A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.     

Catabolism of 5-fluoro-2'-deoxyuridine by isolated rat intestinal epithelial cells

The kinetics of conversion of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) by isolated rat intestinal epithelial cells was investigated. Also, the effects of potential inhibitors of this reaction, which is catalyzed by uridine phosphorylase and thymidine phosphorylase, were determined. A 2.5% suspension of isolated cells was incubated with FdUrd or FUra, and at specific times cells were lysed with perchloric acid and fluoropyrimidines were determined by high-performance liquid chromatography. During a 25-min incubation with either FdUrd or FUra, the amount of drug in the incubation system (total volume 0.8 ml) fell by less than 5%. However, in the presence of FdUrd, the amount of FUra increased linearly over 25 min. The apparent Vmax and Km for FUra formation were 17-27 nmole/mg DNA/min and 1.6-2.5 mM, respectively. With each nucleoside phosphorylase inhibitor, the apparent Km increased but Vmax was unaffected. The apparent Ki values were as follows (in mM): 5-nitrouracil (an inhibitor of both uridine phosphorylase and thymidine phosphorylase), 0.12; 4-thiothymine (a uridine phosphorylase-selective inhibitor), 1.52; and 6-benzyl-2-thiouracil (a thymidine phosphorylase-selective inhibitor), 0.73. It was concluded that intestinal epithelial cells are capable of degrading FdUrd to FUra and that the cells possess both uridine phosphorylase and thymidine phosphorylase activity.     

中国黑粉补遗

裂孢黑粉菌属(Schizonella)是我国新记录属,其主要特征为孢子堆生叶上;黑粉成对,其萌发方式同黑粉菌属(Ustilago)苔草裂孢黑粉菌(Schizonella melanogramma(DC.) Schrot.)寄生于苔草属(Carex sp.)植物上,其孢子堆生叶上,条状,粉质;黑粉孢子卵形,近球形或不规则半球形,常成对,7.5 -12.5 (-14)×5.5-(-12)µm,橄榄褐色.另一我国新记录种是鬼灯檠条黑粉菌(Urocystis rodgersiae (Miyabe) Miyabe ex Zund.),寄生在鬼灯檠(Rodgersia aesculifolia Batal.)植物上.孢子堆生叶下;孢子球27-60(-75)×25-45.5(-57µm,由1-6个黑粉孢子组成;不育细胞10-15×9-l0µm,浅褐色.黑粉孢子14-20×10-14µm,暗褐色.此种孢子球比原产于日本的模式种略大,其它特征相同.     

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of intermediate filaments and other special markers by testicular germ cell tumors. With reference to embryogenesis.

Distribution of intermediate filament proteins (IFs) and several special markers was studied in 39 testicular germ cell tumors and 8 embryos and foetuses. The similarity and difference between development of germ cell tumor and embryogenesis were immunohistochemically investigated. Seminoma and embryonal carcinoma, as tumoral counterparts of undifferentiated germ cells, were characterized by little IF expression. This study revealed that the maturing and differentiating process in germ cell tumor is different from normal embryonal development and the tumor cells showed leaping maturing steps in tumorigenesis. Immunostaining for IFs helped to discover the further differentiation occurring in embryonal carcinoma and to demonstrate heterogeneous elements in non-seminoma germ cell tumors, which sometimes might not be apparent by light microscopical observation of H&E staining section. According to the findings, two patterns in mixed germ cell tumors are suggested; i.e., combined and diffuse types. The mechanism of tumorigenesis of the two types is supposed to be different. Clinically, the prognosis of most patients with testicular germ cell tumor is fairly good because of the improved chemotherapies that are dependent on histological diagnosis.     

The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase.

Unlike autocatalyzed self-splicing reactions, nuclear pre-mRNA splicing requires transacting macromolecules and ATP. A protein encoded by the PRP2 gene of Saccharomyces cerevisiae is required, in conjunction with ATP, for the first cleavage-ligation reaction of pre-mRNA splicing. In this study, we have purified two forms of the PRP2 gene product with apparent molecular weights of 100 kDa and 92 kDa, from a yeast strain overproducing the protein. Both proteins were indistinguishable in their ability to complement extracts derived from a heat-sensitive prp2 mutant. Furthermore, we show that the PRP2 protein is capable of hydrolyzing nucleoside triphosphates in the presence of single-stranded RNAs such as poly(U). However, purified PRP2 by itself did not unwind double-stranded RNA substrates. The fact that an RNA-dependent NTPase activity is intrinsic to PRP2 may account for the ATP requirement in the first catalytic reaction of pre-mRNA splicing.     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

A threshold result for an epidemiological model

A threshold parameter R ₀ is identified for an SIRS epidemiological model which has nonlinear incidence and a distributed delay for transfer out of the removed class. For R ₀ < 1, the disease free equilibrium is proved to be the global attractor for all solutions.Research supplied in part by NSERC A-8965     

Synthesis of oligodeoxyribonucleotides containing degenerate bases and their use as primers in the polymerase chain reaction.

Heptadecaoligodeoxyribonucleotides containing one or more of the bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and hypoxanthine (I) and combinations of P with K and I have been synthesised on a DNA synthesiser. The stability of duplexes containing these basemodified oligomers with P/A, P/G, K/C and K/T; P/A, P/G, I/C, I/T and I/A, I/G, I/C, I/T base pairs were compared by measuring their melting transition (Tm) values. Oligomers containing both P and K and P and I were more stable than those with I alone or with mismatches. These oligomers together with one with a P base at the 3'-end were used as primers in polymerase chain reaction (PCR) experiments. They were all effective primers except one with I alone and a triple mismatch. Thus the use of the degenerate bases P and K in primer design is established.