Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin

Procyanidins are bioactive flavonoid compounds from fruits and vegetables that possess insulinomimetic properties, decreasing hyperglycaemia in streptozotocin-diabetic rats and stimulating glucose uptake in insulin-sensitive cell lines. Here we show that the oligomeric structures of a grape-seed procyanidin extract (GSPE) interact and induce the autophosphorylation of the insulin receptor in order to stimulate the uptake of glucose. However, their activation differs from insulin activation and results in differences in the downstream signaling. Oligomers of GSPE phosphorylate protein kinase B at Thr308 lower than insulin does, according to the lower insulin receptor activation by procyanidins. On the other hand, they phosphorylate Akt at Ser473 to the same extent as insulin. Moreover, we found that procyanidins phosphorylate p44/p42 and p38 MAPKs much more than insulin does. These results provide further insight into the molecular signaling mechanisms used by procyanidins, pointing to Akt and MAPK proteins as key points for GSPE-activated signaling pathways. Moreover, the differences between GSPE and insulin might help us to understand the wide range of biological effects that procyanidins have.     

Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8

Leishmania are intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-β and TNF signalling pathways. Here, we investigated whether MIRNA146A rs2910164 and MIRNA499 rs3746444 variants are associated with the development of L. guyanensis (Lg)-cutaneous leishmaniasis (CL). The variants MIR146A rs2910164 and MIR499A rs3746444 were assessed in 850 patients with Lg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the BioPlex assay. Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadj_age/sex = 1.3 [95%CI 0.9–1.8]; Padj_age/sex 0.14) compared to individuals with the genotype GG (ORadj_age/sex = 0.77 [95%CI 0.56–1.0]; Padj_age/sex 0.14) if exposed to Lg-infection. Heterozygous GC individuals also showed lower odds of developing CL (ORadj_age/sex = 0.77 [95%CI 0.5–1.1]; Padj_age/sex 0.09). Homozygosity for the allele C is suggestive of an association with the development of Lg-CL among exposed individuals to Lg-infection. However, the odds of developing CL associated with the CC genotype was evident only in male individuals (OR_adjage = 1.3 [95% CI = 0.9–2.0]; P_adjage = 0.06). Individuals homozygous for the G allele tend to have higher plasma IL-8 and CCL5. Similarly, for the MIR499A rs3746444, an association with the G allele was only observed among male individuals (OR = 1.4 [1.0–1.9]; P = 0.009). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 40% elevated odds of developing Lg-CL (ORadj_age = 1.4 [1.1–1.9]). Individuals with the GG genotype have higher odds of developing Lg-CL (ORadj_age/sex = 2.0 [95%CI 0.83–5.0]; P_adjage = 0.01. Individuals homozygous for the G allele have higher plasma IL-8. Genetic combinations of both variants revealed that male individuals exposed to Lg bearing three or four susceptible alleles have higher odds of developing Lg-CL (OR = 2.3 [95% CI 1.0–4.7]; p = 0.017). Both MIR146A rs2910164 and MIR499A rs3746444 are associated with the development of Lg-CL and this association is prevalent in male individuals.     

Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2

The effects in vitro of sulphated and desulphated cholecystokinin (CCK)-8, and of gastrin-17 and gastrin-34 were studied at concentrations from 10⁻¹⁴ M to 10⁻⁶ M on several functions of human peripheral blood lymphocytes, i.e.: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), and spontaneous and phytohaemagglutinin (PHA)-mediated proliferation. All peptides, at concentrations from 10⁻¹⁰ M to 10⁻⁸ M, inhibited significantly the mobility capacity and PHA-induced proliferation, and increased the adherence and spontaneous proliferation. A dose-response relationship was observed, with a maximum response of lymphocyte functions at 10⁻¹⁰ M. These peptides induced a significant increase of intracellular cAMP levels at 30 and 60 sec. Because lymphoproliferation requires production of interleukin 2 (IL-2) by lymphocytes, we also measured the IL-2 production in the presence of the CCK and gastrin peptides, finding that this production was higher than in the respective controls. When peptides were added to samples containing PHA, the IL-2 production was significantly decreased with respect to samples incubated with PHA alone. These results suggest that the CCK and gastrin peptides are negative modulators of lymphocyte mobility (spontaneous mobility and chemotaxis), causing an inhibition of these activities through an increase of intracellular cAMP levels, and of PHA-induced lymphoproliferation, which is mediated by a diminution of the IL-2 production by lymphocytes.     

Comparative analysis of the neck region in spermatids and spermatozoa of some orthopteran insects

The ultrastructure of the neck region and the participation of noncovalent and disulphide bonds in the head–tail attachment were analyzed in spermatids and spermatozoa of some orthopteran species from the families Tettigoniidae and Acrididae. This study combined conventional electron microscopy with cytochemical procedures to detect acidic proteins and lysine-rich basic proteins, and with treatments using the disruptive agents sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). The organization of the neck region differs in spermatozoa among species in the families analyzed. In Acrididae the neck consists of the beginning of the axoneme attached to the nuclear envelope and surrounded by the centriolar adjunct material. Tettigoniidae species possess a complex organization in the form of a laminar “connective piece,” which shows a high content of lysine-rich and acidic proteins. Experiments using SDS and DTT demonstrate that in Acrididae the head–tail connection is strong, with both noncovalent and disulphide bonds important in maintenance of the attachment. In Tettigoniidae, however, despite the presence of a morphologically well organized connective piece, the head-tail attachment is biochemically labile in comparison to that of Acrididae. The relationships between the morphology of these neck structures and their stability mediated by noncovalent and/or disulphide bonds are discussed.     

Effect of Ethanol Treatment on Rate and Equilibrium Constants for [3H]Muscimol Binding to Rat Brain Membranes: Alteration of Two Affinity States of the GABAA Receptor

Abstract: Equilibrium binding curves were biphasic in control and ethanol-treated rats. [³H]Muscimol binds to sites of high ( K _DA of ∼10 n M ) and low ( K _DB of ∼0.3–0.4 µ M ) affinity. Chronic ethanol treatment produced a decrease in B _maxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high-affinity site. IC₅₀ and K _i values were calculated for several competing ligands, using membranes from both control and ethanol-treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low-affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol-treated animals for both phases.     

Hydrogen sulphide triggers VEGF-induced intracellular Ca signals in human endothelial cells but not in their immature progenitors

Hydrogen sulphide (H₂S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is central to endothelial proliferation and may be triggered by both VEGF and H₂S. Albeit VEGFR-2 might serve as H₂S receptor, the mechanistic relationship between VEGF- and H₂S-induced Ca²⁺ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H₂S donor, stimulates pro-angiogenic Ca²⁺ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca²⁺]_i in Ea.hy926 cells. NaHS-induced Ca²⁺ signals in Ea.hy926 cells did not require extracellular Ca²⁺ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP₃) signalling pathway. Moreover, the Ca²⁺ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca²⁺ signals were suppressed by dl-propargylglycine (PAG), which blocks the H₂S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca²⁺]_i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca²⁺ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H₂S-evoked elevation in [Ca²⁺]_i is essential to trigger the pro-angiogenic Ca²⁺ response to VEGF in mature endothelial cells, but not in their immature progenitors.     

Mechanical Stability and Reversible Fracture of Vault Particles

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.