Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Simple quantitative measurement of serum choriogonadotropin compared with immunoradiometric, immunoenzymometric, and chemiluminescent assays.

We evaluated a new simplified quantitative method (Tandem Icon QSR; Hybritech) for choriogonadotropin (hCG), which could theoretically be performed in a ward or with outpatients. The method was compared with immunoradiometric (Bioclone Australia), chemiluminescent (Amerlite; Amersham), and immunoenzymometric assays (Stratus; Dade). We analyzed by each of the methods 104 serum samples from pregnant and nonpregnant patients. For quantitative hCG values indicative of pregnancy (greater than 25 int. units/L), excellent correlation was observed between the Tandem Icon method and the other standard laboratory assays (r = 0.995, 0.990, and 0.992, respectively). Occasional problems arose because of the instability of Tandem Icon QSR reagents at room temperature but this was resolved by storing the reagents at 4 degrees C. We conclude that this simplified quantitative method for hCG is reliable and suitable for use outside of the routine immunoassay laboratory.

Pituitary response to LHRH, LH pulsatility and plasma melatonin and prolactin changes in ewe lambs treated with melatonin implants to delay puberty.

Prepubertal ewe lambs were treated with empty or filled melatonin implants. The implants were placed s.c. at birth and pituitary responsiveness to various doses of LHRH, LH/FSH pulsatility and prolactin and melatonin secretion were examined at 10, 19, 28, 36 and 45 weeks of age. Control animals (N = 10) showed no consistent alteration in pituitary responsiveness to LHRH during development. Ewes treated with melatonin (N = 10) had puberty onset delayed by 4 weeks (P less than 0.03) but no effect of melatonin on LH or FSH response to LHRH injection was observed at any stage of development. In the control and melatonin-treated ewe lambs the responses to LHRH injection were lower during darkness than during the day at all stages of development. No consistent differences in LH or FSH pulsatility were observed between treatment groups or during development. Prolactin concentrations, however, failed to decrease at the time of puberty (autumn) in the melatonin-treated group. Melatonin-treated ewe lambs maintained normal rhythmic melatonin production which was superimposed on a higher basal concentration and showed the same increase in melatonin output with age as the control ewes. These results indicate that the delayed puberty caused by melatonin implants is not due to decreased pituitary responsiveness to LHRH or to dramatic changes in basal LH or FSH secretion.

Pinealectomy delays puberty in ewe lambs.

Fifteen pinealectomized and 15 unoperated ewes were exposed to constant light for 3 weeks before and 10 weeks after lambing. Fourteen pinealectomized and 15 unoperated ewes were allowed to lamb outdoors. Five ewe lambs born in constant light to the 2 groups of dams were pinealectomized at 10 weeks of age. Ewes and lambs were then returned to the field. Puberty (determined by weekly progesterone analysis) was significantly delayed (P less than 0.05) in the pinealectomized ewe lambs. Median pubertal age in pineal-intact ewe lambs was 37 weeks compared to 49 weeks in pinealectomized lambs. Constant light during the first 10 weeks of life had no effect upon puberty onset nor did the pineal status of the dam. Control lambs entered seasonal anoestrus at the time pinealectomized ewe lambs were entering puberty. Pinealectomized lambs entered anoestrus at the same time as control lambs were beginning their second breeding season. These results confirm a key role of pineal-mediated hormonal signals in the control of puberty in the sheep.

Effects of melatonin implants in ram lambs.

Sixteen pinealectomized and 19 unoperated ewes were exposed to constant light for about 4 weeks before and 4 weeks after lambing. Six ram lambs born to unoperated ewes were implanted s.c. with melatonin sachets while 8 ram lambs were implanted with empty sachets. The 8 ram lambs born to pinealectomized dams also received empty implants. Ewes and lambs were then returned to the field. Analysis of weekly blood samples indicated that prolactin secretion was significantly decreased in the ram lambs with empty implants between 44-51 weeks of age whereas lambs treated with melatonin failed to show a significant change during development. All 3 groups of rams had elevated LH levels between 7 and 17 weeks of age, and a second period of high LH between 27 and 40 weeks. There were no significant differences between groups in the patterns of FSH secretion; FSH was highest between 7 and 17 weeks of age, and again between 27 and 40 weeks of age. Plasma testosterone levels in all groups increased gradually between 4 and 35 weeks. Between 38 and 48 weeks of age testosterone concentrations were markedly elevated in all groups. Growth was not affected by melatonin treatment. These results indicate that neonatal melatonin treatment has subtle endocrine effects; whether these effects are sufficient to compromise fertility in the ram, however, remains to be established.

Effects of pinealectomy, oestradiol and melatonin on plasma prolactin and LH secretion in ovariectomized sheep.

The study examines whether the pineal gland mediates in the seasonal rhythm in the response of the hypothalamo-pituitary axis to oestrogen in the sheep. Five groups of ewes, each comprised of five sham-pinealectomized ewes and five pinealectomized ewes, were maintained under field conditions over a 2-year period. Group I ewes were ovariectomized and treated with two oestradiol capsules in the first year and one oestradiol capsule in the second year; group 2 ewes were ovariectomized and treated with two oestradiol capsules and three melatonin sachets in the first year and one oestradiol capsule and three melatonin sachets in the second year; group 3 ewes were ovariectomized and treated with three melatonin sachets in the first year and one oestradiol capsule in the second year; group 4 ewes were ovariectomized and treated with empty implants; group 5 had intact ovaries. Blood samples were taken at weekly intervals for determination of plasma prolactin, LH and FSH levels. Cyclic ovarian activity, determined by rams fitted with a marking harness, occurred in both sham-pinealectomized and pinealectomized ewes in group 5 during December to May in both years. A circannual rhythm in plasma prolactin levels was apparent in all ten groups of animals. The levels were highest in spring (September to November) and decreased before the onset of breeding activity in midsummer (December). Ovariectomized ewes with intact pineal glands and treated with oestradiol capsules (group 1) exhibited marked changes in LH and FSH concentrations during the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of melatonin implants in ewe lambs.

Sixteen pinealectomized and 19 unoperated ewes were exposed to constant light for about 4 weeks before and 4 weeks after lambing. Five ewe lambs born to pinealectomized ewes were implanted s.c. with melatonin sachets and 6 ewe lambs implanted with empty sachets. The 6 ewe lambs born to unoperated dams received empty implants. Ewes and lambs were then returned to the field. Puberty (determined by weekly progesterone analysis) was significantly delayed (P less than 0.05) in 4 of the 5 melatonin-treated ewe lambs; mean pubertal age of ewes with empty implants was 44 weeks of age compared to 45, 63, 72, greater than 72, greater than 72 weeks of age for the melatonin-treated animals. Prolactin levels were decreased in the ewes with empty implants in autumn, coincident with the period of cyclic ovarian activity. No significant decrease in prolactin was observed in autumn in melatonin-treated lambs. Growth was not affected by melatonin treatment. These results indicate a role of the pineal gland and melatonin in the control of puberty in sheep.

Effects of melatonin implants on the circadian rhythm of plasma melatonin and prolactin in sheep.

The effects of implanting Silastic capsules containing melatonin on plasma melatonin and prolactin levels were investigated in pinealectomized (Px) and sham-operated sheep (SPx). Prior to implantation, melatonin was found in plasma samples obtained during the night period from SPx sheep (mean value 150 pg/ml), but could not be (less than 25 pg/ml) detected in plasma samples obtained during the day in SPx sheep or in any sample obtained during the night or day period in Px sheep. Following implantation, a constant basal plasma melatonin level of about 165 pg/ml was established in all sheep with a superimposed nighttime rise in SPx animals suggesting no diminution of endogenous melatonin production during the dark period. Following melatonin treatment, there was a marked depression in plasma prolactin levels in both SPx and Px sheep. These results are interpreted to indicate that 1) there is no negative feedback of melatonin upon its own synthesis and release, 2) that there is no circadian change in the rate of metabolism of melatonin and 3) that constant melatonin availability in sheep caused a depression in plasma prolactin levels similar to that found following exposure of animals to a short day.