Differential expression of two linked selection genes (HSVI-tk and Eco.gpt) in transformed teratocarcinoma and in L cells

Upon transfection of (TK-)F9 teratocarcinoma stem-cells and (TK-)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI-tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV-gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk-regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low.     

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

New syntheses of deuterated protoporphyrin-IX derivatives for heme protein nmr studies

An efficient total synthesis of 1,5-di(trideuteromethyl)protoporphyrin-IX (3) dimethyl ester from monopyrrole precursors is described, the synthesis proceeding through crystalline tripyrrene and a,c-biladiene salt intermediates. The 2- and 4-vinyl groups in (3) are formed from the corresponding (2-chloroethyl) substituents by way of base-promoted dehydrochlorination. In protio solvents, this synthetic step is shown to exchange out preferentially deuterons in the 1-methyl group, and this observation is exploited in an efficient synthesis of the 1,3-di(trideuteromethyl)protoporphyrin-IX (22) dimethyl ester from 2,4-diacetyldeuteroporphyrin-IX (20) dimethyl ester (which is in turn accessible from commercially available protoporphyrin-IX (5)). Thus, basic exchange in deuterated solvent of (20) gives the deuterated analog, which after reduction and dehydration gives the 1,3-di(trideuteromethyl)protoporphyrin-IX analog (22), in which the vinyl H₂ and propionic CH₂·CO functions have also become deuterated.     

Differential staining of chromatids reveals substitution of BUdR for thymine in “old” DNA strands

Metaphases collected from cultures grown for three cell cycles in 5-bromodeoxyuridine (BUdR) and then for one or two further cell cycles without BUdR show persistence of differentially FPG-stained chromatids. The cell cycle length is not altered by the presence of BUdR. After removal of BUdR, the cells synthesize DNA and incorporate mainly thymine, as demonstrated by density gradient analysis of DNA. Our observations suggest that chromatids with T-B DNA stain lightly after removal of BUdR, in contrast with their dark staining when cultures are maintained in BUdR. Thus, in any experimental condition, there is a correspondence between the nature (T-DNA or B-DNA) of the “old” DNA strands and the FPG-staining (dark or light) of the chromatids.     

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Abstract

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.     

Effects of Timber Harvests and Silvicultural Edges on Terrestrial Salamanders

Balancing timber production and conservation in forest management requires an understanding of how timber harvests affect wildlife species. Terrestrial salamanders are useful indicators of mature forest ecosystem health due to their importance to ecosystem processes and sensitivity to environmental change. However, the effects of timber harvests on salamanders, though often researched, are still not well understood. To further this understanding, we used artificial cover objects to monitor the relative abundance of terrestrial salamanders for two seasons (fall and spring) pre-harvest and five seasons post-harvest in six forest management treatments, and for three seasons post-harvest across the edge gradients of six recent clearcuts. In total, we recorded 19,048 encounters representing nine species of salamanders. We observed declines in mean encounters of eastern red-backed salamanders (Plethodon cinereus) and northern slimy salamanders (P. glutinosus) from pre- to post-harvest in group selection cuts and in clearcuts. However, we found no evidence of salamander declines at shelterwoods and forested sites adjacent to harvests. Edge effects induced by recent clearcuts influenced salamanders for approximately 20 m into the forest, but edge influence varied by slope orientation. Temperature, soil moisture, and canopy cover were all correlated with salamander counts. Our results suggest silvicultural techniques that remove the forest canopy negatively affect salamander relative abundance on the local scale during the years immediately following harvest, and that the depth of edge influence of clearcuts on terrestrial salamanders is relatively shallow (<20 m). Small harvests (<4 ha) and techniques that leave the forest canopy intact may be compatible with maintaining terrestrial salamander populations across a forested landscape. Our results demonstrate the importance of examining species-specific responses and monitoring salamanders across multiple seasons and years. Long-term monitoring will be necessary to understand the full impacts of forest management on terrestrial salamanders.     

An electrochemical biosensor for 3-hydroxybutyrate detection based on screen-printed electrode modified by coenzyme functionalized carbon nanotubes

3-Hydroxybutyrate, one of the main blood ketone bodies, has been considered as a critical indicator for diagnosis of diabetic ketoacidosis. Biosensors designed for detection of 3-hydroxybutyrate with advantages of precision, easiness and speedy performance have attracted increasing attention. This study attempted to develop a 3-hydroxybutyrate dehydrogenase-based biosensor in which single-walled carbon nanotubes (SWCNT) was used in order to immobilize the cofactor, NAD⁺, on the surface of screen-printed electrode. The formation of NAD⁺–SWCNT conjugates was assessed by electrochemistry and electron microscopy. Cyclic voltammetry was used to analyze the performance of this biosensor electrochemically. The considerable shelf life and reliability of the proposed biosensor to analyze real sample was confirmed by this method. The reduction in the over potential of electrochemical oxidation of NADH to ?0.15 V can be mentioned as a prominent feature of this biosensor. This biosensor can detect 3-hydroxybutyrate in the linear range of 0.01–0.1 mM with the low detection limit of 0.009 mM. Simultaneous application of screen-printed electrode and SWCNT has made the biosensor distinguished which can open new prospects for detection of other clinically significant metabolites.