

Effect of Feeding Millet (Sorghum vulgarie) Protein on Growth,Nucleic Acids and Proteins of Liver and Plasma of Rats

Effect of feeding millet (Sorghum vulgarie) at 5, 10 and 15 per cent protein levels respectively for a period of six weeks to rats on their liver DNA, RNA and proteins of liver, its subcellular fractions and plasma has been studied, and results compared with rats fed casein at 10 per cent level. Both liver DNA and RNA of rats fed millet at 5 per cent protein level were significantly increased. Liver proteins (mg/l00 g body weight) of rats fed millet at 5 and 10 per cent protein level were significantly increased and plasma proteins decreased. Incorporation of leucine-I-¹⁴C into both liver and plasma proteins of rats fed millet was significantly higher than the control.     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.