Altered neural cell fates and medulloblastoma in mouse patched mutants.

The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities also seen in BCNS patients.

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.

Ca2+ uptake by rat brain region synaptosomes following in vivo administration of ovarian steroids.

Effects of progesterone (P) and estradiol-17 beta benzoate (EB), applied s.c. into sexually mature, long-term ovariectomized (OVX) rats, on subsequent depolarization-induced uptake of Ca2+ were studied in synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), nucleus caudatus putamen (NCP) and the hippocampus. In intact animals, synaptosomal Ca2+ uptake differed from region to region: it was lowest in the brain stem and highest in the hippocampus. In comparison to intact animals, ovariectomy resulted in a marked increase of the uptake regardless the structure investigated, suggesting an inhibitory action of ovaries on the uptake of Ca2+ in a considerable portion of rat brain. Single injection of 2 mg P, given to OVX rats 24 h prior to decapitation, evoked a marked decrease in Ca2+ uptake by synaptosomes of the brain stem and MRF and particularly by those of NCP and the hippocampus. Single injection of 5 micrograms EB into OVX animals 72 h prior to the experiment was as effective as P in inhibiting Ca2+ uptake by synaptosomes of the brain stem and MRF, but less effective than P in case of NCP and the hippocampus. This suggests involvement of P and EB in the modulation of synaptic transmission by affecting neuronal Ca2+ uptake.

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)

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.

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)

[Transfusion-infusion methods in the treatment of acute poisoning]. / Transfuziono-infuzione metode u tretmanu akutnih trovanja.

For modern acute poisoning management there are several therapeutic transfusion-induced methods (forced diuresis, haemodialysis, peritoneal dialysis, haemoperfusion, plasmapheresis, exsanguino-transfusion, blood and blood component transfusion, intravenous solution infusion etc), each of them having their advantages, disadvantages and limitations. No one of these methods is so efficacious enough that its application could be justified in all poisonings. However, some of them are considered to be method of choice in the treatment of certain acute poisonings.

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)

The role of neuropeptide Y (NPY) in control of gonadotropin and prolactin release in the rat.

Neuropeptide Y is a peptide found in a variety of hypothalamic loci which is frequently colocalized with catecholamines. It is also secreted into hypophyseal portal vessels. We have previously evaluated the effects of this peptide on FSH, LH and prolactin release. In ovariectomized females neuropeptide Y inhibits LH release. It has similarly been reported to inhibit LH release in intact males; however, estrogen priming of ovariectomized animals converts this inhibitory action into a stimulatory effect. In ovariectomized animals the peptide has a direct stimulatory effect on perifused pituitary cells, enhancing the release of both FSH and LH, an effect which is contrary to that obtained with LH release after intraventricular injection of the peptide. In the present experiments the physiological significance of these effects has been evaluated by the intraventricular injection (3V) of highly specific antiserum directed against the peptide. In ovariectomized and in ovariectomized, estrogen-primed rats, the third ventricular injection of antiserum had no effect on gonadotropin release. In male rats intraventricular injection of the antiserum elevated LH, which indicates that the inhibitory action of the peptide seen in intact males is of physiological significance. However, it has been reported by others that the proestrous type discharge of LH-RH is blocked by intraventricular injection of neuropeptide Y antiserum. Neuropeptide Y might therefore play an essential role in the preovulatory release of LH. On the other hand, others have shown that intraventricular injection of neuropeptide Y in male rats can either stimulate, at low doses, or inhibit, at high doses, the release of prolactin. We have confirmed the inhibitory action, which appears to be of physiological significance since antisera directed against the peptide injected intraventricularly resulted in an elevation of prolactin release. The results of these studies indicate that neuropeptide Y plays a very important and often physiologically significant role in the control of LH and prolactin release by hypothalamic action.

[Fibrin glue in tympanoplasty]. / Fibrinski lepak u timpanoplastici.

In order to achieve more favourable anatomic and functionally more permanent position of the transmission materials, fibrin glue was applied in 68 patients undergoing different microsurgical interventions for the cure of inflammatory processes in the middle ear or improvement of conductive deafness of inflammatory or traumatic origin. Fibrin glue has shown to be equally suitable both in fixation of the biological materials - homografts and autografts and in fixation of alloplastic prostheses. The efficacy of preservation of the best position was demonstrated in all forms of direct collumelas, ossicular graft interposition, closure of the labyrinthine fistulas and reconstruction of the external attic wall.

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)

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.

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.

Effects of unilateral castration on the hypothalamic structures involved in the regulation of gonadal function in rat.

This study was undertaken to evaluate the effects of unilateral gonadectomy on the hypothalamic structures involved in the regulation of gonadal function in adult rats of both sexes. Unilateral gonadectomy was performed; 15 days later stereological parameters of cell activity of both the halves of hypothalamic preoptico-suprachiasmatic area (PO-SC) and arcuate nucleus (NA) were analyzed. Under the same experimental conditions the activities of the FSH and LH immunoreactive cells were analyzed separately in both the halves of the adenohypophysis. The results showed that in the rats of both sexes subjected to unilateral gonadectomy the mean diameter of cell nuclei of the contralateral half of PO-SC was significantly greater than that of the ipsilateral half. However, in the control intact or bilaterally gonadectomized rats, there were no significant differences in the values of the same parameter between two halves of PO-SC. On the other hand, neither in the unilaterally gonadectomized nor in the controls, the values of the mean diameter of NA cell nuclei differed significantly between the two halves of this structure. The FSH and LH pituitary cells behaved like NA cells. Therefore, since in the experimental animals compensatory function was developed, and since nervous signaling was different from the sides of the removed and intact gland, the present results suggest involvement of a pure nervous mechanism, besides hormonal control, in the regulation of the compensatory gonadal function. This mechanism seems to be functional in the rats of both sexes. These results also indicate that PO-SC is the anatomical structure involved in this regulation.

Effects of oestradiol and progesterone on stress-induced secretion of prolactin in ovariectomized and/or adrenalectomized female rats.

Prolactin (Prl) secretion in response to an acute stress was studied in ovariectomized (OVX) and/or adrenalectomized (ADX) adult female rats, nontreated or injected sc with a single dose of 5 micrograms oestradiol-17 beta benzoate (OB) or 2 mg progesterone (P). The stress applied consisted of cutting the tip of tail of conscious animals. Radioimmunoassay was used to measure Prl in the serum prepared from blood collected by decapitation 10 min following the stress, i.e., at the point of maximum recorded Prl response. It was found that the capacity of the animals to secrete large quantities of Prl under stress was, when compared to that in intact controls, markedly reduced in OVX or ADX rats and substantially absent in OVX + ADX rats. A 10-fold increase of basal serum Prl, similar in magnitude to the increase in intact controls, was induced by the stress in OVX animals pretreated with OB. On the contrary, pretreatment of OVX animals with P resulted in a complete block of the Prl response to the stress. The stimulative effect of OB was greatly attenuated in stressed OVX + ADX rats. The results suggest that OB potentiates whereas P attenuates the stress-induced secretion of Prl in the female rat, and that the potentiating effect of OB is dependent on functionally intact adrenals.

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.

[Albumin and its therapeutic use - part I]. / Albumin i njegova terapijska primena -- I deo.

Albumin is most abundant and most studied protein of the circulation. Its biosynthesis is closely dependent on the nutrition, amino acid supply, hormonal millieu, environment, osmotic equilibrium, diseases and some other factors. Albumin is synthetized in the liver on a polyscmes and delivered into the blood streem. Degradation of albumin is practically still unknown and is one of many biological puzzles. Albumin prepared for therapeutic use almost contains dimers, oligomers and polymers which are very important, because they are one of the parameters for evaluation of albumin products quality.

[Albumin and its therapeutic use - Part II]. / Albumin i njegova terapijska primena -- II deo.

Albumin is the most effective oncotic agent and has wide clinical application. In the resuscitation of injured patients with hypovolemic shock (during war and peace) the use of supplemental albumin is purported to be more effective in restoring plasma volume, increasing cardiac output, preventing pulmonary oedema and maintaining organ perfusion than resuscitation without supplemental albumin. Most important in a study of albumin are criterias for appropriate use because it was sometimes unjustified. Therapeutic use of albumin should not be dictated by arbitrary or pragmatic restriction but rather by rational prescribing from physicians well educated in its use.

Effects of aging on prolactin release after stress in female and male rats.

Young adult and elderly male and female intact rats, as well as chronically ovariectomized (OVX) young and elderly female rats, were subjected to an acute stress by cutting the tip of the tail and prolactin (Prl) concentrations were measured in their blood collected by decapitation at various times thereafter. Maximum concentrations of the hormone were markedly lower in all the three groups of elderly rats than those found in the corresponding young animals, and appeared to occur with a delay in the females, but not in the males. In addition, the Prl-response to stress was attenuated in OVX animals regardless of their age. The result of these experiments, performed at two points on the age scale, suggests that in sexually mature rats of both sexes the stress-induced secretion of Prl is inversely related to the age of the animal and that the reverse relationship is retained in OVX females.

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.