Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Incubation of [1-13C]-5-phosphoribosyl pyrophosphate ([1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of [1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik [Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway.     

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.     

The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

在甜椒花药培养中马来酰胼对小孢子发育的抑制作用

选用甜椒的小孢子单核期花药,用100、300、500、2000ppm的马来酰肼溶液浸泡处理,并设对照,进行无激素MS固体培养基培养。分别取样用各种组化方法对花药内部多糖、蛋白质、核酸及ATP酶进行组化反应和形态学上的观察。与对照组相比,处理组花药外部形态和内部结构出现许多变异。小孢子内多糖,蛋白质含量减少;绒毡层无明显变化;两组中,核酸的含量均无明显变化;ATP酶的活性低于对照组。可能,马来酰肼对于花药中ATP酶等产生抑制作用,致使花药败育。     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [³H]leukotriene D₄ (LTD₄) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD₄. The values for fifty percent inhibition of bound [³H]LTD₄ was 1.5 nM for LTD₄, 45 nM for LTC₄ and 24 nm for LTE₄. LTB₄ at 3.0 × 10⁻⁵ M had no effect on [³H]LTD₄ binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC₄ and LTD₄. However, in the presence of 20 nM serine-borate this method was highly specific for LTD₄. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD₄. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient ‘r’ of 0.992. The assay was also validated by quantitation of LTs released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD₄ than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD₄ specifically or LTC₄ plus LTD₄ simultaneously in one cell system.     

Production of leukotrienes and prostaglandins in the rat uterus during peri-implantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC4 and LTB4) and prostaglandins (PGE2 and PGF2 alpha) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC4 or LTB4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE2 and PGF2 alpha showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: vasodilators and agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se.     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.