5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.     

Computerized video time-lapse (CVTL) analysis of cell death kinetics in human bladder carcinoma cells (EJ30) X-irradiated in different phases of the cell cycle

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.     

Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.

We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.     

Factors influencing the incidence of neonatal jaundice.

A retrospective study of 12 461 single births confirmed an association between maternal oxytocin infusion and neonatal jaundice. The effect of oxytocin on jaundice was independent of gestational age at birth, sex, race, epidural anaesthesia, method of delivery, and birth weight, each of which was significantly associated with neonatal jaundice. The effect of oxytocin was, however, small, producing a calculated mean increase in peak plasma bilirubin concentration of 8.6 mumol/1 (0.5 mg/100 ml); this excess was independent of sex and less than the effect of the baby being born one week earlier.     

The genetics of dopa decarboxylase in Drosophila melanogaster

Summary Data on 46 mutations in the structural gene, Ddc. for dopa decarboxylase and 33 mutations in the methyl dopa hypersensitive gene, 1(2)amd, in Drosophila melanogaster are presented including information on their isolation, their effects on DDC activity, and their sensitivity to dietary methyl dopa. Intragenic complementation of both loci is documented, the effects of heteroallelic complementing heterozygosity on DDC activity, in vitro thermolability of DDC, and on temperature sensitive viability are presented. Data are marshalled to support rejection of the hypothesis that Ddc mutations and 1(2)amd, mutations are lesions in a single gene.     

Cloning of the Glutamyl-tRNA Synthetase (gltX) Gene from Pseudomonas aeruginosa

The glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive gltX mutation of Escherichia coli JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from Rhizobium meliloti.