Biomedical subjects

L Milenkovic

Publications and source records attributed to L Milenkovic.

16 recordsLinked to original sources

PubMed2000

Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

3T3 Cells↗

PubMed2000

In vivo functions of the patched protein: requirement of the C terminus for target gene inactivation but not Hedgehog sequestration.

The membrane protein Patched (Ptc) is a key regulator of Hedgehog (Hh) signaling in development and is mutated in human tumors. Ptc opposes Hh-induced gene transcription and sequesters Hh protein. To dissect these functions, we tested partially deleted forms of Ptc in Drosophila. Deletion of either half of Ptc abolishes all function while coexpression of the halves restores nearly full activity. Deletion of the final 156 residues of Ptc permits Hh sequestration but abolishes inhibition of Hh targets. This deletion has dominant-negative activity, promoting target gene activation in a ligand-independent manner. We observe little or no association of full-length or partially deleted Ptc with the membrane protein Smoothened in Drosophila cultured cells.

Animals↗

PubMed1999

Mouse patched1 controls body size determination and limb patterning.

Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1(-)(/)(-) mice obscures the roles of ptc1 in later stages of development. We partially rescued ptc1 homozygous mutant embryos using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allowed survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gave rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly was preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud.

Animals↗

PubMed1996

Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog.

The signaling protein Hedgehog (Hh) controls cell fate and polarizes tissues in both flies and vertebrates. In flies, Hh exerts its effects by opposing the function of a novel transmembrane protein, Patched, while also locally inducing patched (ptc) transcription. We have identified a mouse homolog of ptc which in many tissues is transcribed near cells making either Sonic or Indian hedgehog. In addition, ectopic Sonic hedgehog expression in the mouse central nervous system induces ptc transcription. As in flies, mouse ptc transcription appears to be indicative of hedgehog signal reception. The results support the existence of a conserved signaling pathway used for pattern formation in insects and mammals.

Amino Acid Sequence↗

PubMed1992

Effects of thymosin alpha-1 on pituitary hormone release.

The thymosins are a family of hormone-like products of epithelial cells of the thymus which are important in maintenance and function of the immune system. Thymosin fraction 5, a partially purified extract of calf thymus, can influence pituitary hormone release. We have studied the effects of thymosin alpha 1 (T alpha 1), the first peptide isolated from thymosin fraction 5, on thyrotropin (TSH), adrenocorticotropin (ACTH), prolactin (Prl) and growth hormone (GH) release. To evaluate its effect in vivo we injected the peptide into the third ventricle of conscious male rats and measured the concentration of the pituitary hormones in plasma at different times after the injection. Following third-ventricular injection of T alpha 1, there was a significant decrease in plasma TSH and ACTH concentrations in comparison with values of control groups injected with diluent. The decrease in plasma TSH was of longer duration and was obtained with a lower dose of T alpha 1 than that of ACTH. Also, a significant decrease in plasma Prl was observed, with the same dose as for TSH. On the other hand, there were no significant changes in plasma GH. To examine if there is any direct effect of T alpha 1 at the pituitary level, we incubated hemipituitaries from male rats in vitro with different concentrations of the peptide. In this system T alpha 1 evoked a dose-dependent release of TSH and ACTH, while there was no effect on the release of Prl and GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1992

Effect of thymosin alpha 1 on hypothalamic hormone release.

Thymosin alpha 1 (T alpha 1) is a well-characterized immunopotentiating polypeptide originally isolated from calf thymus. We have recently shown in vivo, probable hypothalamic effects of T alpha 1 to decrease the release of the pituitary hormones, TSH, PRL and ACTH from the pituitary gland. Therefore, in the present study we evaluated the effect of the peptide on the release of hypothalamic regulatory hormones: thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), as well as somatostatin (SRIH), from medial basal hypothalamic (MBH) fragments incubated in vitro. After a preliminary time-course study indicated that a 30-min incubation period was optimal, it was used for all the other experiments. At the end of the incubation the tissue was still able to respond to a depolarizing K+ concentration for 15 min by a 4-fold increase of TRH concentration compared to control basal release during the preceding 30 min. T alpha 1 was shown to inhibit the release of TRH and CRH from MBH fragments incubated in vitro with a minimal effective dose (MED) of 10(-11) M. SRIH and CRH release was also inhibited but the MED for these peptides was 10(-9) M. The relative responsiveness to the action of T alpha 1 was TRH greater than CRH, which was greater than SRIH. This correlated with our previous in vivo results for pituitary hormone release, except in the case of SRIH since we previously did not detect any significant effect of the peptide on growth hormone release. Finally, we evaluated the possible involvement of other neurotransmitters in the effect of T alpha 1 on TRH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Evidence for suppression of Na-dependent Ca2+ efflux from rat brain synaptosomes by ovarian steroids in vivo.

The possibility that intracellular Ca2+, which mediates neurotransmitter release, regulation of membrane permeability, microtubule polymerization and axonal transport, is influenced by gonadal steroids via a Na-Ca exchange mechanism was examined. The resting Ca2+ uptake into synaptosomes was measured using crude synaptosomal pellets (P2 fraction), isolated from the brain stem, mesencephalic reticular formation (MRF), nucleus caudatus (NC) and the hippocampus of intact, long-term ovariectomized (OVX) and OVX plus progesterone (P) or estradiol-17 beta benzoate (EB) treated adult female rats. Irrespective of the brain structure investigated, the uptake was 1) markedly increased in synaptosomes from OVX animals in comparison to intact controls, and 2) reduced to near control values in synaptosomes from OVX rats treated s.c. with a single dose of 2 mg P or 5 micrograms EB. Since Ca2+ influx into synaptosomes was shown earlier to depend on external sodium concentration, which was the same in all experiments described in this work, the results obtained indicate that ovarian steroids modulate basal synaptic activity in the rat brain by suppressing Na-dependent Ca2+ efflux from the nerve cell.

Animals↗

PubMed1991

Therapeutic leukapheresis.

For the majority of leukemic patients, leukapheresis represents emergency treatment aimed at reducing the number of white blood cells and producing an immediate improvement in the clinical picture. We have shown that leukapheresis procedures performed for the therapy of leukocytosis in 4 patients with acute leukemia (2 myelocytic; 1 lymphocytic; 1 monoblastic) resulted in marked reduction in the white blood cell count and a considerable reduction in symptomatology. Repeat removal of white blood cells applied in 20 instances for patients with chronic myelocytic leukemia also produced a significant decrease in the cell count and relief of symptoms such as sweating, malaise, and pain due to splenomegaly. In chronic lymphocytic leukemia (31 patients), intensive and frequent leukapheresis procedures were followed by a marked fall in white blood cell count, regression of splenomegaly/lymphadenopathy and resolution of many symptoms and signs induced by the large number of cells.

Adolescent↗

PubMed1990

Role of monokines in control of anterior pituitary hormone release.

It has long been known that endogenous pyrogen, released as a result of injection of typhoid vaccine or in response to infection, produces fever and increases ACTH secretion. Recent studies have indicated that endogenous pyrogen is, at least in part, IL-1. This monokine has now been shown to activate the release of ACTH by a hypothalamic mechanism with release of CRF and possibly vasopressin, which stimulates the corticotrophs. There may also be a pituitary action to stimulate the release of ACTH directly. In our experiments we showed that IL-1 at low but not higher doses appears to act intrahypothalamically to stimulate GH and PRL release and to inhibit TSH release. In the meantime, another monokine, cachectin, was isolated and its structure determined. We have found that this monokine can act following its third ventricular injection to stimulate ACTH, PRL, and GH release and to inhibit TSH release, at least in part, by release of prostaglandins since indomethacin, an inhibitor of prostaglandin synthesis, produced a blockade of the responses except for those of ACTH. This peptide also has highly potent effects to alter pituitary hormone release by direct action on the pituitary to stimulate ACTH, GH, and TSH and to a slight extent PRL release. These actions appear to involve prostaglandins since indomethacin blocks all of the effects except for the effect on ACTH secretion. This monokine also produces a dose-related lowering of anterior pituitary cyclic AMP levels. When the monokine was incubated along with somatostatin, the lowering of cyclic AMP was reversed, and a potent PRL-releasing effect of the monokine was visible. We have begun studies with a third monokine, gamma interferon, which indicate that it stimulates ACTH release but suppresses plasma GH and TSH levels by a hypothalamic action. It is apparent that these various monokines have powerful effects to alter hypothalamic-pituitary function and that they probably mediate most of the effects of infections on the release of anterior pituitary hormones.

Animals↗

PubMed1990

Role of arachidonic acid or its metabolites in growth-hormone-releasing factor-induced release of somatostatin from the median eminence.

The possible involvement of arachidonic acid (AA) release in growth-hormone-releasing factor (GRF)-induced somatostatin (SRIF) release from the median eminence (ME) of the hypothalamus was evaluated in adult male rats using an in vitro incubation system. The MEs were preincubated with [14C]-AA, then washed and incubated with vehicle or test agents, and the release of SRIF and [14C]-AA into the medium was measured. In the experiments designed only to determine SRIF release, the MEs were first preincubated for 30 min. The medium was then discarded and replaced with fresh buffer or test substances and incubated for 10, 20 and/or 30 min. GRF (10(-10) M) stimulated both AA and SRIF release significantly within 20 min, with maximum release occurring at 30 min. The stimulatory effect of GRF on AA release was coincident with the release of SRIF. A phospholipase A2 inhibitor (10(-6) M, quinacrine) completely abolished the stimulatory effect of GRF on both AA and SRIF release. The release of SRIF induced by GRF was also inhibited by both indomethacin (10(-6) M, a cyclooxygenase inhibitor) and metyrapone (10(-6) M, a cytochrome P-450 inhibitor). On the other hand, nordihydroguaiaretic acid (10(-6) M, a lipoxygenase inhibitor) had no effect on GRF-evoked SRIF release. The data presented here suggest that an important GRF-mediated event leading to SRIF secretion is an elevated release of AA from ME fragments in vitro. In conclusion, our data are suggestive that the stimulatory effect of GRF on SRIF release is due, in part, to the release and subsequent metabolism of AA to one or more metabolites.

Animals↗

PubMed1990

Physiological significance of the negative short-loop feedback of prolactin.

The aim of the present study was to evaluate the physiological significance of the rapid, short-loop, negative feedback of prolactin by passive immunization with antiserum to rat prolactin injected into the third cerebral ventricle (3V) of conscious, freely moving intact or castrated male rats. Blood samples for measurement of plasma prolactin concentrations were removed through implanted external jugular catheters. After injection of 3 microliters of undiluted antiserum, plasma levels of prolactin decreased rapidly (within 5 min) to values undetectable by RIA. Further study revealed that this dose of antiprolactin serum had combined with circulating prolactin, thus rendering it undetectable by RIA. To overcome this problem, we repeated the experiment injecting 2 microliters of diluted antiserum (dilution factors 20, 100, 200, 2,000) into the 3V. When compared to values of plasma prolactin in control rats injected with 2 microliters of normal rabbit serum, none of the dilutions of antiserum induced a significant change in prolactin concentrations for as long as 4 h after injection. Since there was no effect of intraventricularly injected antiprolactin serum on basal prolactin secretion, in the next experiment, intact as well as castrated male rats were subjected to ether stress 30 min after intraventricular injection of antiserum (dilution factor 100). The elevation of plasma prolactin which followed ether stress was significantly higher in male rats pretreated with antiprolactin serum than that which occurred in control rats. A similar enhancement of the increase in plasma prolactin following ether stress, but of longer duration, was obtained in castrated rats injected with antiprolactin serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Physiologically significant effect of neuropeptide Y to suppress growth hormone release by stimulating somatostatin discharge.

Neuropeptide Y (NPY) is a peptide found in a variety of hypothalamic loci which is frequently colocalized with catecholamines. It is also secreted into hypophyseal portal vessels. The injection of NPY into the third ventricle (3V) lowered plasma GH levels in conscious, freely moving male rats. To determine the physiological significance of the hypothalamic inhibitory action of the peptide, highly specific antiserum directed against NPY was injected into the 3V of conscious rats. 3V injection of the antiserum evoked a significant elevation of plasma GH within 2 h on comparison to values in normal rabbit serum-injected, ovariectomized rats. The difference increased and reached a maximum at 6 h after injection. On the other hand, there was no effect of the antiserum in ovariectomized, estrogen, progesterone-blocked rats. Intraventricular injection of the anti-NPY serum also caused a significant elevation of plasma GH within 2 h in normal male rats and the increases above values in normal rat serum-injected control animals became even more significant at 3 and 4 h. To determine the mechanism by which NPY lowers GH after its intraventricular injection, its effect on the release of somatostatin (SRIF) from median eminence fragments incubated in vitro was examined. NPY stimulated SRIF release with a highly significant effect at a concentration of 10(-9) M. Borderline stimulation was observed at doses as low as 10(-11) M. The curve was bell-shaped with a declining release at 10(-8) M and 10(-7) M. The releasing action of NPY was blocked by either the alpha 1-receptor blocker, prazosin (10(-6) M), or the beta-receptor blocker, propranolol (10(-6) M), but was not affected by the alpha 2-receptor blocker, yohimbine (10(-6) M). We conclude that NPY has a physiologically significant inhibitory action within the hypothalamus to suppress GH release in ovariectomized female and intact male rats by stimulation of SRIF release by alpha 1 and beta-adrenergic receptor-mediated mechanisms.

Animals↗

PubMed1990

Physiological role of neuropeptide Y (NPY) in control of anterior pituitary hormone release in the rat.

Neuropeptide Y (NPY) is a peptide originally isolated from porcine brain and subsequently shown to be widely distributed in the body of several species, including man. Neuropeptide Y is a circulating peptide; however, blood levels were higher in portal than peripheral blood of anesthesized rats. Earlier studies in ovariectomized and intact male rats have shown that intraventricular injection of NPY inhibits release of growth hormone (GH) and luteinizing hormone (LH) without producing significant modification of plasma follicle stimulating hormone (FSH) and thyrotropin stimulating hormone (TSH). In the male low doses of NPY elevate prolactin (PRL) whereas high doses suppress its release. To assess the physiologic significance of these actions, we injected a highly specific anti-NPY serum (aNPY) into the third cerebral ventricle (3V) of unrestrained male, ovariectomized, and ovariectomized, estrogen progesterone blocked rats and measured plasma GH, PRL, LH and TSH by blood sampling via indwelling jugular catheters. Third ventricular injection of aNPY (2 microliters of 1:10 dilution) caused a significant elevation of plasma GH levels after 3 and 4 h compared to the values in NRS (1:10)-injected rats. To determine if these changes were due to alterations in pituitary responsiveness to somatostatin, the rats were injected intravenously with a challenge dose of somatostatin (0.5 microgram) 2 h after previous injection of aNPY or NRS, and blood samples were taken every 10 min for 30 min. The responses did not differ in both groups which indicated that the antiserum was not acting directly on the pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Hypothalamic action of cachectin to alter pituitary hormone release.

Cachectin (tumor necrosis factor, TNF) is a macrophage hormone which is released during infection and after injection of bacterial lipopolysaccharides. We have already demonstrated that the peptide has direct action on the pituitary to alter pituitary hormone release in vitro. To evaluate its action in vivo, we injected it into the third ventricle (3V) of conscious, male rats and measured its effect on various anterior pituitary hormones. The peptide produced an elevation in rectal temperature measurable on first measurement at 1 hour postinjection which was maintained for 3 hours. The maximal increase in body temperature was 1-1.5 degrees C and maximal effect was obtained by a dose as low as 1 ng (0.3 pmol) of the peptide. Preinjection of indomethacin into the 3V 1 hour prior to injection of TNF completely blocked the effect on body temperature without producing an alteration in rectal temperature itself which suggests that the elevation in body temperature may be mediated by prostaglandins. Following the intraventricular injection of various doses of TNF, there was no significant effect on plasma adrenocorticotropin (ACTH) except with the highest, 100 ng dose tested, which evoked a small but significant increase in plasma ACTH with a delay of 1 to 2 hours. Thus, the dose necessary to release ACTH was much higher than that required to elevate body temperature. The effect was no longer significant in indomethacin-pretreated animals suggesting a role for prostaglandins in the effect. This highest dose of intraventricularly administered TNF also produced a relatively modest, but significant, delayed increase in plasma GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Role of neuromedin B in the control of the release of thyrotropin in the rat.

Neuromedin B (NB), a bombesin-like peptide, was first isolated from porcine spinal cord and subsequently found in the central nervous systems of rat, cat, and human. Immunocytochemical studies have shown that NB is present in hypothalamus and various other regions of the brain and in thyrotrophs of the anterior pituitary of the rat. The possible physiological role of NB in the hypothalamic-pituitary axis is not known. Therefore, we studied the in vivo effect of this peptide on the plasma levels of thyrotropin (TSH) after administering NB to conscious freely moving adult male rats. When injected i.v., only the highest dose of NB (50 micrograms, 44.2 nmol) significantly lowered plasma TSH levels relative to levels in saline controls. When injected intraventricularly, NB doses of 0.5 and 5 micrograms (0.44 and 4.42 nmol, respectively) lowered plasma TSH levels with a 15-min latency period. Responsiveness of the pituitary to TSH-releasing hormone (TRH) was tested after the effective dose of NB was administered i.v. The increase in plasma TSH levels after the i.v. injection of 1 microgram of TRH was not altered by NB. NB at 10(-9) to 10(-7) M also reduced TSH release from hemipituitaries incubated in vitro without decreasing the response to TRH. Consequently, NB appears to have a direct inhibitory effect on TSH release from thyrotrophs. Since the response to TRH was not depressed, NB appears to act on a separate NB thyrotroph receptor that suppresses TSH release by a TRH-independent mechanism. After antiserum directed against NB was injected into the third ventricle, there was a highly significant elevation of the plasma level of TSH, beginning at 4 hr and increasing at 6 hr, relative to initial levels and levels in normal rabbit serum-injected controls. These results indicate that NB has a tonic suppressive effect on TSH release and is a physiologically significant TSH-release-inhibitory factor.

Animals↗

PubMed1989

Cachectin alters anterior pituitary hormone release by a direct action in vitro.

Cachectin (tumor necrosis factor) is a powerful macrophage hormone released during infection, which circulates in blood to produce diverse effects in the organism. We examined the effect of cachectin on release of anterior pituitary hormones from either hemipituitaries or dispersed pituitary cells incubated in vitro. The action of cachectin on dispersed cells was demonstrable only after 2 hr of incubation. With this incubation time, the protein produced a dose-related stimulation of release of adrenocorticotropin (ACTH), growth hormone (GH), and thyrotropin (TSH), but not of prolactin (Prl), from both hemipituitaries and dispersed cells. The doses required for stimulation were low in the case of hemipituitaries, usually of the order of 10(-12) M, whereas they were higher by one or two orders of magnitude with the dispersed pituitary cells. This may be related either to loss of receptors for the protein during the dispersion procedure or to the fact that in the hemipituitary system cell interactions are facilitated because the cells are close to each other. In the dispersed cell system cachectin evoked a dose-related decrease in cyclic AMP content. Incubation with somatostatin lowered the cyclic AMP content of the cells and depressed GH output without altering output of TSH or Prl. When somatostatin and cachectin were incubated together with the cells, the suppression of cyclic AMP production was abolished; TSH and Prl release were stimulated, but the action of cachectin to stimulate GH release was blocked. The stimulation of Prl release by cachectin in the presence of somatostatin may be related to the elevation of cyclic AMP, a known stimulator of Prl release. The cyclooxygenase inhibitor indomethacin nearly completely blocked the stimulatory effect of cachectin on release of GH and TSH from dispersed pituitary cells but had only a slight and nonsignificant attenuating effect on its ACTH-releasing action. These results suggest that at least part of the stimulatory action of the peptide on pituitary hormone release is brought about by prostaglandins. The failure of indomethacin to block the release of ACTH induced by cachectin suggests that other mechanisms may be involved in the release of ACTH induced by this peptide. Since the concentrations of cachectin required to stimulate pituitary hormone release are similar to those that are encountered in plasma during infection, it is likely that this direct pituitary action has pathophysiological significance.

Adrenocorticotropic Hormone↗