TSC-22D1 isoforms have opposing roles in mammary epithelial cell survival.

Transforming growth factor beta (TGFbeta)-stimulated clone-22 domain family member 1 (TSC-22D1) has previously been associated with enhanced apoptosis in several cell systems. In an attempt to identify novel factors that are involved in the control of cell death during mammary gland involution, we found that the mRNA for isoform 2 of TSC-22D1 was highly upregulated 24 h after forced weaning, when a dramatic increase in cell death occurred, closely following the expression of the known inducer of cell death during involution, TGFbeta3. This was paralleled by strongly increased TSC-22D1 isoform 2 protein levels in the luminal epithelium. In contrast, RNA and protein expression levels of the isoform 1 of TSC-22D1 did not change during development. Whereas isoform 2 induced cell death, isoform 1 suppressed TGFbeta-induced cell death and enhanced proliferation in mammary epithelial cell lines. Furthermore, four distinct forms of isoform 2 protein were detected in the mammary gland, of which only a 15-kDa form was associated with early involution. Our data describe novel opposing functions of the two mammalian TSC-22D1 isoforms in cell survival and proliferation, and establish the TSC-22D1 isoform 2 as a potential regulator of cell death during mammary gland involution.

p73 regulates DRAM-independent autophagy that does not contribute to programmed cell death.

Evading programmed cell death is a common event in tumour development. The p53 family member, p73, is a potent inducer of death and a determinant of chemotherapeutic response, but different to p53, is rarely mutated in cancer. Understanding cell death pathways downstream of p53 and p73 is therefore pivotal to understand both the development and treatment of malignant disease. Recently, p53 has been shown to modulate autophagy--a membrane trafficking process, which degrades long-lived proteins and organelles. This requires a p53 target gene, DRAM, and both DRAM and autophagy are critical for p53-mediated death. We report here that TA-p73 also regulates DRAM and autophagy, with different TA-p73 isoforms regulating DRAM and autophagy to varying extents. RNAi knockdown of DRAM, however, revealed that p73's modulation of autophagy is DRAM-independent. Also, p73's ability to induce death, again different to p53, is neither dependent on DRAM nor autophagy. In contrast to TA-p73, deltaN-p73 is a negative regulator of p53-induced and p73-induced autophagy, but does not affect autophagy induced by amino-acid starvation. These studies, therefore, represent not only the first report that p73 modulates autophagy but also highlight important differences in the mechanism by which starvation, p53 and p73 regulate autophagy and how this contributes to programmed cell death.

The nonlinear decay of complex signals in dissipative media.

The solution of Burgers' equation with random initial conditions is often said to describe "Burgers turbulence." The Burgers equation describes two fundamental effects characteristic of any turbulence-the nonlinear transfer of energy over the spectrum and the dissipation of energy in the small-scale components. Strong interaction between coherent harmonics, associated with the nondispersive nature of the dynamics, leads to the appearance of local self-similar structure. In Burgers' equation, continuous random initial fields are transformed into sequences of regions with regular behavior, with random locations of the shocks separating them. Moreover, the statistical properties of such random fields are also self-similar. It is already known that the merging of the shocks leads to an increase of the external scale of the turbulence, and because of this the energy of a random signal ("noise") decreases more slowly than the energy of simple signals. Here we show that similar behavior takes place for complex regular signals with fractal structure in the coordinate or in the wave-number space. In all these cases, the law of increase of the external scale is determined by the behavior of the structure function of the integral of the initial field-i.e., the structure function of the initial action. (c) 1995 American Institute of Physics.

Effects of active immunization of ram lambs and bull calves against synthetic luteinizing hormone releasing hormone.

Ram lambs and bull calves were immunized against LH-RH by injections given in weeks 0, 6, 12 and 28 (ram lambs, week 0=16 to 20 weeks of age) and weeks 0, 6, 12 and 18 (bull calves, week 0=approximately 4 weeks of age). The testis size of LH-RH-immunized animals was significantly less than that of controls from week 13 onwards in ram lambs and from week 15 onwards in bull calves. When ram lambs were sampled in week 17 and bull calves in week 20, mean plasma gonadotrophin and testosterone concentrations were consistently lower in LH-RH-immunized animals than in controls. Single intravenous injection of synthetic LH-RH or an analogue of LH-RH in week 27 failed to induce LH or testosterone responses in LH-RH-immunized ram lambs. Motile semen samples could not be obtained from any of the LH-RH-immunized ram lambs in weeks 24, 25 and 26 or from 7 of 10 in week 72, but samples of moderate motility were obtained in week 72 from three rams in which LH-RH antibody titres had fallen. No attempt was made to obtain semen from bull calves. After castration there was no increase in plasma LH in LH-RH-immunized rams and only a small increase in LH-RH-immunized bull calves. Mean testis weight was significantly lower in LH-RH-immunized animals than in controls of both species. Thus the normal development of the reproductive system in ram lambs and bull calves was blocked by active immunization against LH-RH. Some evidence was obtained for natural reversal of the effects with time and falling antibody titres. These findings demonstrate the potential of LH-RH immunization as an alternative to castration.

The effects of injecting [D-Ser(But)6] Des Gly-NH2(10) luteinizing hormone releasing hormone ethylamide in early, mid and late seasonal anoestrus on gonadotrophin secretion and luteal function in the ewe.

The ability of the luteinizing hormone releasing hormone (LH-RH) analogue [D-Ser(But)6] Des-Gly-NH2(10) LH-RH ethylamide to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and to induce ovulation and luteal function in seasonally anoestrous ewes was investigated by injecting the analogue at three stages of the anoestrus (day 118, day 182 and day 235 of the year). After injection on day 118, eight of nine ewes ovulated and all of the former secreted progesterone during the subsequent 20 days. After injection on day 182, six of the nine ewes ovulated, of which none showed luteal function. Only two of the nine ewes were not already secreting progesterone on day 235. Both of these responded to the analogue by secreting normal luteal levels of progesterone. The mean LH peak heights in response to injection at the three stages showed no significant differences from one another. The mean FSH peak height after injection on day 182 was significantly lower than the mean FSH peak height associated with the other two challenges (P<0.05). On day 116 of the following year, 20 ewes were treated with the analogue as before. The high progesterone levels confirmed the results of the day 118 challenge in the previous year. However, none of the ewes conceived when inseminated artificially 24 and 36 hours after analogue treatment.

Relative activity, plasma elimination and tissue degradation of synthetic luteinizing hormone releasing hormone and certain of its analogues.

The abilities of three nonapeptide analogues of synthetic luteinizing hormone releasing hormone (LH-RH) to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in anoestrous and cyclic ewes were examined, as were their elimination from the plasma in vivo and degradation by extracts of the hypothalamus, anterior pituitary gland, lung, kidney, liver and plasma in vitro. In all cases, comparisons were made with synthetic LH-RH. When injected i.v. into mature ewes as a single dose, the potencies of the analogues were graded and Des Gly-NH2(10) LH-RH ethylamide was found to be the least potent. It was not possible to demonstrate any significant increase in the potency of this analogue over LH-RH, although a trend was apparent with each parameter examined. [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide had the greatest potency. There were no differences between the responses of anoestrous ewes and those of ewes treated on day 10 of the oestrous cycle. None of the analogues had a rate of elimination from the plasma different from that of LH-RH during either the first or the second components of the biphasic disappearance curve. The incubation of LH-RH with tissue extracts showed that extracts of the hypothalamus and anterior pituitary gland degraded LH-RH to a similar extent. Both the hypothalamic and anterior pituitary gland extracts degraded more LH-RH than did lung extract, which in turn destroyed more LH-RH than did extracts of kidney or liver tissue. The degradative abilities of kidney and liver extracts did not differ from each other. Plasma failed to degrade LH-RH or the analogues. Although LH-RH was rapidly destroyed by hypothalamic extract in vitro, of the analogues, only Des Gly-NH2(10) LH-RH ethylamide was degraded. The anterior pituitary gland and kidney extracts failed to degrade [D-Ser6] Des Gly-NH2(10) LH-RH ethylamide and [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide as rapidly as LH-RH. Extracts of liver and lung were incapable of catabolizing any of the analogues. There was an inverse correlation between the LH- and FSH-releasing potency of an analogue and its rate of degradation by anterior pituitary gland extract. The slower rates of catabolism of certain analogues of LH-RH by the anterior pituitary gland may explain their increased LH- and FSH-releasing potency.

Luteinizing hormone release after two injections of synthetic luteinizing hormone releasing hormone in the ewe.

Anoestrous ewes were given two injections of 30 mug synthetic luteinizing hormone releasing hormone (LH-RH) separated by one of the following intervals: 1-5, 3, 6, 12 or 24 h. The first injection caused an increase in the plasma LH concentration in each animal. The response to the second injection was dependent on the interval between the injections. When the second injection was administered 1-5 h after the first it caused a further increase in the LH concentration to maximal levels which were significantly greater than those induced in the other anoestrous groups. When the second injection was administered 3 h after the first, there was no significant difference between the responses to the two injections although the time to reach the maximal LH concentration was shorter and the height of the LH peak was greater in each animal following the second injection. When the second injection was administered 6, 12 or 24 h after the first, the LH response was significantly less, in terms of height and area of the induced peak, than following the first injection. The LH response to the second injection was particularly low in the 12 and 24 h groups. Two injections of 30 mug synthetic LH-RH were also administered at 1-5 h intervals to ewes on either day 10 of the oestrous cycle or at onset of oestrus. The pattern of LH responses in all these animals was similar to that observed in anoestrous ewes injected at 1-5 h intervals. The total LH release, as assessed in terms of the induced peaks, was significantly greater in the onset of oestrus group than in the day 10 group or any of the anoestrous groups. Presumably the sensitization-desensitization sequence of the pituitary gland to LH-RH which has been demonstrated, together with the effects of sex steroid hormones, must play an important part in the development and decay of the natural preovulatory LH peak.

Comparison of the redox bioassay with other assays for luteinizing hormone.

The cytochemical (redox) bioassay for LH has been compared with established LH assays. Measurements made by redox bioassays were considerably lower than those made by radioimmunoassay in human female plasma samples obtained at mid-cycle. There was no apparent relationship between measurements on incubation media from cultures of sheep pituitary glands made by redox bioassay and the ovarian ascorbic acid depletion (OAAD) assay. After polyacrylamide gel electrophoresis of a crude extract of a human pituitary gland, redox LH measurements were lower than those of the OAAD assay and radioimmunoassay in the cathodal segments of the gel. By contrast, there was reasonable agreement between LH measurements made by radioimmunoassay and redox assay in cathodal fractions from gel electrophroesis of a purified pituitary LH preparation. Follicle-stimulating hormone, and the alpha- and beta-subunits of LH depressed the response of intact LH in the redox assay; this might explain the relatively low levels of LH measured by redox assay in some of the experiments described. Which type of assay best reflects the biological activity of LH in man remains to be determined.

Leteinizing hormone and luteinizing hormone releasing hormone-like immunoreactivity in the jugular venous blood of sheep at various stages of the oestrous cycle.

Luteinizing hormone and LH-RH-like immunoreactivity were measured in the jugular venous plasma of Clun Forest ewes at various stages of the oestrous cycle. Blood samples were collected through jugular venous cannulae every 2 h for at least 20 days from three ewes during the breeding season. The ewes were checked twice daily for oestrus using a vasectomized ram. Plasma LH peaks of apparent height 112-192 ng NIH-LH-S17 equivalents/ml were detected at oestrus with basal levels of 2-15 ng/ml during most of the remainder of the 17-day oestrous cycle. Peaks of LH-RH-like immunoreactivity occurred at various times of the cycle. The apparent maximal level of these peaks was 220 pg/ml compared with basal levels of less than 10 pg/ml. Further ewes (two for each group) were sampled at 4 min intervals for 12 h, (1) from onset of oestrus, (2) 36-48 h after onset of oestrus or (3) on day 10 of the oestrous cycle. In the ewes sampled at oestrus, peaks of LH-RH-like immunoreactivity were detected before, during and after the preovulatory LH peak. Those detected after the LH peak were unassociated with any further increases in the plasma LH level. In the ewes sampled 36-48 h after onset of oestrus and on day 10 of the cycle, several peaks of LH-RH-like immunoreactivity unassociated with any increases in the LH level were detected. These peaks, and those detected at oestrus, had durations of only one or two samples, and in some cases reached levels of several ng/ml compared with basal levels of less than 10 pg/ml. The significance of these results is discussed.

Progesterone levels following treatment of seasonally anoestrous ewes with synthetic LH-releasing hormone.

Plasma progesterone determinations were carried out on blood samples collected daily from Clun Forest ewes during the normal oestrous cycle and also after administration of LH-releasing hormone (LH-RH) during seasonal anoestrus. Levels of plasma progesterone at oestrus ranged from 0.1 to 0.5 ng/ml and luteal phase levels from 3 to 6 ng/ml. Levels found during seasonal anoestrus were within the range of those observed as oestrus. Following treatment with LH-RH, there was in increase in the plasma LH level in a-l cases and ovulation occurred in twenty-three out of twenty-seven treated ewes. In the animals which ovulated, the plasma progesterone concentration either remained basal (eighteen animals) or rose to a lower level (2 ng/ml is greater than) than that found during the luteal phase of the cycle.