The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes.

The 70-kDa heat shock proteins are molecular chaperones that participate in a variety of cellular functions. This chaperone function is stimulated by interaction with hsp40 proteins. The Saccharomyces cerevisiae gene encoding the essential hsp40 homologue, SIS1, appears to function in translation initiation. Mutations in ribosomal protein L39 (rpl39) complement loss-of-function mutations in SIS1 as well as PAB1 (poly(A)-binding protein), suggesting a functional interaction between these proteins. However, while a direct interaction between Sis1 and Pab1 is not detectable, both of these proteins physically interact with the essential Ssa (and not Ssb) family of hsp70 proteins. This interaction is mediated by the variable C-terminal domain of Ssa. Subcellular fractionations demonstrate that the binding of Ssa to ribosomes is dependent upon its C terminus and that its interaction with Sis1 and Pab1 occurs preferentially on translating ribosomes. Consistent with a function in translation, depletion of Ssa protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function. This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.

The cyclin box and C-terminus of cyclins A and E specify CDK activation and substrate specificity.

The cyclins and their catalytic partners, the Cyclin Dependent Kinases (CDKs), are essential for progression through the cell cycle. Cyclin/kinase complexes containing cyclins A or E are active primarily in late G1 to S phase and both have been shown to phosphorylate histone H1 and the retinoblastoma gene product (pRb) in vitro. Despite these similarities, cyclins A and E display differences in CDK activation and substrate specificity. We find that in vitro, cyclin E/CDK2 and cyclin A/CDK2 phosphorylate histone H1 similarly but only cyclin A/CDK2 phosphorylates lamin B. While both cyclin A and cyclin E bind CDK1 efficiently, only cyclin A activates CDK1 kinase activity. Using chimeric proteins between cyclins A and E we find that both the cyclin box and C-terminus of cyclins A and E are required for CDK binding, activation and targeting of substrate specificity.

G1 cyclins control the retinoblastoma gene product growth regulation activity via upstream mechanisms.

Inactivation of the retinoblastoma gene product (pRb) occurs concomitant with the appearance of its hyperphosphorylated form in mid to late G1. Multiple cyclin/CDK complexes are implicated in the cell cycle phosphorylation of pRb. Using in vivo expression systems, we show that cyclins A, E, D1, D2, and D3 each function to phosphorylate and inactivate pRb. In vivo, G1 cyclin/kinase complexes enhance the phosphorylation of pRb, and these effects of cyclin/kinases on pRb can be overcome by the addition of p21, a wide spectrum inhibitor of G1 kinases. Kinases associated with cyclins A, E, and D1 phosporylate pRb indistinguishably in vivo, according to proteolytic maps. Although cyclin D1 has been reported to bind to pRb directly, requiring the pRb-binding motif LXCXE, a mutant D1 lacking the pRb-binding motif remains able to phosphorylate pRb in vivo and in vitro and is also able to reverse the growth-inhibitory properties of pRb in intact cells. Finally, coexpression of G1 cyclins and kinases represses pRb-mediated growth inhibition in Saos-2 cells. The multiplicity of mechanisms for pRb phosphorylation and inactivation suggests that several pathways exist for the regulation of pRb by phosphorylation.

Time-course and steroid specificity of aromatase induction in rat hypothalamus-preoptic area.

We studied the time-course and steroid specificity for aromatase induction in the hypothalamus-preoptic area (HPOA) of the adult male rat. Aromatase activity (AA) was measured in tissue homogenates by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H] androstenedione. We found that by 48 h after administration of testosterone, HPOA AA was significantly (p less than 0.01) greater than control values in castrated rats. In contrast, AA was significantly (p less than 0.01) reduced 12 h after castration, and reached its lowest levels by 4 days after castration. Several other steroids, administered in 3-cm Silastic capsules for 7 days, were tested for their capacity to induce hypothalamic AA. In addition to testosterone, only 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were effective. Neither the stereoisomers of these compounds nor several other steroids, including estradiol, progesterone, and corticosterone, were active. This profile of activity indicates that the induction of HPOA AA is androgen-specific and, together with the demonstrated time-course of induction, lends further support to the hypothesis that androgens regulate AA through a receptor mechanism and the synthesis of new protein.

Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques.

The conversion of androgens to active metabolites by neural tissue is believed to be an essential component in the cellular mechanism of androgen-induced neuroendocrine responses. In this study, we measured the in vitro aromatization and 5 alpha-reduction of androgens in incubations of microdissected brain regions from four intact and five castrated (6 weeks) adult male rhesus monkeys. Individual nuclei were microdissected from 600-microns frozen brain sections and homogenized in a potassium phosphate buffer. Aromatase activity was measured by a radiometric assay that uses the incorporation of tritium from [1 beta-3H]androstenedione into 3H2O as an index of estrogen formation. We estimated 5 alpha-reductase activity by isolating 5 alpha-dihydrotestosterone on two different chromatography systems and measuring the amount of this product formed from [1 alpha,2 alpha-3H]testosterone. We acidified a portion of each homogenate and determined LHRH content by RIA. Between brain nuclei, aromatase activity varied 1500-fold, whereas 5 alpha-reductase activity varied only 3-fold. Both enzyme activities were highest in amygdaloid, medial preoptic, and medial diencephalic nuclei and lowest in the caudate nucleus. Aromatase activities in the supraoptic nucleus, periventricular area, medial preoptic area-anterior hypothalamus, and lateral hypothalamus were significantly (P less than 0.05) lower in castrated males. Castration did not significantly affect 5 alpha-reductase activity, except for an increase in the basolateral amygdala. The highest concentrations of LHRH were in the infundibular nucleus-median eminence and were 30 times greater than amounts measured in preoptic and medial hypothalamic nuclei. The LHRH contents of the infundibular nucleus-median eminence, ventral medial nucleus, and lateral hypothalamus were significantly lower in castrated males (P less than 0.05). In addition, we observed a significant correlation between aromatase activity and LHRH content in the basal hypothalamus of intact males (r = 0.947; P less than 0.05; n = 8), but not in the preoptic-anterior hypothalamus (r = 0.068; P greater than 0.05; n = 10). No correlation was observed between 5 alpha-reductase activity and LHRH content in either area. These data indicate that castration selectively affects androgen metabolism and LHRH content in discrete regions of the brain of male monkeys and suggest that aromatase and 5 alpha-reductase are regulated differentially in the primate brain.

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system.

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.

Experimental induction of estradiol positive feedback in intact male monkeys: absence of inhibition by physiologic concentrations of testosterone.

Estradiol benzoate (E2B) induces an increase in serum luteinizing hormone (LH) in gonadectomized macaques of both sexes and in intact females but not males. To determine the interaction of testosterone (T) with E2B, we treated 6 intact male cynomolgus macaques as follows: 1) with E2B alone; 2) E2B after pretreatment with estradiol-17 beta (E2) for 2 wk; and 3) Treatment 2 plus exogenous T along with the E2. In Experiment 4, 6 female cynomolgus macaques were treated with large doses of T for 10 days prior to E2B. Serum levels of T and E2 were quantified by radioimmunoassay; LH by bioassay. In Experiment 1, LH decreased from 5.20 +/- 0.95 micrograms/ml (mean +/- SEM) to 0.47 +/- 0.04 micrograms/ml. In Experiment 2, E2 pretreatment depressed LH and T, and these animals responded to E2B with an increase in LH. Replacement of T (Experiment 3) did not inhibit positive feedback. Intact females treated with T responded to E2B with an increase in LH. These results suggest that a testicular product other than T is involved in the inhibition of the positive feedback response to E2B in intact male macaques, and that E2 pretreatment overcomes this effect.

Small doses of human chorionic gonadotropin prevent estradiol-induced luteolysis.

The manner in which exogenous 17 beta-estradiol (E2) induces premature luteolysis in primates is unclear. In an effort to determine whether exogenous luteotropic hormone inhibits E2-induced luteolysis, E2 capsules were implanted subcutaneously in 11 cynomolgus macaques during the early luteal phase; six animals received injections of human chorionic gonadotropin (hCG; 7.5, 10, or 15 IU/day) for 10 days, and the remaining monkeys received saline. Blood was collected once daily for measurement of E2, progesterone, and bioactive luteinizing hormone (LH). Peak progesterone concentrations were between 0.7 and 5.0 ng/ml and declined prematurely in monkeys given E2 plus saline; the luteal phase was 11.5 +/- 0.6 days (mean +/- SE). With E2 plus hCG treatment, serum progesterone continued to increase after E2 capsule placement and reached peak levels of 4.0-13.0 ng/ml; the luteal phase was 15.3 +/- 0.5 days. Therefore, E2-induced luteolysis was overcome by concurrent administration of hCG. These results suggest that exogenous tropic hormone circumvents the inhibitory influence of E2 on luteal function, but the details of the interaction remain unknown.

Effects of progesterone on estrogen-induced gonadotropin release in male rhesus macaques.

The effects of progesterone (P) on estrogen (E)-induced gonadotropin release were studied in 10 adult male rhesus macaques castrated more than 2 yr earlier. The intent was to determine whether physiological levels of P (approximately 400 pg/ml) found in the systemic circulation of intact males would block E-induced gonadotropin release and whether the responses of castrated males were similar to those of castrated females with and without pretreatment with 17 beta-estradiol (E2). Different doses of P were administered in Silastic capsules (0.3, 4.0, and 5.0 cm) implanted sc. A 0.3-cm implant maintained serum P levels at about 400 pg/ml (equivalent to physiological levels in intact males); 5.0-cm implants produced serum levels of about 4.0 ng/ml (similar to luteal phase levels in females). In male monkeys treated for approximately 3 weeks with E2, only the highest dose (approximately 4.0 ng/ml) of P blocked FSH induced by estradiol benzoate (E2B). LH was blocked in one third of the animals thus treated. The same P dose was ineffective in blocking E2-induced LH release in spayed females pretreated with E2, but did block FSH release. Gonadectomized males and females not treated beforehand with E2 released LH in response to an E2B challenge, but FSH was not elevated in the peripheral circulation under these experimental conditions. These results suggest that luteal phase levels of P block E2-induced FSH release in gonadectomized males and females. With the same treatment regimens, P blocks E2 action in some males, but all females responded to E2B by releasing LH. These data also suggest that estrogen priming is necessary for FSH, but not LH, release in adult rhesus macaques of both sexes. The prerequisite of E treatment for the induction of positive feedback appears to be associated with the level of gonadotropin suppression before E2B treatment.

The boys in the club.

A historical perspective of men in dietetics. The pioneer spirit of Lenna Cooper is exemplified in the gradual emergence of the male dietitian. It was not an easy task for the early female dietitian to accept the concept of male membership. From the acceptance of the first known qualified male dietitian in 1921 and of the first male intern in 1934 to the final acceptance of male dietitians in the U.S. Army in the early 1950s, there is recorded female resistance. As the status of the profession grew, so did men's interest in it.

Generalization of aggressive behavior in adolescent delinquent boys.

The generalization of conditioned aggressive and nonaggressive responses in a group of six adolescent delinquent boys was investigated. Responses were reinforced in card games where a token reinforcement system with money as a back-up rinforcer was used. Conditioning of responses was rapid. Generalization, measured in terms of frequency of physical contact, was tested in a group game for which no reinforcement was given. Generalization occurred during aggressive contingencies. During nonaggressive contingencies, responses did not return completely to the baseline level.