Effects of methylglyoxal and pyridoxamine in rat brain mitochondria bioenergetics and oxidative status

Advanced glycation end products (AGEs) and methylglyoxal (MG), an important intermediate in AGEs synthesis, are thought to contribute to protein aging and to the pathogenesis of age-and diabetes-associated complications. This study was intended to investigate brain mitochondria bioenergetics and oxidative status of rats previously exposed to chronic treatment with MG and/or with pyridoxamine (PM), a glycation inhibitor. Brain mitochondrial fractions were obtained and several parameters were analyzed: respiratory chain [states 3 and 4 of respiration, respiratory control ratio (RCR), and ADP/O index] and phosphorylation system [transmembrane potential (ΔΨ_m), ADP-induced depolarization, repolarization lag phase, and ATP levels]; hydrogen peroxide (H₂O₂) production levels, mitochondrial aconitase activity, and malondialdehyde levels as well as non-enzymatic antioxidant defenses (vitamin E and glutathione levels) and enzymatic antioxidant defenses (glutathione disulfide reductase (GR), glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) activities). MG treatment induced a statistical significant decrease in RCR, aconitase and GR activities, and an increase in H₂O₂ production levels. The administration of PM did not counteract MG-induced effects and caused a significant decrease in ΔΨ_m. In mitochondria from control animals, PM caused an adaptive mechanism characterized by a decrease in aconitase and GR activities as well as an increase in both α-tocopherol levels and GPx and MnSOD activities. Altogether our results show that high levels of MG promote brain mitochondrial impairment and PM is not able to reverse MG-induced effects.     

TDP-43 Inclusion Bodies Formed in Bacteria Are Structurally Amorphous,Non-Amyloid and Inherently Toxic to Neuroblastoma Cells

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.     

Accumulation of Polyphosphate in Lactobacillus spp. and Its Involvement in Stress Resistance

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.     

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30–150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.     

18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.     

Intrapulmonary Vascular Dilatation Evaluated by 99mTc-MAA Scintigraphy and Its Association with Portal Hypertension in Schistosomiasis

Background

Portal hypertension is responsible for various complications in patients with schistosomiasis, among them intrapulmonary vascular dilations (IPVD). In cirrhotic patients the presence of IPVD is a sign of poor prognosis, but in patients with hepatosplenic schistosomiasis (HSS) there are no studies assessing the significance of this change. The aim of this study was to evaluate the occurrence of IPVD through ^99mTc-MAA scintigraphy in patients with HSS and its relationship with clinical, laboratory, endoscopic and ultrasound parameters.

Methods

Cross-sectional study evaluating 51 patients with HSS. Patients were diagnosed with IPVD when the brain uptake of ^99mTc-MAA was higher than 6%. Subsequently, they were divided according to presence (G1) or absence (G2) of IPVD and variables were compared between groups.

Results

Overall, 51 patients with mean age of 56±12 years were assessed. IPVD was observed in 31 patients (60%). There was no statistically significant differences between groups when clinical, laboratory and endoscopic parameters were compared. Regarding ultrasound parameters, the splenic vein diameter was smaller in G1 (0.9±0.3 cm) compared to G2 (1.2±0.4 cm), p = 0.029.

Conclusion

In patients with HSS, the occurrence of IPVD by ^99mTc-MAA scintigraphy was high and was associated with lower splenic vein diameter, which can be a mechanism of vascular protection against portal hypertension. However, more studies are needed to determine the clinical significance of the early diagnosis and natural evolution of IPVD in this population.     

Synthesis,conformation, and membrane modifying properties of the trikoningin KB lipopeptaibols: Effect of hydrophobicity and chirality in position 1

Summary We synthesized by solution-phase methods the naturally occurring, 10-amino acid residue lipopeptaibol antibiotics trikoningins KBI and KBII, and the [l-Iva¹] KB analogue, in which the amino acid in position 1 is different, with the aim at investigating the effect of hydrophobicity and chirality in that position. A solution conformational analysis, using FT-IR absorption and CD techniques, indicated that all of the three decapeptides are predominantly helical in a membrane-mimetic environment. Permeability measurements showed an increase of the activity from the [Aib¹] peptide to the more hydrophobic [Iva¹] peptides. Conversely, the effect of a change in chirality, obtained by replacingd-Iva¹ withl-Iva, turned out to be of minor significance.     

Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads

Two commercial porous styrene-divinylbenzene beads (Diaion HP20LX and MCI GEL CHP20P) have been evaluated as supports to immobilize lipase B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immobilized the enzyme, permitting a very high loading capacity: around 110 mg CALB/wet g of support compared to the 50 mg obtained using decaoctyl Sepabeads. Although enzyme specificity of the enzyme immobilized on different supports was quite altered by the support used in the immobilization, specific activity of the enzyme immobilized on MCI GEL CHP20P was always higher than those found using decaoctyl Sepabeads for all assayed substrates. Thus, a CALB biocatalyst having 3-8 folds (depending on the substrate) higher activity/wet gram of support than the commercial Novozym 435 was obtained. Half-live of CAL-Diaion HP20LX at 60 °C was 2-3 higher than the one of Novozym 435, it was 30-40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10 M hydrogen peroxide.Results indicate that styrene-divinylbenzene supports may be promising alternatives as supports to immobilize CALB.     

Nitrogen Form Alters Hormonal Balance in Salt-treated Tomato (Solanum lycopersicum L.)

Mixed nitrate/ammonium fertilization can partially alleviate the negative effects of salinity on growth of some plant species compared to all-nitrate or all-ammonium fertilization. To gain insights about the mechanisms involved, tomato (Solanum lycopersicum L. cv Moneymaker) plants were grown hydroponically for 3 weeks with two NO₃ ⁻/NH₄ ⁺ fertilization regimes (6/0.5 and 5/1.5; N_total = 6.5 mM) in the absence (control) or presence of salt stress (100 mM NaCl). Ammonium enrichment had no effect on growth and other parameters under control conditions. Under salinity, however, ammonium enrichment improved shoot and root biomass by 20% and maintained leaf PSII efficiency close to control levels. These changes were related to higher leaf K⁺, NO₃ ⁻, and NH₄ ⁺ concentrations and activities of the N-assimilatory enzymes glutamate synthase (GOGAT) and glutamine synthase (GS) in the leaves. Ammonium enrichment also attenuated the salt-induced increase in leaf abscisic acid (ABA) concentration and decrease in leaf concentrations of indole 3-acetic acid (IAA) and the cytokinins trans-zeatin (tZ) and trans-zeatin riboside (tZR). Enhanced cytokinin status was probably due to maintenance of root-to-shoot cytokinin transport and decreased leaf induction of the cytokinin-degrading enzyme cytokinin oxidase/dehydrogenase (CKX) under ammonium-enriched conditions. It is concluded that nitrogen form modifies salinity-induced physiological responses and that these modifications are associated with changes in plant hormone status.