Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.     

Does alligator testis produce estradiol? A comparison of ovarian and testicular aromatase

Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 microl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 +/- 1.6 pmol mg-1 h-1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg-1 h-1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.     

Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal

Summary. Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem. Received July 23, 2001 Accepted October 11, 2001     

Organization,expression and evolution of a disease resistance gene cluster in soybean

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.     

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.     

Effect of exercise at three exercise intensities on salivary cortisol

Changes in cortisol concentration in response to exercise at 3 different intensities were quantified. Ten apparently healthy, recreationally active males participated. On 4 separate occasions, subjects were assigned a random order of 1-hour cycle ergometer bouts of exercise at 44.5 +/- 5.5%, 62.3 +/- 3.8%, and 76.0 +/- 6.0% (mean +/- SD) of VO2peak and a resting control session. Saliva samples were collected before exercise at 10, 20, 40, and 59 minutes of exercise and at 20 minutes of recovery. Differences in cortisol concentration were assessed via multivariate analysis of variance (alpha = 0.05) Tukey post hoc analysis when indicated. During the highest-intensity exercise session, cortisol was significantly higher at 59 minutes of exercise (p = 0.004) and at 20 minutes of recovery (p = 0.016) than at those same time points during the resting control session. No significant differences in cortisol concentration were noted among resting, low-, and moderate-intensity exercise. Exercise <40 minutes in duration elicited no significant differences at any intensity. These data indicate that only exercise of high intensity and long duration results in significant elevations of salivary cortisol.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.