The cyclic GMP-protein kinase G pathway regulates cytoskeleton dynamics and motility in astrocytes

We have previously demonstrated that inflammatory compounds that increase nitric oxide (NO) synthase expression have a biphasic effect on the level of the NO messenger cGMP in astrocytes. In this work, we demonstrate that NO-dependent cGMP formation is involved in the morphological change induced by lipopolysaccharide (LPS) in cultured rat cerebellar astroglia. In agreement with this, dibutyryl-cGMP, a permeable cGMP analogue, and atrial natriuretic peptide, a ligand for particulate guanylyl cyclase, are both able to induce process elongation and branching in astrocytes resulting from a rapid, reversible and concentration-dependent redistribution of glial fibrillary acidic protein (GFAP) and actin filaments without significant change in protein levels. These effects are also observed in astrocytes co-cultured with neurons. The cytoskeleton rearrangement induced by cGMP is prevented by the specific protein kinase G inhibitor Rp-8Br-PET-cGMPS and involves downstream inhibition of RhoA GTPase since is not observed in cells transfected with constitutively active RhoA. Furthermore, dibutyryl-cGMP prevents RhoA-membrane association, a step necessary for its interaction with effectors. Stimulation of the cGMP-protein kinase G pathway also leads to increased astrocyte migration in an in vitro scratch-wound assay resulting in accelerated wound closure, as seen in reactive gliosis following brain injury. These results indicate that cGMP-mediated pathways may regulate physio-pathologically relevant responses in astroglial cells.     

Complex formation regulates the glycosylation of the reversibly glycosylated polypeptide

Reversible glycosylated polypeptides (RGPs) are highly conserved plant-specific proteins, which can perform self-glycosylation. These proteins have been shown essential in plants yet its precise function remains unknown. In order to understand the function of this self-glycosylating polypeptide, it is important to establish what factors are involved in the regulation of the RGP activity. Here we show that incubation at high ionic strength produced a high self-glycosylation level and a high glycosylation reversibility of RGP from Solanum tuberosum L. In contrast, incubation at low ionic strength led to a low level of glycosylation and a low glycosylation reversibility of RGP. The incubation at low ionic strength favored the formation of high molecular weight RGP-containing forms, whereas incubation at high ionic strength produced active RGP with a molecular weight similar to the one expected for the monomer. Our data also showed that glycosylation of RGP, in its monomeric form, was highly reversible, whereas, a low reversibility of the protein glycosylation was observed when RGP was part of high molecular weight structures. In addition, glycosylation of RGP increased the occurrence of non-monomeric RGP-containing forms, suggesting that glycosylation may favor multimer formation. Finally, our results indicated that RGP from Arabidopsis thaliana and Pisum sativum are associated to golgi membranes, as part of protein complexes. A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.     

The influence of interleukin-1beta on oxytocin signalling in primary cells of human decidua

OBJECTIVE: Oxytocin (OT) and its corresponding receptor (OTR), synthesized within the pregnant uterus, play a key role in the process of (preterm) labor as part of a paracrine system that regulates symmetrical contractility. In the setting of intrauterine infection, a major cause of preterm labour and birth, decidua serves as a major source of cytokine production. The present study evaluates the time-dependent effect [0-24 h] of the inflammatory cytokine Interleukin-1beta (IL-1beta) treatment on OT signalling and OT stimulated prostaglandin release in primary cultures of human decidua. STUDY DESIGN: Primary cultures of human decidua (n=6) were treated with IL-1beta [5 ng/ml] for 0-24h and or indomethacin [100 microM]--an inhibitor of the prostaglandin synthesis--for 0-24 h or for 24 h. OT peptide expression, OTR binding, Inositol triphosphate production (IP(3)), and arachidonic acid (AA) as well as prostaglandin (PGE(2)) release were measured. RESULTS: IL-1beta transiently reduced cytoplasmic OT peptide at 4-6 h of IL-1beta incubation, while its secretion into the media was increased after 6 h of stimulation. The later was completely blocked by indomethacin. A decrease in OTR mRNA expression and a loss of OTR binding were detected after 8 h and 16 h of IL-1beta treatment, respectively. IL-1beta also decreased IP(3) production and AA release, but significantly enhanced OT mediated PGE(2) production. This effect was completely suppressed by the cyclooxygenase-2 (COX-2) inhibitor NS-398. CONCLUSION: Our data suggest, that IL-1beta indirectly increases OT secretion in primary cultures of human decidua in a time dependent fashion through the production of prostaglandins through COX-2 and that this increase in OT peptide may secondarily down-regulate the OTR and its signalling cascade. These findings might explain the poor effectiveness of oxytocin receptor antagonists as tocolytic agents in the setting of intrauterine infection.     

Angiotensin-(1 7) stimulates the phosphorylation of JAK2, IRS-1 and Akt in rat heart in vivo: role of the AT1 and Mas receptors

Angiotensin (ANG) II exerts a negative modulation on insulin signal transduction that might be involved in the pathogenesis of hypertension and insulin resistance. ANG-(1-7), an endogenous heptapeptide hormone formed by cleavage of ANG I and ANG II, counteracts many actions of ANG II. In the current study, we have explored the role of ANG-(1-7) in the signaling crosstalk that exists between ANG II and insulin. We demonstrated that ANG-(1-7) stimulates the phosphorylation of Janus kinase 2 (JAK2) and insulin receptor substrate (IRS)-1 in rat heart in vivo. This stimulating effect was blocked by administration of the selective ANG type 1 (AT(1)) receptor blocker losartan. In contrast to ANG II, ANG-(1-7) stimulated cardiac Akt phosphorylation, and this stimulation was blunted in presence of the receptor Mas antagonist A-779 or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The specific JAK2 inhibitor AG-490 blocked ANG-(1-7)-induced JAK2 and IRS-1 phosphorylation but had no effect on ANG-(1-7)-induced phosphorylation of Akt, indicating that activation of cardiac Akt by ANG-(1-7) appears not to involve the recruitment of JAK2 but proceeds through the receptor Mas and involves PI3K. Acute in vivo insulin-induced cardiac Akt phosphorylation was inhibited by ANG II. Interestingly, coadministration of insulin with an equimolar mixture of ANG II and ANG-(1-7) reverted this inhibitory effect. On the basis of our present results, we postulate that ANG-(1-7) could be a positive physiological contributor to the actions of insulin in heart and that the balance between ANG II and ANG-(1-7) could be relevant for the association among insulin resistance, hypertension, and cardiovascular disease.     

Modulation of polyamine balance in Lotus glaber by salinity and arbuscular mycorrhiza.

In this work we investigated the involvement of Glomus intraradices in the regulation of plant growth, polyamines and proline levels of two Lotus glaber genotypes differing in salt tolerance, after longterm exposure to saline stress. The experiment consisted of a randomized block design with three factors: (1) mycorrhizal treatments (with or without AM fungus); (2) two salinity levels of 0 and 200mM NaCl; and (3) L. glaber genotype. Experiments were performed using stem cuttings derived from L. glaber individuals representing a natural population from saline lowlands. One of the most relevant results was the higher content of total free polyamines in mycorrhized plants compared to non-AM ones. Since polyamines have been proposed as candidates for the regulation of root development under saline situations, it is possible that AM plants (which contained higher polyamine levels and showed improved root growth) were better shaped to cope with salt stress. Colonization by G. intraradices also increased (Spd+Spm)/Put ratio in L. glaber roots. Interestingly, such increment in salt stressed AM plants of the sensitive genotype, was even higher than that produced by salinization or AM symbiosis separately. On the other hand, salinity but not mycorrhizal colonization influenced proline levels in both L. glaber genotypes since high proline accumulation was observed in both genotypes under salt stress conditions. Our results suggest that modulation of polyamine pools can be one of the mechanisms used by AM fungi to improve L. glaber adaptation to saline soils. Proline accumulation in response to salt stress is a good indicator of stress perception and our results suggest that it could be used as such among L. glaber genotypes differing in salt stress tolerance.     

A girl with del(4)(q33) and occipital encephalocele: clinical description and molecular genetic characterization of a rare patient

We present clinical and developmental data on a girl with a de novo terminal deletion of the long arm of chromosome 4, del(4)(q33). The patient was evaluated at birth and followed up until 5 years of age. She showed facial and digital dysmorphism, a complex congenital heart defect, a large occipital encephalocele, and postnatal growth deficiency. Her neuropsychomotor milestones were delayed, and she developed learning difficulties. Apart from standard Giemsa banding, a molecular genetic analysis was performed using a comparative genomic hybridization (CGH) array. This revealed a terminal deletion at the band 4q32.3, which is directly adjacent to 4q33. The clinical findings in our patient differ from those described previously in patients with del(4)(q33) and del(4)(q32), respectively. In particular, the prominent occipital encephalocele has not been observed before in a terminal 4q deletion.     

E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

The plasma membrane Ca²⁺?ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF_x), aluminium fluoride (AlF_x) and magnesium fluoride (MgF_x) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P_i product states. Our results show that the phosphate analogues BeF_x, AlF_x and MgF_x inhibit PMCA Ca²⁺?ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca²⁺?ATPase inhibition by AlF_x and BeF_x depended on Mg²⁺ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF_x and BeF_x but not MgF_x apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E?→?E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA.In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.     

Phytochemical Findings Evidencing Botanical Origin of New Propolis Type from North‐West Argentina

Propolis samples from north‐west Argentina (Amaicha del Valle, Tucumán) were evaluated by palynology, FT‐IR spectra, and RP‐HPTLC. In addition, the volatile fraction was studied by HS‐SPME‐GC/MS. The botanical species most visited by Apis mellifera L. near the apiaries were collected and their RP‐HPTLC extracts profiles were compared with propolis samples. In addition, GC/MS was performed for volatile compounds from Zuccagnia punctata Cav. (Fabaceae). FT‐IR spectra and RP‐HPTLC fingerprints of propolis samples showed similar profiles. In RP‐HPTLC analyses, only Z. punctata presented a similar fingerprint to Amaicha propolis. The major volatile compounds present in both were trans‐linalool oxide (furanoid), 6‐camphenone, linalool, trans‐pinocarveol, p‐cymen‐8‐ol, and 2,3,6‐trimethylbenzaldehyde. Potential variations for the Amaicha del Valle propolis volatile fraction as consequence of propolis sample preparation were demonstrated.     

Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth‐promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress‐related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col‐0 and aba2‐1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro‐grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild‐type Col‐0 and on the mutant aba2‐1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col‐0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.