Genotype-phenotype correlations in 5-fluorouracil metabolism: a candidate DPYD haplotype to improve toxicity prediction.

5-Fluorouracil is among the most widely used anticancer drug, but a fraction of treated patients develop severe toxicity, with potentially lethal injuries. The predictive power of the available pretreatment assays, used to identify patients at risk of severe toxicity, needs improvements. This study aimed to correlate a phenotypic marker of 5-fluorouracil metabolism (the individual degradation rate of 5-fluorouracil-5-FUDR) with 15 functional polymorphisms in the dihydropyrimidine dehydrogenase gene (DPYD). Single SNP (single-nucleotide polymorphism) analysis revealed that the SNPs rs1801160, rs1801265, rs2297595 and rs3918290 (splice site variant IVS14+1G>A) were significantly associated with a decreased value of 5-FUDR, and the rs3918290 causing the larger decrease. Multi-SNP analysis showed that a three-SNP haplotype (Hap7) involving rs1801160, rs1801265 and rs2297595 causes a marked decrease in 5-FUDR, comparable to that caused by the splice site variant rs3918290, which is the main pharmacogenetic marker associated with severe fluorouracil toxicity. The similar effect played by Hap7 and by the splice site variant rs3918290 upon individual 5-FUDR suggests that Hap7 could also represent a similar determinant of fluorouracil toxicity. Haplotype assessment could improve the predictive value of DPYD genetic markers aimed at the pre-emptive identification of patients at risk of severe 5-fluorouracil toxicity.The Pharmacogenomics Journal advance online publication, 28 July 2015; doi:10.1038/tpj.2015.56.

Cinnabarinic acid, an endogenous metabolite of the kynurenine pathway, activates type 4 metabotropic glutamate receptors.

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

Gene variants with suicidal risk in a sample of subjects with chronic migraine and affective temperamental dysregulation.

BACKGROUND: Risk factors for suicide are at least partially heritable and functional polymorphisms of targeted genes have been suggested to be implicated in the pathogenesis of this phenomenon. However, other studies examining the association between specific gene variants and suicide revealed inconsistent findings. We aims to evaluate the possible association between MAO-A3, CYP1A2*1F and GNB3 gene variants, hopelessness and suicidal risk in a sample of subjects with chronic migraine and affective temperamental dysregulation. METHODS: 56 women were genotyped for MAO-A3, CYP1A2*1F and GNB3 gene variants. Participants were also assessed using Beck Hopelessness Scale (BHS), the Temperament Evaluation of the Memphis, Pisa, Paris and San Diego-Autoquestionnaire (TEMPS-A), and the Suicidal History Self-Rating Screening Scale (SHSS). RESULTS: Patients with higher total scores on affective dysregulated temperaments are more likely to have higher BHS (11.27+/=5.54 vs. 5.73+/=3.81; t19.20 = -3.57; p < 0.01) and higher SHSS total scores (4.79+/=3.31 vs. 1.05±2.31; t17.74 = -3.90; p < 0.001) than those with lower total scores. 67% of patients in the dysregulated group has BHS total scores >= 9 indicating high levels of hopelessness. No association was found between MAO-A3, CYP1A2*1F and GNB3 gene variants and suicidal risk as assessed by BHS and SHSS. CONCLUSIONS: This study did not sustain the association between MAO-A3, CYP1A2*1F and GNB3 gene variants and increased suicidal risk in patients with chronic migraine and affective temperamental dysregulation. Further studies investigating the gene-environment interaction or focusing on other genetic risk factors involved in suicidal behaviour are needed.  

Proteomic profiles in hyperandrogenic syndromes.

BACKGROUND: Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. METHODS: Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. RESULTS: Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. CONCLUSION: Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p'-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, alpha-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-beta isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

Proteomic analysis of peripheral T lymphocytes, suitable circulating biosensors of strictly related diseases.

T lymphocytes and/or their subpopulations from peripheral blood may represent molecular sensors to be used for the evaluation of gene expression modification in physiological and pathological conditions, providing a unique and easily available biological model for integrated studies of gene expression in humans. In this study, a proteomic approach was applied to evaluate the association between changes in T cell protein expression patterns and specific diseased conditions. In particular, two hyperandrogenic syndromes were studied, sharing many clinical and biochemical signs: polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH). Comparison of proteomic maps of T lymphocytes derived from patients affected by PCOS or CAH with those derived from healthy subjects showed that 14 proteins are expressed differentially in both PCOS and CAH, 15 exclusively in PCOS and 35 exclusively in CAH. Seventeen of these proteins have been identified by mass spectrometry analysis. Furthermore, proteomic data mining by hierarchical clustering was performed, highlighting T lymphocytes competence as a living biosensor system.

Purification and characterization of bioactive peptides from skin extracts of Rana esculenta.

The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Arg9-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.

Amphibian skin: a promising resource for antimicrobial peptides.

Amphibian skin is a rich source of biologically active compounds that are assumed to have diverse physiological and defence functions. In addition to the range of pharmacologically active peptides present, some of which have mammalian homologues, skin secretions contain a broad spectrum of antimicrobial peptides. As yet, such peptides from only a few species have been studied, and screening of other species is expected to yield further new antimicrobial activities. Natural antimicrobial peptides isolated from amphibian skin could provide lead structures for either the chemical, or rDNA synthesis of novel antimicrobials.

Novel antimicrobial peptides from skin secretion of the European frog Rana esculenta.

Three antimicrobial peptides were isolated from skin secretion of the European frog, Rana esculenta. Two of them show similarity to brevinin-1 and brevinin-2, respectively, two antimicrobial peptides recently isolated from a Japanese frog [Morikawa, N., Hagiwara, K. and Nakajima, T. (1992) Biochem. Biophys. Res. Commun. 189, 184-190]. The third one, named esculentin, is 46 residues long and represents a different type of peptide. All these peptides have as a common motif an intramolecular disulfide bridge located at the COOH-terminal end. The peptides from R. esculenta show distinctive antibacterial activity against representative Gram-negative and Gram-positive bacterial species. In particular, esculentin is the most active against Staphylococcus aureus, and has a much lower hemolytic activity.

Gene-encoded peptide antibiotics and innate immunity. Do 'animalcules' have defence budgets?

Gene-encoded peptide antibiotics have been isolated from plants, animals and microbes. Their protective role has been related to innate immunity, which has gradually become accepted across the biomedical community. The evidence for the immune function of peptide antibiotics has been convincingly demonstrated by a combination of both in vitro and in vivo data for plants and insects, but for vertebrates in vivo data are scarce. Using frogs as model systems, it has been shown that the genes for antibacterial peptides are down-regulated by glucocorticoids, while IkappaB alpha is clearly up-regulated. Experimental infections with frog bacteria have shown that the normal capacity to control the natural flora is lost after treatment with glucocorticoids. A low-specificity immune mechanism is cost-effective, something that may have been of importance during animal evolution.

The primary structure of ornithine aminotransferase. Identification of active-site sequence and site of post-translational proteolysis.

Tentative assignments of functional residues in rat liver mitochondrial ornithine aminotransferase have recently been made using the amino acid sequence deduced from a cDNA clone [(1985) J.Biol.Chem. 260, 12993-12997]. Partial sequences obtained using the pure mature protein demonstrate that one of these assignments, that of Lys 292 as the residue that binds the coenzyme pyridoxal phosphate, is correct. However, the identification of the Glu 34-Gln 35 bond as the site of post-translational proteolysis is in error. This cleavage occurs instead at Ala 25-Thr 26.

Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-kappaB and NF-IL6 binding sites in its promoter.

The sequence of a gene from Bombina orientalis was determined which codes for antibacterial peptides. The gene comprises two exons separated by a large intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for two identical bombinins and one bombinin H. The promoter region of the bombinin gene contains putative recognition sites for nuclear factors, such as NFkappaB and NF-IL6. In vivo experiments on B. orientalis have shown that a short contact with bacteria is sufficient to induce a marked increase in the amount of antibacterial peptides in the skin secretion of frogs. This increase was suppressed by pretreatment with glucocorticoids. In the latter case, a significant increase of I kappaB alpha in the secretion is also detectable.

Primary structure of porcine Cu,Zn superoxide dismutase.

The complete amino acid sequence of Cu,Zn superoxide dismutase from porcine erythrocytes has been determined. Comparison of the sequence with that of the bovine enzyme shows an overall high degree of homology with conservation of the crucial residues and the presence of two regions prone to variation. In one of these hypervariable regions the insertion of one residue with respect to the bovine enzyme and evidence of structural microheterogeneity has been observed. On the basis of the three-dimensional structure of the bovine enzyme no obvious relationship is apparent between a specific amino acid replacement and the unique pH-dependence pattern of the activity of the porcine enzyme.

Identification and characterization of two dermorphins from skin extracts of the Amazonian frog Phyllomedusa bicolor.

Skin extracts of South American hylid frogs of the subfamily Phyllomedusinae contain dermorphins and deltorphins, opioid heptapeptides highly selective for either mu or delta receptors. In all these peptides, a D-amino acid is present in the second position. The structure of the precursors for Ala-deltorphins was recently deduced from cloned cDNAs derived from skin of Phyllomedusa bicolor (Richter et al. (1990) Proc. Natl. Acad. Sci. USA 87, 4836-4839). From the amino acid sequence of these precursors, the existence of three peptides related to dermorphin could be predicted. From methanol extracts of skin of Ph. bicolor we have isolated two of these peptides, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH. The biological activity of these new dermorphins and their amidated counterparts is presented.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin.

Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-kappaB is an important transcription factor for many immunity-related mammalian proteins and also for insect immune genes. The activity of NF-kappaB is regulated by the interaction with an inhibitor, I kappaB. It was recently demonstrated that glucocorticoids induce the synthesis of I kappaB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-kappaB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of I kappaB alpha. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-kappaB-regulated transcription.

Rohdei-litorin: a new peptide from the skin of Phyllomedusa rohdei.

The bombesin-litorin family of peptides is characterized by the common amino acid sequence-Gly-His-X-Met-NH2 at the C-terminus, where X is a hydrophobic or aromatic residue. A new member of this family, rohdei-litorin, has been isolated from amphibian skin and its structure shown to be: Glp-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2. This new peptide displayed a greater affinity than other members of the family for rat urinary bladder receptors. A litorin-like peptide, with high affinity for this kind of receptor, has already been described in mammalian spinal cord and named neuromedin B. Rohdei-litorin shares with neuromedin B the entire C-terminal octrapeptide and may be considered the amphibian counterpart of this mammalian neuropeptide.

The structure of syringomycins A1, E and G.

By a combination of 1D and 2D 1H- and 13C-NMR, FAB-MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser-Ser-Dab-Dab-Arg-Phe-Dhb-4(Cl)Thr-3(OH)Asp with the beta-carboxy group of the C-terminal residue closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acylated by 3-hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3-hydroxydecanoic and 3-hydroxytetradecanoic acid.

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae.

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.

Characterization and analysis of D-amino acids.

The need to screen a large number of natural extracts, with the aim of detecting D-amino acids or isolating and characterizing peptides containing them, has stimulated the development of novel and improved procedures for the analysis of amino acid enantiomeric mixtures, with special attention paid to automation. Different methods for the analysis of D-amino acids are described and discussed.

Occurrence and function of D-amino acid-containing peptides and proteins: antimicrobial peptides.

Antimicrobial peptides are widely distributed in living organisms, where they represent a constitutive defence system acting as a first line of response against infections. The number of such peptides discovered has increased rapidly in the last few years, and more than 100 have been described from different sources. So far, antimicrobial peptides containing a D-amino acid have only been found in the skin secretions of frogs belonging to the genus Bombina. In the second position of the sequence of the mature peptides either D-alloisoleucine or D-leucine were detected. The D-amino acids are derived from the corresponding L forms by an as yet unknown posttranslational reaction.