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.

Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2.

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

Kynurenine metabolism in multiple sclerosis.

Objective--Excitatory amino acid receptors are involved in the normal physiology of the brain, and may play a role in the pathogenesis of neurological disorders such as Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, etc. It has been demonstrated that the blockade of one of these receptors ameliorates the symptoms of experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS). In a recent study, a decreased level of kynurenic acid was found in the cerebrospinal fluid of patients with MS. The only known endogenous excitotoxin receptor antagonist is the tryptophan metabolite kynurenic acid. Another metabolite is quinolinic acid, which exerts different action: it is an excitotoxin receptor agonist. The ratio of these two metabolites is determined by the activities of kynurenine aminotransferase I and II (KAT I and KAT II). In this study, we measured the activities of these enzymes and the concentration of kynurenic acid in the red blood cells (RBC) and in the plasma of patients with MS. KAT activities were detected both in the RBC and in the plasma. As compared with the control subjects, the KAT I and KAT II activities were significantly higher in the RBC of the patients. The concentration of kynurenic acid is elevated in the plasma of MS patients, and there is a tendency to an elevation in the RBC. These changes may indicate a compensatory protective mechanism against excitatory neurotoxic effects. Our data demonstrate the involvement of the kynurenine system in the pathogenesis of MS, which may predict a novel therapeutic intervention.

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.

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.

Identification of protein substrates for transglutaminase in Caenorhabditis elegans.

Transglutaminase-dependent cross-linking of proteins leads to protein polymerisation that confers stability as well as resistance to mechanical disruption and chemical attack. Various transglutaminases have been implicated in a wide range of biological phenomena occurring in both extracellular and intracellular compartments, but further clarification of the physiological role of these enzymes requires identification of possible substrate molecules. Here we report the detection, purification, and identification of two proteins, enolase and ATP synthase alpha subunit as glutamine donor protein substrates for the transglutaminase of the nematode Caenorhabditis elegans.

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.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.

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.

Electrospray mass spectrometry of phosphoramidites, a group of acid-labile compounds

In contemporary solid-phase synthesis of oligonucleotides, phosphoramidites containing O-beta-cyanoethyl and N,N -diisopropyl groups are the most widespread monomer units. The N,N -diisopropyl phosphoramidite group can be activated by mild acidic treatment and then it easily reacts with nucleophiles (alcohols, water, etc.) to furnish the required phosphodiester linkage efficiently and cleanly. Owing to these properties, these compounds cannot be investigated using classical electrospray ionization. Their mass spectometric analysis is further hampered by the fact that they are often transiently protected with acid-sensitive groups (4, 4'-dimethoxytrityl, 4-monomethoxytrityl or trityl), which give intense signals in the spectra. Nanoelectrospray measurements from non-aqueous solvents (e.g. acetonitrile, methanol, tetrahydrofuran) were carried out in order to eliminate the nucleophilic water. Different types of alkali metal salts were used to form adduct ions. Among these salts, lithium chloride was found to be the most suitable for the analysis of amidites. Fairly abundant [M+Li](+) and [M+Cl](-) ions are formed in the positive and negative ion mode, respectively. These ions represent the base peaks in most cases whereas the intensities of the peaks corresponding to the protecting group are reduced by approximately 20%. This method is a powerful tool for the mass spectrometric identification of phosphoramidites. Copyright 1999 John Wiley & Sons, Ltd.

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.

Identification of oxytocin and vasopressin from neurohypophyseal cell culture.

Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Capillary chromatography/microelectrospray mass spectrometry used for the identification of putative cyclin-dependent kinase inhibitory protein in Medicago.

A home-built capillary chromatography/microelectrospray system was used for peptide mass mapping of a putative cyclin-dependent kinase inhibitor protein with molecular mass of 8.5 kDa. The masses of identified tryptic fragments were then input to a protein database search routine. Daughter ion scans were done only on the most abundant tryptic fragments. On the basis of protein database search results and tandem mass spectrometric measurements the bioactive protein was identified as ubiquitin.

High-performance separation methods in the analysis of a new peptide family: the galanins.

An analytical investigation of a new peptide family, the human galanins and their fragments, was carried out by reversed-phase HPLC, capillary zone electrophoresis (CZE) at different pH values and micellar electrokinetic capillary chromatography (MECC) in phosphate-borate-sodium dodecyl sulphate buffer. None of the methods seems to be superior to the others. The complementary nature of the electrophoretic methods is obvious when the profiles of peptides are compared; impurities not separated by HPLC are separated by CZE or MECC and vice versa. With these three different separation methods, a more complex analytical control of the synthetic work can be achieved.

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.

Evaluation of biological activities of new LH-RH antagonists (T-series) in male and female rats.

A series of new highly potent LH-RH antagonists (T-series) has been synthesized in our laboratory. Among these analogs, antagonists [Ac-D-Nal(2), D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Car)2)6, D-Ala10]LH-RH (T-140); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Ac)2)6, D-Ala10]LH-RH (T-148); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(For)2)6, D-Ala10]LH-RH (T-151) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2bu(For)2)6, D-Ala10]LH-RH (T-159) were the most powerful. Antagonists T-140, T-148 and T-151 produced a complete blockade of ovulation in normal cycling rats at a dose of 1.5 micrograms/rat and antagonist T-159 at a dose of only 0.75 micrograms/rat. The inhibitory effects of compounds T-148, T-151 and T-159 on gonadotropin and sex steroid secretion were investigated in male and female rats. To determine their effect on LH levels in castrated male and ovariectomized female rats, T-148, T-151 and T-159 were injected subcutaneously in doses of 0.625 and 2.5 micrograms/rat. Blood samples were taken at different intervals for 48 h. All three compounds at either dose caused a significant (P < 0.01) decrease in LH levels for more than 6 h. Significant (P < 0.01) inhibition of LH lasted for more than 24 h following a dose of 2.5 micrograms sc of all 3 antagonists in both male and female rats. Serum FSH levels were also suppressed significantly for more than 48 h in castrated male rats by all three antagonists at a dose of 5 micrograms/rat sc.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-chain analogs of luteinizing hormone-releasing hormone containing cytotoxic moieties.

Five hexapeptide and heptapeptide analogs of luteinizing hormone-releasing hormone (LH-RH) were synthesized for use as carriers for cytotoxic compounds. These short analogs were expected to enhance target selectivity of the antineoplastic agents linked to them. Native LH-RH-(3-9) and LH-RH-(4-9) containing D-lysine and D-ornithine at position 6 were amidated with ethylamine and acylated on the N terminus. The receptor-binding affinity of one hexapeptide carrier AJ-41 (Ac-Ser-Tyr-D-Lys-Leu-Arg-Pro-NH-Et) to human breast cancer cell membranes was similar to that of [D-Trp6]LH-RH. Alkylating nitrogen mustards (melphalan, Ac-melphalan), anthraquinone derivatives including anticancer antibiotic doxorubicin, antimetabolite (methotrexate), and cisplatin-like platinum complex were linked to these peptides through their omega-amino group at position 6. The hybrid molecules showed no LH-RH agonistic activity in vitro and in vivo but had nontypical antagonistic effects on pituitary cells in vitro at the doses tested. These analogs showed a wide range of receptor-binding affinities to rat pituitaries and cell membranes of human breast cancer and rat Dunning prostate cancer. Several of these conjugates exerted some cytotoxic effects on MCF-7 breast cancer cell line.

Potent agonists of growth hormone-releasing hormone. II.

Analogs of the 29-amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) or Lys-NH2 in position 29 have been synthesized by the solid-phase method, purified, and tested in vitro. Except for one peptide, all analogs contained desaminotyrosine (Dat) in position 1. All contained Nle27 in order to avoid oxidation of Met27. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27 and/or 28. Analogs [Dat1, Ala15, Nle27, Asn28]GH-RH(1-28)Agm (II, [Asn28]-Mz-2-51); [Dat1, Ala15, D-Lys21, Nle27, Asn28]GH-RH(1-28)Agm (III, MZ-3-125); and [Dat1, D-Asn8, Ala15, D-Lys21, Nl27, Asn28]GH-RH(1-28)Agm(IV, MZ-3-129) were 5.7, 2.8, and 3.9 times more potent in vitro, respectively, than GH-RH(1-29)NH2. However, if we compare the potencies of peptides II and III (analogs of the bovine sequence) with those of the analogs of human GH-RH (XII and XIII) [Dat1, Ala15, Nle27]GH-RH(1-28)Agm; [Dat1, Ala15, D-Lys21, Nle27]GH-RH(1-28)Agm, respectively, the GH-releasing potency was decreased by 50% and 33%, respectively, by the incorporation of Asn28. Our studies indicate that Lys-NH2 at the C-terminus of GH-RH(1-29) and/or beta-Ala, GABA (gamma-aminobutyric acid), and Phe in position 15 are disadvantageous, but potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.