Artificial Induction and Evaluation of a Mild Isolate of Tomato Spotted Wilt Virus

A severe isolate (BL) of tomato spotted wilt virus (TSWV) that originated from Hawaii was treated with nitrous acid in an effort to obtain mild mutants. The standardized procedure used in mutation experiments was: extracting infected Gomphrena globosa L. leaf tissue in 0.01 M Na₂SO₃, 0.125 M sodium acetate and 0.4 M sodium nitrite at pH 5.5 and incubating the extract for 20 min at room temperature. The extract was inoculated to tobacco (Nicotiana tabacum L. cv. Havana 423) and local lesions were subsequently transferred to lettuce (Lactuca sativa L. cv. Minetto). One isolate (R27G) that incited mild symptoms in lettuce was obtained out of 868 local-lesion-transfers. Under greenhouse conditions, the isolate induced mild symptoms on tomato (Lycopersicon esculentum Mill.) but was severe on peppers (Capsicum annuum L.). The effect of the R27G isolate on growth of potted tomatoes kept outdoors was variable. In one trial, only 15 % of the fruit had symptoms versus 67 % in another trial. R27G fully protected Datura stramonium L. plants that were challenge inoculated with the severe parent BL isolate. Less effective cross protection was observed against a severe isolate from Oklahoma.     

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

记真盔甲鱼类两新属——兼论真盔甲鱼类系统发育关系

本文记述了早泥盆世真盔甲鱼类两新属:憨鱼属(Nochelaspis)和翼角鱼属(Pterogonaspis).真盔甲鱼类是盔甲鱼类的一个单系类群,现有七属十二种,文中运用分支系统学原理对其系统发育关系进行了初步探讨.     

γ-氨基丁酸、荷包牡丹碱和L-谷氨酸钠对猫和家兔同侧和对侧图形视觉诱发电位的影响

在猫和家兔大脑半球一侧视区17/18交界处施加γ—氨基丁酸(GABA)、荷包牡丹碱和L—谷氨酸钠,以及用氯化钾和冷冻阻遏的方法,记录对侧和同侧皮层相应处图形视觉诱发电位(PVEP)的变化。讨论了GABA、荷包牡丹碱和L—谷氨酸钠对猫和兔的对侧和同侧PVEP的影响。     

Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography

Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

Construction of Killer Wine Yeast Strain

A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the killer factor also inhibited the growth of killer-sensitive cells satisfactorily when it was grown in grape juice.     

Synthesis of a nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II and its heavy metal-binding properties

A nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II was synthesized by the fragment condensation method. The Cd-binding ability of the beta-fragment was much stronger than that of cysteine as thionein and synthetic alpha-fragment corresponding to the C-terminal sequence 30-61 of human liver metallothionein II. Both the alpha- and beta-fragments bound preferentially to Cu ions rather than Cd ions.