The genotypic response of mouse embryos to multiple freezing variables

Four- and eight-cell embryos from 3 mouse genotypes were cryopreserved to study the relationship of genetics and freezing variables on post-thaw survival. Embryos from outbred N:NIH(S), N:NIH(S)-B and inbred (C57BL/6N) mice were exposed to 1 of 2 cryoprotectants (glycerol [GLYC] versus dimethyl sulfoxide [DMSO]) and stored in 1 of 2 containers (ampules [AMP] versus straws [STR]). Containerized embryos were cooled at a rate of 0.5 degrees C/min to a minimum of -35 degrees C, transferred into liquid nitrogen, and later thawed and cultured in vitro. Genotypic differences (p less than 0.05) were noted for 4 interrelated embryo characteristics including post-thaw survival (PTS), embryo degeneration rate (EDR), and quality grade (QG) and viability grade (VG) ratings. The PTS for outbred embryos was greater (p less than 0.05) in GLYC than DMSO, whereas inbred C57BL/6N embryos survived similarly (p greater than 0.05) in either cryoprotectant. Compared to DMSO counterparts, embryos from GLYC-treated outbred groups had improved QG and VG ratings and reduced EDR (p less than 0.05), but comparable results were observed between GLYC- AND DMSO-treated embryos in the C57BL/6N group. Between genotypes, type of container affected PTS and EDR (p less than 0.05) but not QG or VG ratings (p greater than 0.05). Within genotypes, PTS generally ranged 15 to 20% higher (p less than 0.01) in AMP than STR groups. Increased PTS was noted in outbred mouse x GLYC x AMP groups; however, based on the degrees of difference, the inbred C57BL/6N strain appeared less affected by this 3-way interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis

Summary The influence of different primary aliphatic alcohols on the activities of two key enzymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of ¹⁴C- and ³H-labelled substrates the enzymes 3-hydroxy-3-methylglutaryl-CoA-reductase and squalene-hopenecyclase were detected with activities of 1.6 pmol x (min x mg protein)^-1 and 2.3 pmol x- (min x mg protein)^-1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Furthermore, 3-hydroxy-3-methylglutaryl-CoA-reductase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alcohol at a concentration of 6% (v/v). This corresponded with a similar increase of hopanoid content of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent hopanoid biosynthesis of Z. mobilis.Abbreviation HMG-CoA-reductase 3-hydroxy-3-methylglutaryl-coenzyme A-reductase     

Physiologic characterization of transformed and cloned rat granulosa cells

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.     

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Immunological detection of phytoene desaturase in algae and higher plants using an antiserum raised against a bacterial fusion-gene construct

Immunological characterization of phytoene desaturase, a key enzyme of carotenoid biosynthesis, is reported. For this purpose, a phytoene-desaturase fusion protein has been employed. For its construction 921 base pairs of the crtI gene were fused to the N-terminal region of the Escherichia coli lacZ gene. Plasmid pGABX2 resulted from insertion of a BglI - XhoI fragment from the Rhodobacter capsulatus carotenoid biosynthesis gene cluster, carrying the crtI, crtA and crtB genes, into pBR322. A 968-base-pair SalI-fragment from pGABX2 was cloned into pUR288 at the 3' end of the lacZ gene. Isopropyl-beta-D-thio-galactopyranoside-dependent activation of the lacZ fusion gene resulted in expression of a stable 150-kDa protein. After electroelution from SDS/polyacrylamide slab gels, the protein was used for antibody production. The heterogenic antiserum obtained was tested by Western blotting against proteins from Rhodobacter capsulatus and several different photoautotrophic organisms including higher plants. Apparent molecular masses of immunoreactive proteins from Rhodobacter, Aphanocapsa, rape and spinach were around 64 kDa. In Bumilleriopsis a 55-kDa protein was found instead. The antibody also inhibited in vitro desaturation of phytoene when detergent-solubilized membranes were employed.