Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration.

The thiomethylation of Bacillus subtilis tyrosine transfer ribonucleic acid (tRNATyr) (i6A) has been shown to occur during the slowing-down of growth. The extent of this modification in stationary-phase cells grown in defined medium has been determined in parallel with the sporulation frequency. We observed that the presence of phosphate repressed sporulation and also inhibited the thiomethylation of tRNATyr (i6A) of B. subtilis W168. These effects were partially eliminated by decreasing the glucose concentration until it was growth limiting. In the case of strain W23S, in which sporulation is insensitive to glucose repression, sporulation and tRNATyr thiomethylation were not inhibited by nonlimiting concentrations of phosphate. These results suggest that both sporulation and tRNATyr hyper-modification share some common regulatory process.     

Cervical transfer of deep frozen cattle embryos

Cattle blastocysts were collected from 29 donors 7-8 days after estrus and frozen and stored in liquid nitrogen up to several months. Two procedures were used for freezing and thawing: After thawing, the embryos were cultured from 8 to 12 hours before transfer; 36% of the embryos continued normal development during culture; both procedures resulted in a high pregnancy rate (procedure A: 10 15 ; procedure B: 11 15 ) after single cervical transfer of the frozen thawed embryos which developed normaly in vitro . However the overall survival rate was low (25%) and varied between donors, indicating that progress must be made before the technique of freezing can be extended to applied conditions.     

Placental expression of major histocompatibility complex class I in bovine somatic clones

Abnormally increased placental expression of major histocompatibility complex class I (MHC-I) molecules at the trophoblastic surface has been suggested previously to be the cause of early fetal loss in nuclear transfer (NT) bovine pregnancies. Here, we report the lack of expression of MHC-I at the trophoblastic surface at D30 and D60 and in placentomes from D60 to term in placentas obtained by NT from three different genotypes and by artificial insemination, whatever the outcome of the pregnancy. MHC-I expression was assessed by immunohistochemistry using four different antibodies, including a novel beta2-microglobulin antibody. The MHC-I type of the clones was established using reference strand-mediated conformation analysis (RSCA); however, since it proved problematic to type the recipient animals in the same way, outcome of pregnancy could not be related to MHC compatibility. In conclusion, the present study provides no evidence to support abnormal expression of MHC-I on the trophoblastic surface in clones as a major cause of fetal loss during pregnancy after NT.     

Pyrazole diaminopyrimidines as dual inhibitors of KDR and Aurora B kinases

In an effort to identify kinase inhibitors with dual KDR/Aurora B activity and improved aqueous solubility compared to the Abbott dual inhibitor ABT-348, a series of novel pyrazole pyrimidines structurally related to kinase inhibitor AS703569 were prepared. SAR work provided analogs with significant cellular activity, measureable aqueous solubility and moderate antitumor activity in a mouse tumor model after weekly ip dosing. Unfortunately these compounds were pan-kinase inhibitors that suffered from narrow therapeutic indices which prohibited their use as antitumor agents.     

Comparison of cloned and non-cloned Holstein heifers in muscle contractile and metabolic characteristics

Muscle contractile and metabolic characteristics were studied on nine cloned and eight non-cloned (control) heifers. The animals were submitted to repeated biopsies of the semitendinosus (ST) muscle at the ages of 8, 12, 18 and 24 months. The contractile type was determined from the proportion of the different myosin heavy chain (MyHC) isoforms separated by electrophoresis. Glycolytic metabolism was assessed by lactate dehydrogenase (LDH) activity, and oxidative metabolism was assessed by isocitrate dehydrogenase (ICDH), cytochrome-c oxidase (COX) and β-hydroxyacyl-CoA dehydrogenase (HAD) activities. In cloned heifers at 8 months of age, there was a greater proportion of MyHC I (slow oxidative isoform) and MyHC IIa (fast oxido-glycolytic isoform), a lower proportion of MyHC IIx (fast glycolytic isoform), greater COX and HAD activity and a lower LDH/ICDH ratio compared with control heifers. Thus, young cloned heifers had slower muscle types associated with a more oxidative muscular metabolism than control heifers. From 12 months of age onwards, no significant differences were observed between cloned and control heifers. A delay in muscle differentiation and maturation in cloned heifers is hypothesised and discussed.     

Acute norepinephrine reuptake inhibition decreases performance in normal and high ambient temperature.

Combined inhibition of dopamine (DA)/norepinephrine (NE) reuptake improves exercise performance and increases core temperature in the heat. A recent study demonstrated that this effect may primarily be related to increased DA activity. NE reuptake inhibition (NERI), however, has received little attention in humans, certainly in the heat, where central fatigue appears to be a main factor influencing performance. Therefore the present study examines the effect of NERI (reboxetine) on exercise capacity, thermoregulation, and hormonal response in normal and high temperature. Nine healthy well-trained male cyclists participated in this study. Subjects ingested either placebo (Pla; 2 x 8 mg) or reboxetine (Rebox; 2 x 8 mg). Subjects exercised in temperate (18 degrees C) or warm (30 degrees C) conditions and cycled for 60 min at 55% W(max) immediately followed by a time trial (TT; Pla18/Rebox18; Pla30/Rebox30) to measure exercise performance. Acute NERI decreased power output and consequently exercise performance in temperate (P = 0.018) and warm (P = 0.007) conditions. Resting heart rate was significantly elevated by NERI (18 degrees C: P = 0.02; 30 degrees C: P = 0.018). In Rebox18, heart rate was significantly higher than in the Pla18, while in the heat no effect of the drug treatment was reported during exercise. In Rebox30, all hormone concentrations increased during exercise, except for growth hormone (GH), which was significantly lower during exercise. In Rebox18, prolactin (PRL) concentrations were significantly elevated; GH was significantly higher at rest, but significantly lower during exercise. In conclusion, manipulation of the NE system decreases performance and modifies hormone concentrations, thereby indicating a central NE effect of the drug. These findings confirm results from previous studies that predominantly increased DA activity is important in improving performance.     

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.     

Preliminary results on variability in oocyte recovery and developmental competence in cattle derived from embryonic cloning: work in progress

To investigate female gamete developmental competence and variability in cloned cattle, we performed ovum pick-up and in vitro fertilization in four sets of cloned heifers (n = 10, two sets of triplets and two sets of twins), and four groups of non-genetically related control animals (n = 13). A total of 304 OPU were performed and 1798 oocytes were recovered. Mean oocyte production per female per OPU (+/-S.D.) was similar for clone or control animals (5.7+/-2.9 versus 6.1+/-4.5, respectively), however, in two sets of clones variance for the number of oocytes recovered differed significantly (7.1 versus 23.9 and 7.3 versus 26.7, respectively P<0.001) between clone groups and their respective controls, cloned animals being more homogenous. After in vitro maturation, fertilization with semen from the same bull, and culture, the proportion of oocytes from cloned animals that developed into blastocysts was 35.0+/-29.2% and was not significantly different from controls (29.4+/-30.9). The CV for oocyte recovery, and blastocyst rates was lower in all groups of cloned animals than in controls. Nevertheless, within each set of clones, CV values indicated some degree of variability between animals, thus confirming that cloned cattle are not the exact phenotypic copy of each other. Despite the large number of oocytes analyzed, results should be interpreted with caution due to the limited number of cloned animals.