Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Flavonoids from Artemisia ludoviciana var. ludoviciana

Nineteen flavonoids were isolated from Artemisia ludoviciana var. ludoviciana, including a new 2′- hydroxy- 6-methoxyflavone, 5,7,2′,4′-tetrahydroxy-6,5′-dimethoxyflavone. The known compounds include quercetagetin 3,6,3′,4′-tetramethyl ether, eupatilin, 5,7-dihydroxy-3,6,8,4′-tetramethoxyflavone, luteolin 3′,4′-dimethyl ether, jaceosidin, 5,7,4′-trihydroxy-3,6-dimethoxyflavone, tricin, hispidulin, chrysoeriol, kaempferol 3-methyl ether, apigenin, axillarin, eupafolin, selagin and luteolin together with three flavones which were previously isolated for the first time from Artemisia frigida: 5,7,4′-trihydroxy-6, 3′,5′-trimethoxyflavone, 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone and 5,7,3′,4′-tetrahydroxy-6,5′- dimethoxyflavone.     

Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases.

DNA molecules isolated from bacteriophage P4 are mostly linear with cohesive ends capable of forming circular and concatemeric structures. In contrast, almost all DNA molecules isolated form P4 tailless capsids (heads) are monomeric DNA circles with their cohesive ends hydrogen-bonded. Different form simple DNA circles, such P4 head DNA circles contain topological knots. Gel electrophoretic and electronmicroscopic analyses of P4 head DNA indicate that the topological knots are highly complex and heterogeneous. Resolution of such complex knots has been studied with various DNA topoisomerases. The conversion of highly knotted P4 DNA to its simple circular form is demonstrated by type II DNA topoisomerases which catalyze the topological passing of two crossing double-stranded DNA segments [Liu, L. F., Liu, C. C. & Alberts, B. M. (1980) Cell, 19, 697-707]. The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNA topoisomerases.     

Iatrogenic hyponatraemia of the newborn due to maternal fluid overload: a prospective study.

Over five weeks 136 out of 246 deliveries were studied. Maternal plasma sodium concentrations were normal at admission. At delivery no significant difference was found between maternal and infant cord plasma sodium concentrations. Twenty-four of the 41 mothers who had received only oral fluids during labour had infants whose cord plasma sodium concentrations were normal. Of the 95 mothers who had been given intravenous fluids, however, only 14 infants with normal plasma sodium concentrations, 31 had a concentrations of 130 mmol (mEq)/1 or less and nine of these had a concentration of 125 mmol/1 or less. There was a highly significant inverse relation between cord plasma sodium concentration and rate of fluid administration, suggesting that hyponatraemia was due to intravenous treatment with predominantly sodium-free solutions. Endogenous antidiuretic activity probably increases during labour, and synthetic oxytocin in large doses has been shown to have an antidiuretic effect. The dose used in this study did not appear to have such an effect. Glucose solutions are often used as a vehicle for oxytocin; 83% of all fluid intake in this study was 5% or 10% glucose in water. Fluid balance in labour should be supervised closely, and oxytocin should be given in a more concentrated solution.     

Ly-m19: The Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with 70'/3 cells, an established pre-B cell line, were fused with cells of the nonsecretor myeloma line NS.1. One established hybridoma cell line (clone K10.6) continuously secreted antibody that recognized a new antigenic specificity tentatively named Ly-m19. This newly found antigen is detectable on both T and B cells. Cytotoxicity assays reveal that 75 percent of the spleen and lymph-node cells, 35 percent of bone-marrow cells, and 15 percent of thymus cells reacted with antibody of clone K10.6. Strains expressing the specificity Ly-m19.1 are characterized by negative reactions and include the strains AKR, CE/J, RF/J, GR/A, SJL, P/J, BDP/J, and LG/J. All other strains so far tested are Ly-m19.2. This strain distribution pattern distinguishes Ly-m19 from any known murine lymphocyte alloantigen, but it parallels the Lyb-2 ^c haplotype. Linkage test of a set of AKXL recombinant inbred strains revealed close linkage of Ly-m19 and Lyb-2 loci on mouse chromosome 4.Abbreviations used in this paper LPS lipopolysaccharide - B6 C57BL/6 - Con-A concanavalin A - MLC mixed-lymphocyte culture The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

The effect of prostaglandins A2,E1,E2,15 methyl E2, 16, 16 dimethyl E2 and F2α on erythropoiesis

Prostaglandins A₂, E₁, E₂, methylated E₂s and F₂α affected erythropoiesis and/or erythropoietin (Ep) production. This action is indicated in the exhypoxic, polycythemic mouse where radioiron incorporations into RBC increased after administration of these compounds. The kidney and liver have been indicated through previous studies, to actively participate in Ep production. By the removal of one of these active sites in a murine system treated with prostaglandins it is shown that a response is reflected in Ep levels. Interference of the action of prostaglandins (PG) is altered by the removal of one of these target sites of Ep production. The erythropoietic responses elicited by PGA₂, E₁, and perhaps the methylated PGE₂s act through the liver whereas PGE₂ may operate through a renal pathway for its response. PGF_2α reveals no effect on erythropoietic activity and is no different than that observed for vehicle-treated controls. The prostaglandins tested appear to act primarily through the kidney or liver but the possibility exists that some yet undetermined organ site may also be involved.     

UPRE-lac Z为报告基因评价酵母UPR响应初步研究 *

目的: 未折叠蛋白质反应UPR是酵母最重要蛋白质质量控制机制之一,研究UPR响应规律有助于优化异源蛋白分泌途径合成和应对酸醇等胁迫因子的细胞自我保护。方法: 选择实验室菌株W303-1A和工业菌株An-a,以UPRE启动子控制下的Lac Z为报告基因,利用CRISPR/Cas9技术构建得到指示菌株W303-1A (leu 2::UPRE-lac Z)和An-a (leu 2::UPRE- lac Z),分别简称WZ和AZ。结果: 生长曲线测定显示WZ和AZ与亲本菌株的生长接近;添加下述试剂孵育4h后测定β-半乳糖苷酶酶活:1μg/ml衣霉素、8%(v/v)乙醇、0.3%(v/v)乙酸、5%(v/v)乙醇+0.1%(v/v)乙酸;菌株AZ的比酶活分别是对照值的8.2、26.4、1.1和7.9倍,而菌株WZ则分别为12.6、2.4、1.0和1.0倍;进一步以YEplac195为载体表达β-葡萄糖苷酶,AZ和WZ转化子在2%纤维二糖中生长24h的β-葡萄糖苷酶酶活值分别为0.35和6.12U/ml,相应的LacZ则分别为对照值的3.1和5.4倍。结论: 两个菌株显示了在抑制物和异源蛋白表达UPR响应和调控能力上的显著差异,为其改造利用提供了方向;研究也为分析抑制物耐受性和异源蛋白表达关键制约因素、优化酵母ER和UPR信号通路的调控奠定了初步方法基础。     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.