Effect of nano-sized, elemental selenium supplement on the proteome of chicken liver.

The nano-sized (100-500 nm) selenium has higher bioavailability and relatively lower toxicity compared to other selenium forms. The objective of the present study was to compare liver proteome profiles of broiler chicken fed with control diet without Se supplementation and diet supplemented with nano-Se with 4.25 mg/kg DM. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography-mass spectrometry (LC-MS). Seven hundred and eight spots were detected, and 18 protein spots showed significant difference in their intensity (p < 0.05) between the two groups. In response to nano-Se supplementation, the expression of 8 proteins was higher, and 5 proteins were lower in nano-Se supplemented group compared to control group. The functions of the differentially expressed proteins indicate that the high dose of selenium supplementation induced a dietary stress. Selenium supplementation may influence the metabolism of fatty acids and carbohydrates and antioxidant system, and increase the quantity of cytoskeletal actin and the expression of actin regulatory protein as well.

Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Kynurenine administered together with probenecid markedly inhibits pentylenetetrazol-induced seizures. An electrophysiological and behavioural study.

The kynurenine pathway converts tryptophan into various compounds, including l-kynurenine, which in turn can be converted to the excitatory amino acid receptor antagonist kynurenic acid, which may therefore serve as a protective agent in such neurological disorders as epileptic seizures. Kynurenic acid, however, has a very limited ability to cross the blood-brain barrier, whereas kynurenine passes the barrier easily. In this study, we tested the hypothesis that kynurenine administered systemically together with probenecid, which inhibits kynurenic acid excretion from the cerebrospinal fluid, results in an increased level of kynurenic acid in the brain that is sufficiently high to provide protection against the development of pentylentetrazol-induced epileptic seizures. CA3 stimulation-evoked population spike activity was recorded from the pyramidal layer of area CA1 of the rat hippocampus, and in another series of behavioural experiments, water maze and open-field studies were carried out to test the presumed protective effect of kynurenine + probenecid pre-treatment against pentylenetetrazol-induced seizures. This study has furnished the first electrophysiological proof that systemic kynurenine (300 mg/kg, i.p.) and probenecid (200 mg/kg, i.p.) administration protects against pentylenetetrazol-induced (60 mg/kg, i.p.) epileptic seizures.

Biological half-life and organ distribution of [3H]8-arginine-vasopressin in the rat.

The biological half-life of synthetic, radiochemically pure, biologically active [3H]8-arginine-vasopressin ([3H]AVP), the distribution of radioactivity among the organs and the in-vivo metabolism of the hormone were studied in the rat. The half-life calculated from the [3H]AVP radioactivities isolated from the blood was found to be 1.74 +/- 0.22 (S.D.) min in the fast phase, and 16.98 +/- 1.01 min in the slow phase. The half-lives of total radioactivity were longer in both phases. The radioactivity accumulated to the greatest extents in the adenohypophysis and small intestine. The radioactive substance was accumulated more by the kidney than by the liver, but the hormone underwent inactivation more quickly in the liver.

Biological half-life and organ distribution of [3H]1-deamino-8-D-arginine-vasopressin in the rat.

The biological half-life of synthetic, radiochemically pure, biologically active [3H]1-deamino-8-D-arginine-vasopressin (dDAVP) in rats was found to be 5.33 +/- 0.28 (S.E.M.) min in the initial, transitional, fast phase and 56.28 +/- 3.27 min in the second, slow phase. The substance accumulated to the greatest extent in the kidney and small intestine and only slightly in the adenohypophysis. The results have suggested that the extended biological half-life may play a role in the more marked and longer antidiuretic effect of dDAVP. The explanation of the poor accumulation in the adenohypophysis may be that dDAVP does not possess an effect similar to that of corticotrophin releasing factor.

Mechanism of restoration of ACTH release in rats with long-term lesions of the paraventricular nuclei.

The effects of lesions in the paraventricular nucleus (PVN) on the adrenocortical response to ether stress were investigated in neurohypophysectomized and intact rats. During the first 4 days after placement of lesions in the PVN, the corticosterone response to ether stress was almost completely inhibited. It then gradually increased and, within 4-6 weeks of surgery, was restored to about 60% of that in sham-operated rats. Basal plasma concentrations of corticosterone were low in rats after placement of lesions in the PVN and/or after neurointermediate lobectomy (NILX). Corticosterone responses to ether stress were similar in groups submitted to PVN lesions and/or NILX, and lower than those in the appropriate sham-operated groups. In all lesioned groups, plasma ACTH concentrations after a combination of stressors (ether plus laparotomy) were also lower than those in the sham-operated groups. Six weeks after lesioning of the PVN, immunoreactive rat corticotrophin-releasing factor-41 (rCRF-41) concentrations in stalk-median eminence (SME) extract fell to about 5% of that in sham-operated rats, while immunoreactive arginine vasopressin (AVP) concentrations did not change. Immunohistochemistry revealed a substantial decrease in rCRF-41 immunostaining of the median eminence 6 weeks after lesioning of the PVN, though randomly located clusters of stained terminals were still seen in the whole rostro-caudal extent of the median eminence. A mixture containing synthetic rCRF-41 and AVP, in proportions similar to those in SME extracts from sham-operated rats, caused significantly less release of ACTH from anterior pituitary cell cultures than did SME extracts from sham-operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short term cardiotoxicity of ddC is partially based on ROS-mediated signalling through poly- and mono-ADP-ribosylation reactions and depression of HSP70 levels, whose processes represent a new mtDNA independent mechanism for ddC-induced cell damage.

Antitumor effects of analogs of LH-RH and somatostatin: experimental and clinical studies.

Many clinical approaches for the treatment of hormone-sensitive tumors are being developed based on analogs of LH-RH and somatostatin. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of LH-RH agonists like [D-Trp6]-LH-RH and new LH-RH antagonists free of edematogenic effects such as [Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6,D-Ala10]-LH -RH (SB-75). Agonists and antagonists of LH-RH have been used in patients with prostate cancer and might be also beneficial for the treatment of breast cancer and ovarian, endometrial and pancreatic carcinomas. Some of the effects of LH-RH analogs can be due to direct action since LH-RH receptors have been found in these cancers. The use of sustained delivery systems based on microcapsules of PLG, makes the treatment more efficacious. Octapeptide analogs of somatostatin such as D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) and related analogs were designed specifically for antitumor activity. These somatostatin analogs, by virtue of having a wide spectrum of activities appear to inhibit various tumors through multiple mechanisms. Direct antiproliferative actions of somatostatin analogs appear to be mediated by specific receptors located on tumor cells. High affinity binding sites for RC-160 and related analogs have been found in human pancreatic, prostate, breast and ovarian cancers and brain tumors such as meningiomas. In vivo administration of analog RC-160 inhibits the growth of Dunning R-3327 prostate cancers in rats, MXT mammary tumors in mice and BOP-induced ductal pancreatic cancers in hamsters. Combination of microcapsules of RC-160 with [D-Trp6]-LH-RH results in synergistic potentiation of the inhibition of these cancers. Somatostatin analog RC-160 and LH-RH antagonist SB-75 are the object of further experimental studies and clinical trials aimed at the exploration of their inhibitory effects on the processes of malignant growth.

Biological half-lives and organ distribution of tritiated 8-lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin in Brattleboro rats.

The biological half-lives and organ distribution of tritiated 8-lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin were determined in R-Amsterdam rats and in homozygous and heterozygous Brattleboro rats with hereditary central diabetes insipidus. It was found that the biological half-lives of [3H]LVP and [3H]dDAVP in the Brattleboro rats did not differ significantly from that found in the control R-Amsterdam rats. The half-life of [3H]dDAVP proved longer than that of [3H]LVP in all three groups of animals. In the case of [3H]LVP the highest radioactivities were observed in the neurohypophyses, adenohypophyses, and kidneys of both the R-Amsterdam and Brattleboro rats. The accumulation of tritiated material was higher in the small intestine of the Brattleboro rats than in that of the R-Amsterdam animals. In all three groups of rats, [3H]dDAVP was accumulated to the greatest extent in the kidney and the small intestine. The kidney and small intestine contained less radioactivity in homozygous Brattleboro rats than in the controls. There was only a slight radioactivity accumulation in the adenohypophysis and neurohypophysis. From the results it was concluded that the decrease in the rate of enzymatic decomposition may play a role in the increased duration of antidiuretic action of dDAVP. The results have led to the conclusion that the accelerated elimination of vasopressin and its pathologic organ accumulation are probably not involved in the water metabolism disturbance of Brattleboro rats with hereditary diabetes insipidus.

Synthesis of two peptide scorpion toxins and their use to investigate the aortic tissue regulation.

The 37 amino acid residue polypeptides iberiotoxin and charybdotoxin, which contain three disulfide bridges, were chemically synthesized and characterized. The physiological effectiveness of these peptides was tested on rabbit aorta in vitro.

Identification of factor C protein from Streptomyces griseus by microelectrospray mass spectrometry.

Factor C, an extracellular signal protein of cellular differentiation, was studied and significant homology was found to several zinc finger-type regulatory proteins. The complete amino acid sequence, deduced from the gene, that encodes the protein, did not support the hypothesis that this protein might be a zinc finger-type regulatory protein. However, a theoretical single nucleotide insertion in the gene can result in another similarly sized protein containing about 20 His residues, which would be responsible for the high zinc affinity of factor C. The protein sample was reduced, alkylated and then in-gel digested with trypsin. The peptide fragments were then separated by capillary chromatography and identified by microelectrospray mass spectrometry. Peaks of higher intensity were sequenced by tandem mass spectrometry. The identified peptide fragments and the measured molecular mass of factor C protein also confirmed the original sequence of protein, as there was no shift in the open reading frame.

Integration of mass spectrometry into early-phase discovery and development of central nervous system agents.

The early-phase discovery and development of useful central nervous system (CNS) agents present ample opportunities to exploit mass spectrometry and provide detailed compound/mixture characterization, or to make the process faster and/or more economic. Neuropeptide FF antagonists and centrally active thyrotropin-releasing hormone analogues were used as specific examples in this work. We evaluated the characterization of focused libraries of peptide derivatives by electrospray ionization, tandem mass spectrometry and liquid chromatography/tandem mass spectrometry on a quadrupole ion trap and nanoelectrospray on a Fourier transform ion cyclotron resonance mass spectrometer. Immobilized artificial-membrane chromatography was employed as a model to predict/rank new agents against lead compounds for their potential to reach the central nervous system in pharmacologically significant amounts. Measuring brain concentrations in rodents after the intravenous administration of test compounds was used as an in vivo approach, and we took advantage of microdialysis sampling that furnished samples without interfering tissue matrix and afforded the estimation of extracellular concentrations in a localized part of the brain. Overall, making atmospheric-pressure ionization mass spectrometry an integral part of the process has played a major role in increasing throughput, selectivity, specificity and detection sensitivity and thereby providing useful information about the extent or mechanism of transport and metabolic activation/inactivation in early-phase discovery and development of CNS agents.

Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures.

The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats. The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP). The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation. Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration. This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP. The application of B after DA agonists proved ineffective. The results indicate that NH hormone production can be directly influenced by the DA-ergic system. The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Effects of beta-endorphin2-9 on arginine-8-vasopressin and oxytocin levels in hypothalamic and limbic brain regions.

Immunoreactive arginine-8-vasopressin (AVP) and oxytocin (OXT) were measured in rat hypothalamic and limbic brain regions after the intracerebroventricular administration of beta-endorphin fragment 2-9 (beta E2-9). The peptide decreased the AVP content of the hippocampus and the OXT levels in the septum and amygdala. The present data favor the view that beta E2-9 interacts with limbic AVP- and OXT-systems.