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.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Protection by salvianolic acid A against adriamycin toxicity on rat heart mitochondria.

It was found that salvianolic acid A (Sai A) has potent antioxidant activity. The effects of Sai A on adriamycin-induced heart mitochondrial toxicity of rats in vitro and on adriamycin antitumor activity are investigated in this article. Malondialdehyde (MDA) formation and membrane rigidification of rat heart mitochondria intoxicated with adriamycin were significantly reduced by Sai A. In the electron spin resonance (ESR) studies, Sai A has no significant effect on the formation of adriamycin semiquinone radicals (AQ.), while hydroxyl radicals generated by electron transfer from AQ. to H2O2 were scavenged by Sai A dose-dependently. On the other hand, Sai A was shown to have no effects on the antitumor activity of adriamycin in cultured L1210 ascitic tumor cells and in mice with P388 ascite tumor. These results indicate that Sai A protects against adriamycin induced heart mitochondrial toxicity of rats, while Sai A has no antagonizing effect on the antitumor activity of adriamycin.     

Diploid and triploid offspring of triploid agamosporous fernDryopteris pacifica

A cytological and reproductive study of the diploid and triploid agamosporousDryopteris pacifica was made to elucidate the origin of its infraspecific cytotypes. Some triploids produced 16 spore mother cells (SMCs) sometimes with n=41II+41I chromosomes, in addition to eight SMCs with n=123II, in each sporangium. In the former case the 16 SMCs usually underwent abnormal meiosis to give rise to some 50 spores, some of which were regular-shaped; in the latter the eight SMCs multiplied into 32 spores by normal meiosis. We found that spores from one of the triploid plants developed into either diploid or triploid gametophytes, which further apogamously produced diploid or triploid sporophytes, respectively. This novel mechanism of ploidy reduction is discussed in relation to the origin of diploid agamosporous ferns, the taxonomic complexity of the species, and the correlation of agamospory with polyploidy. The mechanism is also compared to that operating in agamospermous angiosperms.     

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.