Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time. © 1996 Wiley-Liss, Inc.     

Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells

In this report, we explore the mechanisms of targeting of p300 to the interleukin-2 (IL-2) promoter in response to mitogenic and oncogenic molecular signals. Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element (CD28RE)-TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation, and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax. The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter, and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE, the IL-2 promoter, and endogenous IL-2 gene expression. Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter. These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells.     

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.     

An oxysterol-derived positive signal for 3-hydroxy- 3-methylglutaryl-CoA reductase degradation in yeast

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.     

Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogen-activated protein kinase activation in the mouse mammary gland

MUC1 is a large (>400 kDa), heavily glycosylated transmembrane protein that is aberrantly expressed on greater than 90% of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast cancer line as well as mouse mammary glands with epidermal growth factor results in the co-immunoprecipitation of MUC1 with a tyrosine-phosphorylated protein of approximately 180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail deleted (DeltaCT), or tandem repeat deleted (DeltaTR)-human MUC1 under the control of the mouse mammary tumor virus promoter to further examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that full-length transgenic MUC1 physically associates with all four erbB receptors, and co-localizes with erbB1 in the lactating gland. Furthermore, we detected a sharp increase in ERK1/2 activation in MUC1 transgenic mammary glands compared with Muc1 null and wild-type animals. These results point to a novel function of increased MUC1 expression, potentiation of erbB signaling through the activation of mitogenic MAP kinase pathways.     

Partial cytochrome b sequences for six Hymenoptera of the eastern United States

Mitochondrial DNA (mtDNA) haplotypes have been commonly used to determine honeybee subspecies relationships. To see if these markers would also be useful for comparisons of other Hymenoptera, we collected workers of six local species: Vespa crabro, the European hornet; Bombus impatiens, a bumblebee; Vespula germanica, the German yellow jacket; Polistes fuscatus, a paper wasp; Halictus ligatus, an alkali bee; and an unspecified Megachile, a leafcutting bee. MtDNA was isolated and digested with six endonucleases (AvaI, BglII, EcoRI, HindIII, HinfI, XbaI). The digested DNA was electrophoresed and visualized on agarose gels with comparison to a standard fragment marker and similarly treated honeybee mtDNA. The fragments obtained were also purified and sequenced. Phylogenetic relationships between six wasp and bee species, Apis mellifera, and several other similar aculeate Hymenoptera were determined. Newly defined DNA sequences were posted to GenBank (AF281169-AF281174).     

Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.     

Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1

We examined the effects of epidermal growth factor (EGF) on MDA-MB-468 cells to understand its mechanism of action in an EGF receptor-rich breast cancer cell line. EGF inhibited the growth of MDA-MB-468 cells with an IC50 of 1.5 +/- 0.5 nM, as determined by measurements of DNA content of cells in culture over a period of 4 to 6 days. This growth inhibition included apoptosis 24 h after EGF addition, as detected by an enzyme-linked immunosorbent assay (ELISA) and Hoechst 33342 staining. In EGF-treated cells, peak activities of two key enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), were reduced by 57% and 83%, respectively. EGF treatment also caused a 30 to 50% decrease in cellular putrescine at all time points tested (12 to 48 h). EGF-induced inhibition of DNA synthesis was also partially reversed by the addition of putrescine or spermidine, but not by spermine. Western blot analysis of cell cycle regulatory proteins showed that EGF-mediated growth inhibition was associated with the induction of p21, an inhibitor of cyclin-dependent kinases. However, EGF had no significant effect on the expression of cyclin D1 or cyclin E. Furthermore, putrescine reversal of EGF effects was associated with the down-regulation of EGF-induced p21. These results suggest that the mechanism of growth inhibition by EGF in MDA-MB-468 cells include a down-regulation of polyamine biosynthesis and the induction of p21. Identification of growth regulatory pathways in breast cancer cells might be useful in the development of novel targets for therapeutic intervention.     

Retinoids and cell growth in the cardiovascular system

Deficiency of vitamin A and its retinoid metabolites has been associated with a number of developmental abnormalities in the cardiovascular system. Many of these effects are mimicked by targeted deletion of retinoic acid receptors in the embryo. Retinoids also display anti-growth activity in fully differentiated cardiac and vascular cells. These activities together with favorable effects on the clotting mechanism suggest that retinoids may prove useful in the management of hypertrophic/hyperproliferative disorders of the heart and vascular wall.