Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.     

Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.     

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.     

Distinct Glycolysis Inhibitors Determine Retinal Cell Sensitivity to Glutamate-Mediated Injury

In this study, we analyzed how distinct glycolysis inhibitors influenced the redox status of retinal cells, used as a neuronal model. Three different approaches were used to inhibit glycolysis: the cells were submitted to iodoacetic acid (IAA), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase, to 2-deoxy-glucose (DG) in glucose-free medium, which was used as a substitute of glucose, or in the absence of glucose. The redox status of the cells was evaluated by determining the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). By the analysis of dose-response curves of MTT reduction, IAA showed values of IC₅₀ = 7.02 × 10^–5 M, whereas DG showed values of IC₅₀ = 7.42 × 10^–4 M. Upon 30 min-incubation, glucose deprivation, per se, did not significantly affect MTT reduction. We also evaluated the reduction of MTT as an indicator of cell injury by exposing the cells to 100 M glutamate during the decrement of glycolysis function. In the presence of glutamate, for 2 h, there was a decrease in MTT reduction, which was potentiated in the presence of DG (10-20% decrease), in the presence of IAA (about 30% decrease) or in glucose-free medium (about 30% decrease). Major changes observed by the MTT assay, upon exposure to glutamate, indicative of changes in the redox status of retinal cells, were concomitant with variations in intracellular ATP. Under glucose deprivation, endogenous ATP decreased significantly from 38.9 ± 4.4 to 13.3 ± 0.7 nmol/mg protein after exposure to 100 M glutamate. The results support a different vulnerability of retinal cells after being exposed to distinct forms of glycolysis inhibition.     

Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein

Analysis of the expression of the Herbaspirillum seropedicae nifA promoter in Escherichia coli and Herbaspirillum seropedicae, showed that nifA expression is primarily dependent on NtrC but also required NifA for maximal expression under nitrogen-fixing conditions. Deletion of the IHF (integration host factor)-binding site produced a promoter with two-fold higher activity than the native promoter in the H. seropedicae wild-type strain but not in a nifA strain, indicating that IHF controls NifA auto-activation. IHF is apparently required to prevent overexpression of the NifA protein via auto-activation under nitrogen-fixing conditions in H. seropedicae.     

Monitoring yeast regulated cell death: trespassing the point of no return to loss of plasma membrane integrity

Apoptosis - Acetic acid and hydrogen peroxide are the most common stimuli to induce apoptosis in yeast. The initial phase of this cell death process is characterized by the maintenance of plasma...     

Mitochondrial dysfunction in Huntington's disease: the bioenergetics of isolated and in situ mitochondria from transgenic mice

Mitochondrial dysfunction is believed to participate in Huntington's disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knock-in) and wild-type littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2+-loading capacity of isolated mitochondria by steady Ca2+-infusion. Mitochondria from R6/2 mice (12-13 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knock-in mice (15-17 weeks), exhibit increased Ca2+-loading capacity when compared with respective wild-type littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full-length mutant huntingtin (in Hdh150 knock-in heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy-demanding stimuli (NMDA-receptor activation in pyruvate-based media to accentuate the mitochondria role in Ca2+-handling), Hdh150 neurons are more vulnerable to Ca2+-deregulation than neurons from their wild-type littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context.     

Carriers of the Complex Allele HFE c.[187C>G;340+4T>C] Have Increased Risk of Iron Overload in S?o Miguel Island Population (Azores,Portugal)

Iron overload is associated with acquired and genetic conditions, the most common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. Here, we conducted a hospital-based case-control study of 41 patients from the São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I pseudodominant inheritance, and 35 unrelated individuals fulfilling the biochemical criteria of iron overload compatible with HH type-I. For this purpose, we analyzed the most common HFE mutations– c.845G>A [p.Cys282Tyr], c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the family’s HH pseudodominant pattern is due to consanguineous marriage of HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that is a -c.187G carrier. Regarding unrelated patients, six were homozygous for c.845A, and three were c.845A/c.187G compound heterozygous. We then performed sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate alternative splicing by HFE exon 2 skipping and consequently, a protein missing the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, we investigated HFE mutations configuration with iron overload by determining haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G allele also carry -c.340+4C, suggesting in-cis configuration. This data is corroborated by the association analysis where carriers of the complex allele HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI = 1.40−2.94, p<0.001). Therefore, homozygous for this complex allele are at risk of having iron overload because they will produce two altered proteins—the p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has a role, as genetic predisposition factor, on iron overload in the São Miguel population. Independent replication studies in other populations are needed to confirm this association.     

Trolox protection of myelin membrane in hydrogen peroxide-treated mature oligodendrocytes

Oligodendrocytes have the highest rate of metabolic activity in the brain and are highly vulnerable to oxidative stress. In this work we determined the protective effect of Trolox, a water-soluble analogue of vitamin E, and insulin, a peptide shown to be neuroprotective, in oligodendrocyte lesion induced by hydrogen peroxide (H₂O₂). Exposure of primary cultures of rat oligodendrocytes to H₂O₂ dose-dependently decreased their reducing capacity, as determined by the MTT assay. H₂O₂ (100 μM) had no effect on Bax levels, active-caspase-3, DNA fragmentation or lactate dehydrogenase (LDH) leakage. Nevertheless, under these conditions, H₂O₂ decreased the levels of myelin basic protein (MBP), used as a marker for oligodendrocyte myelin membrane. Treatment with insulin alone increased MBP levels, but no changes were observed in the presence of insulin plus H₂O₂. In contrast, incubation with Trolox completely prevented H₂O₂-induced decrease in MBP expression, suggesting that vitamin E analogues may prevent against oligodendrocyte oxidative damage.