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.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

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.     

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150^Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150^Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150^Glued complex interactions.     

Anatomy of flower and fruit of <i>Vassobia breviflora</i> (Solanaceae) in the south of the southern Yungas (Argentina)

Solanaceae is a family with nearly 2400 species of cosmopolitan distribution. Vassobia breviflora is the only species of the genus present in Argentina. The goal of this work was to review and characterize the anatomy of the flower and fruit of V. breviflora from samples collected in populations of Yungas in the argentine Northwest. Conventional anatomical techniques were applied. The results showed that most flower, fruit and seed structures did not differ from those previously reported regarding the structural organization described for other species of the Solanaceae family. However, for the first time, we described the androecium, fruit, seed, floral and fruit pedicels, five types of tricomes and five types of stomata in the perianth. We found some differences in the shape of the transmission tissue and in the type of ovule with respect to that previously reported. Also, we located the parenchyma and the epidermic secretory cells of the nectary. In the context of the family Solanaceae, we discussed the function and diagnostic value of the described structures.     

Behavioral actions of urotensin-II

Urotensin-II (U-II) and urotensin-II-related peptide (URP) have been identified as the endogenous ligands of the orphan G-protein-coupled receptor GPR14 now renamed UT. The occurrence of U-II and URP in the central nervous system, and the widespread distribution of UT in the brain suggest that U-II and URP may play various behavioral activities. Studies conducted in rodents have shown that central administration of U-II stimulates locomotion, provokes anxiety- and depressive-like states, enhances feeding activity and increases the duration of paradoxical sleep episodes. These observations indicate that, besides the endocrine/paracrine activities of U-II and URP on cardiovascular and kidney functions, these peptides may act as neurotransmitters and/or neuromodulators to regulate various neurobiological activities.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

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.