Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage.

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC(-/-)) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC(-/-) mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC(-/-) mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC(-/-) mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC(-/-) mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury.

Elements of functional genital asymmetry in the cow.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

Spanish human proteome project: dissection of chromosome 16.

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

Brain-derived neurotrophic factor G196A polymorphism and clinical features in Parkinson's disease.

OBJECTIVES: Parkinson's disease (PD) is characterized by the dopaminergic neuronal death in substantia nigra, and genetic factors appear to be involved in the pathophysiology of this disease. Brain-derived neurotrophic factor (BDNF) is widely expressed in the central nervous system and is necessary for the survival of dopaminergic neurons in substantia nigra. G196A, a common polymorphism of the BDNF gene, not only affects cognitive and motor processes, but also is associated with various psychiatric disorders. We evaluated whether G196A polymorphism is associated with PD and/or modifies clinical manifestations in PD patients. METHODS: We included 193 PD patients and 300 control subjects. G196A polymorphism was screened by restriction fragment length polymorphism analysis. Clinical features of each patient were examined in detail. The possible association between genotype and clinical characteristics were determined by bivariate and multivariate analyses. RESULTS: The distribution of G196A allele and genotype frequency was similar between PD and control subjects. Clinical characteristics, including Hoehn-Yahr stage, motor symptoms, non-motor symptoms (depression, cognitive dysfunction, psychiatric dysfunctions, and sleep behavior disorder), and dyskinesias, were not associated with this polymorphism. CONCLUSIONS: G196A polymorphism is not a risk factor for PD and does not seem to modify clinical features in PD patients studied here.

Prevalence and clinical features of LRRK2 mutations in patients with Parkinson's disease in southern Spain.

BACKGROUND AND PURPOSE: Mutations in leucine-rich repeat kinase 2 (LRRK2) gene are associated with both familial and idiopathic Parkinson's disease (PD), whereas mutations in PARK2 (PARKIN) gene result in early onset recessive PD. Here, the objectives were to determine the frequency of LRRK2 G2019S and R1441G mutations in a PD population from southern Spain; to search for LRRK2 mutations in familial PD cases and to study the effect of PARKIN mutations on clinical features of LRRK2-associated; PD. METHODS: We included 187 PD patients (172 idiopathic, 15 familial) and 287 control subjects from southern Spain. LRRK2 and PARKIN mutations were screened, and clinical features of LRRK2-associated PD were examined. RESULTS: Three (1.7%) idiopathic PD patients carried the G2019S, whereas another three (1.7%) had the R1441G. A novel polymorphism D1420N was found in two (13.3%) familial PD patients. One G2019S carrier also had a homozygous PARKIN deletion, who had early onset PD with clinical symptoms similar to those with PARKIN-associated PD. The remaining LRRK2-asscociated patients had clinical manifestations similar to those with idiopathic PD. CONCLUSIONS: G2019S and R1441G are common LRRK2 mutations in PD patients in this region. PARKIN mutations override clinical features in LRRK2-associated PD.

Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury.

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Hypermethioninaemia due to methionine adenosyltransferase I/III (MAT I/III) deficiency: diagnosis in an expanded neonatal screening programme.

The Expanded Newborn Screening Program (MS/MS) in the region of Galicia (NW Spain) was initiated in 2000 and includes the measurement of methionine levels in dried blood spots. Between June 2000 and June 2007, 140 818 newborns were analysed, and six cases of persistent hypermethioninaemia were detected: one homocystinuria due to cystathionine ß-synthase (CßS) deficiency, and five methionine adenosyltransferase I/III (MAT I/III) deficiencies. The five cases of MAT I/III deficiency represent an incidence of 1/28 163 newborns. In these five patients, methionine levels in dried blood spots ranged from 50 to 147 µmol/L. At confirmation of the persistence of the hypermethioninaemia in a subsequent plasma sample, plasma methionine concentrations were moderately elevated in 4 of the 5 patients (mean 256 µmol/L), while total homocysteine (tHcy) was normal; the remaining patient showed plasma methionine of 573 µmol/L and tHcy of 22.8 µmol/L. All five patients were heterozygous for the same dominant mutation, R264H in the MAT1A gene. With a diet not exceeding recommended protein requirements for their age, all patients maintained methionine levels below 300 µmol/L. Currently, with a mean of 2.5 years since diagnosis, the patients are asymptomatic and show developmental quotients within the normal range. Our results show a rather high frequency of hypermethioninaemia due to MAT I/III deficiency in the Galician neonatal population, indicating a need for further studies to evaluate the impact of persistent isolated hypermethioninaemia in neonatal screening programmes.

Chemical modification of jute fibers for the production of green-composites.

Natural fiber reinforced composites is an emerging area in polymer science. Fibers derived from annual plants are considered a potential substitute for non-renewable synthetic fibers like glass and carbon fibers. The hydrophilic nature of natural fibers affects negatively its adhesion to hydrophobic polymeric matrices. To improve the compatibility between both components a surface modification has been proposed. The aim of the study is the chemical modification of jute fibers using a fatty acid derivate (oleoyl chloride) to confer hydrophobicity and resistance to biofibers. This reaction was applied in swelling and non-swelling solvents, pyridine and dichloromethane, respectively. The formation of ester groups, resulting from the reaction of oleoyl chloride with hydroxyl group of cellulose were studied by elemental analysis (EA) and Fourier Transform infrared spectroscopy (FTIR). The characterization methods applied has proved the chemical interaction between the cellulosic material and the coupling agent. The extent of the reactions evaluated by elemental analysis was calculated using two ratios. Finally electron microscopy was applied to evaluate the surface changes of cellulose fibers after modification process.

A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients.

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway.

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (C12) in order to study the development of structure on elongation of the polypeptide in solution. We present an extensive biophysical characterization of ten fragments using different conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (1-40) has non-native local hydrophobic clusters, but nevertheless does not bind 8-anilinonaphthalene-1-sulphonate (ANS). Hydrophobic regions in longer fragments become gradually more capable of binding ANS as the chain grows to completion, with a tendency to form native structures. Major changes in secondary structure and accessibility to hydrophobic sites occur in parallel, between (1-40) and (1-53), together with changes in hydrodynamic volume and flexibility. NMR studies of (1-53), the first fragment displaying tertiary interactions, show that a subcore is fully formed and the alpha-helix (residues 12 to 24) is of fluctuating structure. Fragments (1-53) and (1-60) share many properties with molten globule-like structures, with varying degrees or order. Fluorescence properties of the native fold are gradually recovered from fragments (1-60) to full-length C12, together with a decrease in hydrophobic exposure. A small degree of co-operativity of formation of structure appears when residue 60 is added, gradually increasing as residue 62 is added, but a full two-state co-operative transition appears only on addition of Arg62 and Val63. We believe this is the result of correct side-chain packing of the hydrophobic core, capping the major elements of secondary structure in C12 at this late stage, which is probed by the complete recovery of the fluorescence of the unique Trp5. The structures that develop as the polypeptide chain increases in length parallel the structural features present in the nucleus for the folding of intact protein, which develops in the transition state. The folding nucleus consists of much of the helix and the interactions made by Ala16 in the helix with residues in the core, especially with Leu49 and Ile57, with the rest of the structure being formed only very weakly in the transition state.

Rotenone induces aggregation of gamma-tubulin protein and subsequent disorganization of the centrosome: relevance to formation of inclusion bodies and neurodegeneration.

Neurodegenerative disorders are characterized by progressive loss of specific neurons in the central nervous system. Although they have different etiologies and clinical manifestations, most of them share similar histopathologic characteristics such as the presence of inclusion bodies in both neurons and glial cells, which represent intracellular aggregation of misfolded or aberrant proteins. In Parkinson's disease, formation of inclusion bodies has been associated with the aggresome-related process and consequently with the centrosome. However, the significance of the centrosome in the neurodegenerative process remains obscure. In the present study, the morphological and functional changes in the centrosome induced by rotenone, a common insecticide used to produce experimental Parkinsonism, were examined both in vitro and in vivo. Aggregation of gamma-tubulin protein, which is a component of the centrosome matrix and recently identified in Lewy bodies of Parkinson's disease, was observed in primary cultures of mesencephalic cells treated with rotenone. Rotenone-treated neurons and astrocytes showed enlarged and multiple centrosomes. These centrosomes also displayed multiple aggregates of alpha-synuclein protein. Neurons with disorganized centrosomes exhibited neurite retraction and microtubule destabilization, and astrocytes showed disturbances of mitotic spindles. The Golgi apparatus, which is closely related to the centrosome, was dispersed in both rotenone-treated neuronal cells and the substantia nigra of rotenone-treated rats. Our findings suggested that recruitment of abnormal proteins in the centrosome contributed to the formation of inclusion bodies, and that rotenone markedly affected the structure and function of the centrosome with consequent induction of cytoskeleton disturbances, disassembly of the Golgi apparatus and collapse of neuronal cells.

Influence of impaired liver methionine metabolism on the development of vascular disease and inflammation.

Methionine (Met) metabolism involves the sequential formation of S-adenosylmethionine (SAM, the main biological methyl donor), S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hcy can be remethylated to Met or catabolized through the trans-sulfuration pathway. In mammals, as much as 48% of Met metabolism and up to 85% of all transmethylation reactions occur in the liver. These figures underscore the central role played by this organ in Met metabolism. Maintaining the homeostasis of this metabolic cycle has proved to be essential for the preservation of liver function up to the point of preventing its neoplastic transformation. However, an adequate hepatic metabolism of Met is not only important for the liver parenchymal cell. Evidence has accumulated over the past few years supporting the involvement of Met-derived metabolites in the triggering or attenuation of pathological processes with systemic implications. This is best illustrated by the fact that a deteriorated liver function has emerged as a major factor in the development of hyperhomocysteinemia. Elevated plasma levels of Hcy have been related to several disorders including cardiovascular and cerebrovascular diseases. On the other end, liver damage also leads to deficient SAM synthesis. Among the consequences of impaired SAM synthesis in liver tissue are the enhanced production of pro-inflammatory cytokines and mediators. In this review, we will address the mechanisms and consequences of abnormal Met metabolism in liver injury, the systemic implications of such impairment and finally the potential therapeutic interventions.

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes.

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.

How is rat liver S-adenosylmethionine synthetase regulated?

The in vivo regulation of S-adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed.

Analysis of the 5' non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme.

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The Mr of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl-Sepharose column. The length of the mRNA 5' non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver.

Creation of a functional S-nitrosylation site in vitro by single point mutation.

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.

Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: Part I. Biochemical implications.

The energy-dependent conversion of methionine to S-adenosyl-L-methionine (SAMe) is catalysed by S-adenosyl-L-methionine synthetase (SAMe-synthetase) in the liver. In the hepatocyte, an equilibrium exists between the high and low molecular weight forms of SAMe-synthetase, which consist of a tetramer and a dimer, respectively, of a 48.5 kilodalton subunit. The 2 enzymic forms differ in their affinity for methionine and sensitivity to inhibition by pyrophosphate; 2 of the sulfhydryl groups of SAMe-synthetase have been identified as essential for the normal functioning of the enzyme. In patients with liver cirrhosis, a marked reduction in the utilisation of the high molecular weight SAMe-synthetase and displacement of the equilibrium occur, the molecular mechanism of which has yet to be established. This loss of activity is associated with a delay in methionine clearance and impairment of the trans-sulphuration pathway, which normally eliminates excess methionine by oxidising homocysteine to sulphate anion. It is hypothesised that in normal liver function the essential sulfhydryl groups of SAMe-synthetase are protected from oxidation by glutathione, a by-product of the trans-sulphuration pathway. However, glutathione levels are reduced in liver cirrhosis, and this may result in increased oxidation of the essential sulfhydryl groups, and consequent inactivation of the enzyme. Thus, the trans-sulphuration pathway may play an important role in the maintenance of normal SAMe-synthetase activity.