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.

Head-to head comparison of mGlu1 and mGlu5 receptor activation in chronic treatment of absence epilepsy in WAG/Rij rats.

Acute treatment with positive allosteric modulators (PAMs) of mGlu1 and mGlu5 metabotropic glutamate receptors (RO0711401 and VU0360172, respectively) reduces the incidence of spike-and wave discharges in the WAG/Rij rat model of absence epilepsy. However, from the therapeutic standpoint, it was important to establish whether tolerance developed to the action of these drugs. We administered either VU0360172 (3 mg/kg, s.c.) or RO0711401 (10 mg/kg, s.c.) to WAG/Rij rats twice daily for ten days. VU0360172 maintained its activity during the treatment, whereas rats developed tolerance to RO0711401 since the 3rd day of treatment and were still refractory to the drug two days after treatment withdrawal. In response to VU0360172, expression of mGlu5 receptors increased in the thalamus of WAG/Rij rats after 1 day of treatment, and remained elevated afterwards. VU0360172 also enhanced mGlu5 receptor expression in the cortex after 8 days of treatment without changing the expression of mGlu1a receptors. Treatment with RO0711401 enhanced the expression of both mGlu1a and mGlu5 receptors in the thalamus and cortex of WAG/Rij rats after 3-8 days of treatment. These data were different from those obtained in non-epileptic rats, in which repeated injections of RO0711401 and VU0360172 down-regulated the expression of mGlu1a and mGlu5 receptors. Levels of VU0360172 in the thalamus and cortex remained unaltered during the treatment, whereas levels of RO0711401 were reduced in the cortex at day 8 of treatment. These findings suggest that mGlu5 receptor PAMs are potential candidates for the treatment of absence epilepsy in humans.

The SNPs in the human genetic blueprint era.

The analysis of human genetic variability can lead to the comprehension of medical issues and to the development of personalized therapeutic protocols. Single nucleotide polymorphisms, are the most common type of human genetic variation and have been associated to disease development and phenotype forecasting. The recent technologies for DNA sequencing and bioinformatic analysis are now giving the opportunity to develop new diagnostic and prevention approaches also through health promotion protocols. The genetic data management is at the same time underlining technical limitations and old ethical issues.

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.  

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.

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.

Alpha-defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection.

The alpha-defensin genes promoter regions contain a putative nuclear factors of activated T cells (NFAT)-binding site and it is known that hepatitis C virus (HCV) core protein activates the interleukin (IL)-2 gene transcription through the NFAT pathway. The aims of this study were to investigate if HCV affects the alpha-defensin expression in peripheral human mononuclear cells (PBMCs) and to evaluate the existence of a correlation between alpha-defensins and liver damage in patients with chronic hepatitis C. Ninety patients with chronic hepatitis C, 30 with chronic hepatitis B and 25 healthy controls were enrolled. Alpha-defensins were identified and quantified in PBMCs by mass spectrometry, enzyme-linked immunosorbent assay, antibacterial activity and mRNA levels. PBMCs from three patients and controls were stimulated with HCV core protein, hepatitis B virus core antigen and the alpha-defensin mRNAs level was quantified. We found that HCV core protein activates in vitro the alpha-defensin transcription. Alpha-defensin levels in patients with chronic hepatitis C (mean +/- SD = 1.103 +/- 0.765 ng/10(6) cells), chronic hepatitis B (0.53 +/- 0.15) and healthy controls (0.217 +/- 0.09) resulted significantly different (P < 0.001). In patients with chronic hepatitis C, the alpha-defensin levels and antibacterial activity correlate with the liver fibrosis. Our data suggest that HCV induces alpha-defensin expression. The high linear correlation of alpha-defensin levels with advancing fibrosis makes the measure of these peptides a reliable marker to evaluate fibrosis stage.

Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles.

Temporins are a novel family of small (10-13 residues) cationic antimicrobial peptides recently isolated from the skin of the European red frog Rana temporaria. Although recently acquired evidence shows that temporins have the potential to kill bacteria by permeabilizing the cytoplasmic membrane, the molecular mechanisms of membrane selectivity and permeabilization are largely unknown. In this study, it was found that temporins cause the release of fluorescent markers entrapped in phosphatidylcholine liposomes in a manner that depends significantly on the size of the solute. Temporins were also shown to lack a detergent-like effect on lipid vesicles, indicating that marker leakage caused by these peptides is not due to total membrane disruption but to perturbation of bilayer organization on a local scale. Binding of temporins to liposomes did lead to a small increase in lipid hydrocarbon chain mobility, as revealed by EPR spectroscopy of nitroxide-labeled fatty acids incorporated in the bilayer. Reference experiments were conducted using the bee venom peptide melittin, whose properties and behavior in natural and model membrane systems are well known. Our findings for temporins are discussed in relation to the models proposed to date to account for the action of antimicrobial peptides on membranes.

Involvement of Rel factors in the expression of antimicrobial peptide genes in amphibia.

Genes coding for antimicrobial peptides in amphibia reveal a remarkably high number of structural motifs for response elements, previously identified in the genes of insect antimicrobial peptides and in those of the mammalian acute phase response. This study focuses on the functional analysis of the bombinin gene promoter in a Drosophila blood cell line, and the identification of kappaB-binding factors in skin secretions of the frog Bombina orientalis. Transfection experiments demonstrated that the bombinin gene promoter was activated in a lipopolysaccharide-dependent manner, and that insect Rel factors target specific sequences in the amphibian gene promoter. After bathing frogs in bacteria, their skin secretions contained kappaB-specific binding complexes, indicating that Rel factors are crucial components in the response against gram-negative bacteria in this species. These results suggest that a common ancestral control mechanism governs the expression of the first line host-defence from insects to vertebrates.

Structure-function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions.

Skin secretions of amphibia of the Bombina genus contain two families of antimicrobial peptides, the bombinins (bombinin-like peptides) and the bombinins H (H for hydrophobic and hemolytic). The latter family includes a number of peptides containing a D-amino acid in the second position, in addition to their corresponding all L-isomers. The antimicrobial activity of three pairs of bombinin H isomers, H2/H4, H6/H7 and GH-1D/GH-1L, has been investigated. The first two pairs of peptides were actually isolated from the secretion, whereas the third was synthesized according to the sequence deduced from a gene coding for a bombinin-like peptide in Bombina orientalis.

Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7.

The structure of a gene coding for bombinin-like peptides (BLP) in Bombina orientalis was determined. It comprises two exons separated by a 1337 bp intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for BLP-7 and a bombinin H-type peptide (GH-2). The promoter region contains putative recognition sites for nuclear factors, such as NF-IL6 and NF-kappaB. The analysis of the structure of this gene, compared with that of the previously reported BLP-3 gene sequence, suggests the occurrence of a gene duplication event, rather than an alternative splicing mechanism, which leads to the generation of both inter- and intra-families variability in this class of cytolytic peptides. Furthermore, chromosome walking analysis indicates that this gene family is not densely clustered.

Structure-function relationships of temporins, small antimicrobial peptides from amphibian skin.

Temporins, antimicrobial peptides of 10-13 residues, were isolated from secretions of Rana temporaria [Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem. 242, 788-792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis.

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.

Expression and activity of cyclic and linear analogues of esculentin-1, an anti-microbial peptide from amphibian skin.

Esculentin-1 is a potent anti-microbial peptide present in minute amounts in skin secretions of Rana esculenta. It contains 46 amino-acid residues and a C-terminal disulfide bridge. We have explored the possibility of producing analogues of this peptide by recombinant expression in Escherichia coli of a fusion protein which is sequestered in inclusion bodies. The peptide of interest has been inserted at the N-terminus of the protein, from which it can be released by cyanogen bromide cleavage. The anti-microbial activities of the recombinant peptide as well as that of a mutant linear form devoid of the disulfide bridge are presented. The recombinant analogues retain the biological activity of the natural peptide, as tested with an inhibition zone assay against a variety of microorganisms. However, experiments on the rate of bacterial killing show that gram-negative bacteria are more sensitive to the peptides than the gram-positive bacterium, the effect of the cyclic peptide being in all cases faster than that of the linear molecule. Moreover, the activity against gram-negative bacteria for both peptides is not affected by salts, whereas the activity against Staphylococcus aureus is lost at high salt concentration.

Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues.

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756-10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.

Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora.

Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora. Their skin glands produce a secretion containing 20-30 different peptides, some antimicrobial some neurotrophic. As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short periods of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo-3, we isolated a spontaneous and stable mutant (Bo-3N), resistant to nalidixic acid, here used to follow the host-microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequenced and cloned. We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IkappaBalpha was up-regulated. Experimental mouth infections with 20 million cells of A. hydrophila Bo-3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 microl 'saliva'. An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min. Using different enzyme-linked immunosorbent assays (ELISAs) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 10 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one-third of that of the non-bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila. The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate. One such function is involved in the control of the natural flora.

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.