A grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) genetic map integrating the position of 139 expressed genes

Grapevine molecular maps based on microsatellites, AFLP and RAPD markers are now available. SSRs are essential to allow cross-talks between maps, thus upgrading any growing grapevine maps. In this work, single nucleotide polymorphisms (SNPs) were developed from coding sequences and from unique BAC-end sequences, and nested in a SSR framework map of grapevine. Genes participating to flavonoids metabolism and defence, and signal transduction pathways related genes were also considered. Primer pairs for 351 loci were developed from ESTs present on public databases and screened for polymorphism in the “Merzling” (a complex genotype Freiburg 993–60 derived from multiple crosses also involving wild Vitis species) × Vitis vinifera (cv. Teroldego) cross population. In total 138 SNPs, 108 SSR markers and a phenotypic trait (berry colour) were mapped in 19 major linkage groups of the consensus map. In specific cases, ESTs with putatively related functions mapped near QTLs previously identified for resistance and berry ripening. Genes related to anthocyanin metabolism mapped in different linkage groups. A myb gene, which has been correlated with anthocyanin biosynthesis, cosegregated with berry colour on linkage group 2. The possibility of associating candidate genes to known position of QTL is discussed for this plant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Marzia Salmaso and Giulia Malacarne contributed equally to the present work.     

The GPI-anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.     

Independent influence of insulin, catecholamines, and thyroid hormones on metabolic syndrome

The objective of this study was to examine whether metabolic syndrome, defined according to adult treatment panel III criteria, is associated with insulin, catecholamines, and thyroid hormones, independently of age and gender. A cohort of 651 euthyroid overweight and obese patients, 440 women and 211 men, aged 18-68 years, were examined. Central fat accumulation (indirectly measured by waist circumference), fasting thyroid-stimulating hormone (TSH), FT(3), FT(4), insulin, glucose, and lipid (cholesterol, HDL-cholesterol, and triglyceride) serum concentrations, 24-h urinary catecholamines, and the level of insulin resistance (estimated by homeostasis model assessment for insulin resistance (HOMA(IR))) were measured. Patients with metabolic syndrome showed higher insulin (P < 0.001) and FT(3) (P < 0.001) serum levels and higher 24-h urinary noradrenaline (P < 0.001) than subjects without this syndrome. The number of metabolic syndrome parameters was directly associated with insulin (P < 0.001) and FT(3) (P < 0.05) serum levels, and with 24-h urinary noradrenaline (P < 0.001) in the whole population. When a multiple regression analysis was performed with the metabolic syndrome as the dependent variable, and age, gender, and insulin, and TSH, FT(3), FT(4) serum levels, and 24-h urinary noradrenaline and adrenaline as independent variables, the metabolic syndrome maintained an independent positive association with age (P < 0.001), male sex (P < 0.001), insulin (P < 0.001), and 24-h urinary noradrenaline (P < 0.001). In conclusion, this study suggests that insulin and noradrenaline cooperate independently to the development of the metabolic syndrome.     

The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output.     

Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics

The L-type Ca current (I_Ca,L), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I_Ca,L can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I_Ca,L, modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM₁₂₃₄) in rabbit ventricular myocytes to inhibit Ca-dependent I_Ca,L inactivation, combined with the ATP-sensitive K current agonist pinacidil or I_Ca,L blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I_Ca,L blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca_i transient alternans but maintained a normal Ca_i transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca_i transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I_Ca,L can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.     

Hot-spot residues at the E9/Im9 interface help binding via different mechanisms

Protein-protein association involves many interface interactions, but they do not contribute equally. Ala scanning experiments reveal that only a few mutations significantly lower binding affinity. These key residues, which appear to drive protein-protein association, are called hot-spot residues. Molecular dynamics simulations of the Colicin E9/Im9 complex show Im9 Glu41 and Im9 Ser50, both hot-spots, bind via different mechanisms. The results suggest that Im9 Ser50 restricts Glu41 in a conformation auspicious for salt-bridge formation across the interface. This type of model may be helpful in engineering hot-spot clusters at protein-protein interfaces and, consequently, the design of specificity. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 916-920, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Effects of nomegestrol acetate administration on central and peripheral beta-endorphin and allopregnanolone in ovx rats

The aim of this study was to investigate the effects of nomegestrol acetate (NOMAc) on the central nervous system by analyzing the neurosteroid allopregnanolone and the opioid beta-endorphin (beta-endorphin). 104 Wistar female rats were used in this study; one group of fertile and one group of ovariectomized rats were used as control. The others were ovariectomized and they underwent a 2-week oral treatment of NOMAc (0.05, 0.1, 0.2, 0.5, 1mg/kg/day), alone or with 0.05 mg/kg/day of estradiol valerate (E2V). Allopregnanolone and beta-endorphin were assessed in different brain areas and in circulation. Ovariectomy decreased allopregnanolone anywhere except in the adrenal gland and E2V reversed the effects of ovariectomy. 0.5 and 1mg/kg/day of NOMAc increased allopregnanolone levels in hippocampus. Combined administration of 1mg/kg/day of NOMAc plus E2V induced a further increase of allopregnanolone levels in hippocampus, hypothalamus, and anterior pituitary. NOMAc (1mg/kg/day) decreased the adrenal content of allopregnanolone, both by itself and associated with E2V. NOMAc increased hippocampal and hypothalamic content of beta-endorphin at the highest doses, and it increased positively E2V action, at 1mg/kg/day, also in anterior pituitary and plasma. These findings reinforce the clinical data regarding the capability of NOMAc to modulate the pathways involved in mood and behaviour. In fact, due to the NOMAc action on hippocampus, hypothalamus, and anterior pituitary, our results highlight the selectivity of NOMAc on part of the limbic system and the anterior pituitary, regarding both allopregnanolone and beta-endorphin.