Phospholipase D2:A Pivotal Player Modulating RBL-2H3 Mast Cell Structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells. (J Histochem Cytochem 60:386-396, 2012)

In Vitro Analysis of Bacterial Morphology by Atomic Force Microscopy of Low Level Laser Therapy 660, 830 and 904 nm

Objective: The objective of this study was to analyze the bacterial morphology by atomic force microscopy (AFM) after the application of low-level laser therapy (LLLT) in in vitro culture of Staphylococcus aureus ATCC 29213. Background data: Infections caused by S. aureus are among the highest occurring in hospitals and can often colonize pressure ulcers. LLLT is among the methods used to accelerate the healing of ulcers. However, there is no consensus on its effect on bacteria. Materials and methods: After being cultivated and seeded, the cultures were irradiated using wavelengths of 660, 830, and 904 nm at fluences of 0, 1, 2, 3, 4, 5, and 16 J/cm(2). Viable cells of S. aureus strain were counted after 24 h incubation. To analyze the occurrence of morphological changes, the topographical measurement of bacterial cells was analyzed using the AFM. Results: The overall assessment revealed that the laser irradiation reduced the S. aureus growth using 830 and 904 nm wavelengths; the latter with the greatest inhibition of the colony-forming units (CFU/mL) (331.1 +/- 38.19 and 137.38 +/- 21.72). Specifically with 660 nm, the statistical difference occurred only at a fluence of 3 J/cm(2). Topographical analysis showed small changes in morphological conformity of the samples tested. Conclusions: LLLT reduced the growth of S. aureus with 830 and 904 nm wavelengths, particularly with 904 nm at a fluence of 3 J/cm(2), where the greatest topographical changes of the cell structure occurred.

Distribution of Rickettsia rickettsii in ovary cells of Rhipicephalus sanguineus (Latreille1806) (Acari: Ixodidae)

Abstract Background Considering the fact that the dog tick, Rhipicephalus sanguineus, has a great potential to become the vector of Brazilian Spotted Fever (BSF) for humans, the present study aimed to describe the distribution of the bacterium Rickettsia rickettsii, the etiological agent of BSF, in different regions of the ovaries of R. sanguineus using histological techniques. The ovaries were obtained from positive females confirmed by the hemolymph test and fed in the nymph stage on guinea pigs inoculated with R. rickettsii. Results The results showed a general distribution of R. rickettsii in the ovary cells, being found in oocytes in all stages of development (I, II, III, IV and V) most commonly in the periphery of the oocyte and also in the cytoplasm of pedicel cells. Conclusions The histological analysis of the ovaries of R. sanguineus infected females confirmed the presence of the bacterium, indicating that the infection can interfere negatively in the process of reproduction of the ticks, once alterations were detected both in the shape and cell structure of the oocytes which contained bacteria.

Anomalous diffusion evidenced by particle tracking at high frame rates.

Diffusion is a common phenomenon in nature and generally is associated with a system trying to reach a local or a global equilibrium state, as a result of highly irregular individual particle motion. Therefore it is of fundamental importance in physics, chemistry and biology. Particle tracking in complex fluids can reveal important characteristics of its properties. In living cells, we coat the microbead with a peptide (RGD) that binds to integrin receptors at the plasma membrane, which connects to the CSK. This procedure is based on the hypothesis that the microsphere can move only if the structure where it is attached move as well. Then, the observed trajectory of microbeads is a probe of the cytoskeleton (CSK), which is governed by several factors, including thermal diffusion, pressure gradients, and molecular motors. The possibility of separating the trajectories into passive and active diffusion may give information about the viscoelasticity of the cell structure and molecular motors activity. And also we could analyze the motion via generalized Stokes-Einstein relation, avoiding the use of any active techniques. Usually a 12 to 16 Frames Per Second (FPS) system is used to track the microbeads in cell for about 5 minutes. Several factors make this FPS limitation: camera computer communication, light, computer speed for online analysis among others. Here we used a high quality camera and our own software, developed in C++ and Linux, to reach high FPS. Measurements were conducted with samples for 10£ and 20£ objectives. We performed sequentially images with different intervals, all with 2 ¹s exposure. The sequences of intervals are in milliseconds: 4 5 ms (maximum speed) 14, 25, 50 and 100 FPS. Our preliminary results highlight the difference between passive and active diffusion, since the passive diffusion is represented by a Gaussian in the distribution of displacements of the center of mass of individual beads between consecutive frames. However, the active process, or anomalous diffusion, shows as long tails in the distribution of displacements.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

Relationship between B-Cell-specific moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III beta (topoIII beta) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII beta, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA1 (p=0.003), H2AX (p=0.024) and topoIII beta (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Development of Plurimetallic Electrocatalysts Prepared by Decomposition of Polymeric Precursors for EtOH/O-2 Fuel Cell

This work aimed to develop plurimetallic electrocatalysts composed of Pt, Ru, Ni, and Sn supported on C by decomposition of polymeric precursors (DPP), at a constant metal: carbon ratio of 40:60 wt.%, for application in direct ethanol fuel cell (DEFC). The obtained nanoparticles were physico-chemically characterized by X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX). XRD results revealed a face-centered cubic crystalline Pt with evidence that Ni, Ru, and Sn atoms were incorporated into the Pt structure. Electrochemical characterization of the nanoparticles was accomplished by cyclic voltammetry (CV) and chronoamperometry (CA) in slightly acidic medium (0.05 mol L-1 H2SO4), in the absence and presence of ethanol. Addition of Sn to PtRuNi/C catalysts significantly shifted the ethanol and CO onset potentials toward lower values, thus increasing the catalytic activity, especially for the quaternary composition Pt64Sn15Ru13Ni8/C. Electrolysis of ethanol solutions at 0.4 V vs. RHE allowed determination of acetaldehyde and acetic acid as the main reaction products. The presence of Ru in alloys promoted formation of acetic acid as the main product of ethanol oxidation. The Pt64Sn15Ru13Ni8/C catalyst displayed the best performance for DEFC.

Oxygen reduction reaction catalyzed by epsilon-MnO2: Influence of the crystalline structure on the reaction mechanism

The oxygen reduction reaction (ORR) was studied in KOH electrolyte on carbon supported epsilon-manganese dioxide (epsilon-MnO2/C). The epsilon-MnO2/C catalyst was prepared via thermal decomposition of manganese nitrate and carbon powder (Vulcan XC-72) mixtures. X-ray powder diffraction (XRD) measurements were performed in order to determine the crystalline structure of the resulting composite, while energy dispersive X-ray analysis (EDX) was used to evaluate the chemical composition of the synthesized material. The electrochemical studies were conducted using cyclic voltammetry (CV) and quasi-steady state polarization measurements carried out with an ultra thin layer rotating ring/disk electrode (RRDE) configuration. The electrocatalytic results obtained for 20% (w/w) Pt/C (E-TEK Inc., USA) and alpha-MnO2/C for the ORR, considered as one of the most active manganese oxide based catalyst for the ORR in alkaline media, were included for comparison. The RRDE results revealed that the ORR on the MnO2 catalysts proceeds preferentially through the complete 4e(-) reduction pathway via a 2 plus 2e(-) reduction process involving hydrogen peroxide as an intermediate. A benchmark close to the performance of 20% (w/w) Pt/C (E-TEK Inc., USA) was observed for the epsilon-MnO2/C material in the kinetic control region, superior to the performance of alpha-MnO2/C, but a higher amount of HO2- was obtained when epsilon-MnO2/C was used as catalyst. The higher production of hydrogen peroxide on epsilon-MnO2/C was related to the presence of structural defects, typical of this oxide, while the better catalytic performance in the kinetic control region compared to alpha-MnO2/C was related with the higher electrochemical activity for the proton insertion kinetics, which is a structure sensitive process. (C) 2012 Elsevier Ltd. All rights reserved.

The Influence of Osteoblast Differentiation Stage on Bone Formation in Autogenously Implanted Cell-Based Poly(Lactide-Co-Glycolide) and Calcium Phosphate Constructs

We tested the hypothesis that the osteoblast differentiation status of bone marrow stem cells (BMSCs) combined with a three-dimensional (3D) structure modulates bone formation when autogenously implanted. Rat BMSCs were aspirated, expanded, and seeded into a 3D composite of poly(lactide-co-glycolide) and calcium phosphate (PLGA/CaP) to produce a hybrid biomaterial. Calvarial defects were implanted with (1) scaffold without cells (SC/NC), (2) scaffold and BMSCs (SC + BMSC), (3) scaffold and osteoblasts differentiated for 7 days (SC + OB7), and (4) for 14 days (SC + OB14). After 4 weeks, there was more bone formation in groups combining scaffold and cells, SC + BMSC and SC + OB7. A nonsignificant higher amount of bone formation was observed on SC + OB14 compared with SC/NC. Additionally, more blood vessels were counted within all hybrid biomaterials, without differences among them, than into SC/NC. These findings provide evidences that the cell differentiation status affects in vivo bone formation in autogenously implanted cell-based constructs. Undifferentiated BMSCs or osteoblasts in early stage of differentiation combined with PLGA/CaP scaffold favored bone formation compared with plain scaffold and that one associated with more mature osteoblasts.

Spectroscopic characterization and crystal structure of cis-Bis(N-(2-benzoyl)-N ',N '-diphenylthioureato-k O-2,S)nickel(II)

The title compound [Ni(C20H15N2OS)(2)] is prepared by the reaction of metal acetate with the corresponding acylthiourea derivative. The complex is characterized by elemental analysis, IR, H-1 and C-13 NMR, and its structure is determined by single crystal X-ray diffraction. The Ni(II) ion is coordinated by the S and O atoms of two N-benzoyl-N',N'-diphenylthiourea ligands in a slightly distorted square-planar coordination geometry. The two O and two S atoms are mutually cis to each other. The substance crystallizes triclinic (P-1 space group) with cell dimensions a = 10.7262(9) , b = 12.938(3) , c = 14.2085(12) , alpha = 74.650(4)A degrees, beta = 78.398(4)A degrees, gamma = 68.200(5)A degrees, and two formula units in the unit cell. The structure is very close to the related N-(2-furoyl) Ni complex reported previously.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Structure Elucidation and Anticancer Activity of 7-Oxostaurosporine Derivatives from the Brazilian Endemic Tunicate Eudistoma vannamei

The present study reports the identification of two new staurosporine derivatives, 2-hydroxy-7-oxostaurosporine (1) and 3-hydroxy-7-oxostaurosporine (2), obtained from mid-polar fractions of an aqueous methanol extract of the tunicate Eudistoma vannamei, endemic to the northeast coast of Brazil. The mixture of 1 and 2 displayed IC50 values in the nM range and was up to 14 times more cytotoxic than staurosporine across a panel of tumor cell lines, as evaluated using the MTT assay.

Invariant Natural Killer T Cell Agonist Modulates Experimental Focal and Segmental Glomerulosclerosis

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis ( FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T ( iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin( ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1 alpha, IL-1 beta, IL-17, TNF-alpha, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-beta analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-beta could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-beta, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-beta through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.