NMR imaging of fluid pathways during drainage of softwood in a pressure membrane chamber

An experiment was implemented to study fluid flow in a pressure media. This procedure successfully combines nuclear magnetic resonance imaging with a pressure membrane chamber in order to visualize the non-wetting and wetting fluid flows with controlled boundary conditions. A specially designed pressure membrane chamber, made of non-magnetic materials and able to withstand 4 MPa, was designed and built for this purpose. These two techniques were applied to the drainage of Douglas fir sapwood. In the study of the longitudinal flow, narrow drainage fingers are formed in the latewood zones. They follow the longitudinal direction of wood and spread throughout the sample length. These fingers then enlarge in the cross-section plane and coalesce until drainage reaches the whole latewood part. At the end of the experiments, when the drainage of liquid water in latewood is completed, just a few sites of percolation appear in earlywood zones. This difference is a result of the wood anatomical structure, where pits, the apertures that allow the sap to flow between wood cells, are more easily aspirated in earlywood than in latewood. (C) 2007 Elsevier Ltd. All rights reserved.

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E-2 and D-2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs` synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo. (C) 2008 Elsevier Ltd. All rights reserved.

Dietary glutamine supplementation affects macrophage function, hematopoiesis and nutritional status in early weaned mice

Background Et aims: To investigate the effect that early weaning associated with the ingestion of either a glutamine-free or supplemented diet has on the functioning of peritoneal. macrophages, hematopoiesis and nutritional status of mice. Methods: Swiss Webster mice were early weaned on their 14th day of life and distributed to two groups, being fed either a glutamine-free diet (-GLN) or a glutamine-supplemented diet (+GLN). Animals belonging to a control group (CON) were weaned on their 21st day of life. Results: The -GLN and +GLN groups had a lower lean body mass, carcass protein and ash content, plasma glutamine concentration and lymphocyte counts both in the peripheral blood and bone marrow when compared to the CON group (P < 0.05). Dietary supplementation with glutamine reversed both the lower concentrations of protein and DNA in the muscle and liver, as well. as the reduced capacity of spreading and synthesizing nitric oxide, hydrogen peroxide, TNF-alpha, IL-1 beta and IL-6 in cultures of peritoneal. macrophages obtained from the -GLN group (P < 0.05). Conclusion: These data indicate that the ingestion of glutamine modulates the function of peritoneal macrophages in early weaned mice. However, a glutamine-supplemented diet cannot substitute maternal milk in respect to immunological and metabolic parameters. (C) 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Glutamine in vitro supplementation partly reverses impaired macrophage function resulting from early weaning in mice

Objective: Glutamine is one of the most abundant amino acids found in maternal milk, and its concentration increases throughout lactation. Because glutamine is essential for macrophage functionality, it is hereby suggested that early weaning in conjunction with the absence of glutamine consumption impairs the functioning of macrophages, which could in turn be reversed with an in vitro supplementation with glutamine. Methods: Swiss Webster mice were early weaned at 14 d of age (EW group) or at 21 d of age (control group, n = 8 per group). The EW group was fed a glutamine-free diet from days 14 to 21 of life. Results: Mice in the EW group presented a significant decrease in plasma and muscle concentrations of glutamine and an increase in the activity of glutamine synthetase in the muscle. Peritoneal macrophages obtained from animals in the EW group presented a significant increase in the production of interleukin (IL)-10 and a significant decrease in the synthesis of IL-1 beta, IL-6, tumor necrosis factor-a, nitric oxide, and hydrogen peroxide and in their ability to adhere, spread, phagocytize, and kill fungi. Glutamine in vitro supplementation reversed the decrease in IL-6, nitric oxide, and hydrogen peroxide synthesis and the decrease in the capacity to adhere, spread, and phagocytize in animals of the EW group. Conclusion: These new. data may imply that a lack of glutamine intake in early weaned mice hampers the functioning of peritoneal macrophages. (C) 2008 Elsevier Inc. All rights reserved.

Is serum amyloid A an endogenous TLR4 agonist?

Serum amyloid A (SAA), a classical acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.

Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264 7 macrophages with LDL(-) for 24 11 resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-): incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells CD95 (Fas), CD95 ligand (FasL). CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages. (C) 2009 Elsevier B V. All rights reserved

B-1 cells modulate oral tolerance in mice

Although the origin and functions of B-1 cells are controversial, they are considered as a cellular element of innate immunity due to their ability to produce natural autoantibodies of the IgM type. These antibodies are encoded by a relatively limited repertoire of V genes, and their resulting diversity is smaller than that produced by conventional B cells. B-1 cells constitute the larger fraction of B cells in the peritoneal cavity and migrate to non-specific inflammation sites. In addition, they contribute to the production of IgA antibodies in the intestinal lamina propria. It has been demonstrated that they participate in the induction and maintenance of peripheral tolerance. Herein, the participation of B-1 cells in inducing oral tolerance is evaluated. Unexpectedly, BALB/Xid mice, the animals deficient in B-1 cells, are not tolerized to OVA but instead are responsive to oral immunization. Conversely, BALB/c mice respond to oral tolerance to this antigen. We used these biological characteristics of these animals to investigate whether BA cells are involved in the induction of oral tolerance to OVA. Results show that B-1 cells from BALB/c mice, treated orally with OVA and adoptively transferred to BALB/Xid mice were able to suppress local hypersensitivity reaction and lymphoproliferative cellular response observed in BALB/.Xid mice. These data demonstrate that B-1 cells have regulatory properties and are involved in the induction of oral tolerance. (C) 2009 Elsevier B.V. All rights reserved.

Protein-Energy Malnutrition Modifies the Production of Interleukin-10 in Response to Lipopolysaccharide (LPS) in a Murine Model

Malnutrition modifies resistance to infection by impairing a number of physiological processes including hematopoesis and the immune response. In this study, we examined the production of Interleukin-4 (IL-4) and IL-10 in response to lipopolysaccharide (LPS) and also evaluated the cellularity of the blood, bone marrow, and spleen in a mouse model of protein-energy malnutrition. Two-month-old male Swiss mice were subjected to protein-energy malnutrition (PEM) with a low-protein diet (4%) as compared to the control diet (20%). When the experimental group lost approximately 20% of their original body weight, the animals from both groups received 1.25 mu g of LPS intravenously. The Cells ill the blood, bone marrow, and spleen were counted, and circulating levels of IL-4 and IL-10 were evaluated in animals stimulated with LPS. Cells from the spleen, bone marrow, and peritoneal cavity of non-inoculated animals were collected for Culture to evaluate the production of IL-4 and IL-10 after stimulating these cells with 1.25 mu g of LPS in vitro. Malnourished animals presented leucopenia and a severe reduction in bone marrow, spleen, and peritoneal cavity cellularity before and after Stimulus with LPS. The circulating levels of IL-10 were increased in malnourished animals inoculated with LPS when compared to control animals, although the levels of IL-4 did not differ. In cells cultured with LPS, we observed high levels of IL-10 in the bone marrow cells of malnourished animals. These findings suggest that malnourished mice present a deficient immune response to LPS. These alterations may be partly responsible for the immunodeficiency observed in these malnourished mice.

Intrinsic Dissolution as a Tool for Evaluating Drug Solubility in Accordance with the Biopharmaceutics Classification System

The Biopharmaceutics Classification System (BCS) is a tool that was created to categorize drugs into different groups according to their solubility and permeability characteristics. Through a combination of these factors and physiological parameters, it is possible to understand the absorption behavior of a drug in the gastrointestinal tract, thus contributing to cost and time reductions in drug development, as well as reducing exposure of human subjects during in vivo trials. Solubility is attained by determining the equilibrium under conditions of physiological pH, while different methods may be employed for evaluating permeability. On the other hand, the intrinsic dissolution rate (IDR), which is defined as the rate of dissolution of a pure substance under constant temperature, pH, and surface area conditions, among others, may present greater correlation to the in vivo dissolution dynamic than the solubility test. The purpose of this work is to discuss the intrinsic dissolution test as a tool for determining the solubility of drugs within the scope of the Biopharmaceutics Classification System (BCS).

Permeation of Large Antineoplastics into Wild Strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been used in genotoxicity and cytotoxicity assays for several years before the Ames Test approach. However the cell permeability of yeast has been considered a limitant factor to this kind of assay and many researchers have been introducing genetic modifications into wild strains to improve the sensitivity to chemical compounds. In our study, we used Saccharomyces cerevisiae ATCC 9763, well known and very common strain in antibiotic assays, and we evaluated the cytotoxicity of some antineoplastic agents (etoposide, epirubicin, carboplatin, cisplatin and mitoxantrone). Each culture was observed under the light of microscope and photographed. Neither genetic modification nor addition of permeation inducers, as dimethylsulfoxide (DMSO), were introduced during the assays and the cells presented good sensitivity to those compounds, demonstrating that other potential strains and characteristics of cells should be reconsidered to improve these assays apart from the cellular permeability.

Comparison of Bidirectional Lamivudine and Zidovudine Transport Using MDCK, MDCK-MDR1, and Caco-2 Cell Monolayers

Bidirectional transport studies were conducted using Caco-2, MDCK, and MDCK-MDR1 to determine P-gp influences in lamivudine and zidovudine permeability and evaluate if zidovudine permeability changes with the increase of zidovudine concentration and/or by association of lamivudine. Transport of lamivudine and zidovudine separated and coadministrated across monolayers based on these cells were quantified using LC-MS-MS. Drug efflux by P-gp was inhibited using GG918. Bidirectional transport of lamivudine and zidovudine was performed across MDCK-MDR1 and Caco-2 cells. Statistically significant transport decrease in B -> A direction was observed using MDCK-MDR1 for zidovudine and MDCK-MDR1 and Caco-2 for lamivudine. Results show increased transport in B -> A and A -> B directions as concentration increases but data from P(app) increase in both directions for both drugs in Caco-2, decrease in MDCK, and does not change significantly in MDCK-MDR1. Zidovudine transport in A -> B direction increases when coadministrated with increasing lamivudine concentration but does not change significantly in B -> A direction. Zidovudine and lamivudine are P-gp substrates, but results assume that P-gp does not affect significantly lamivudine and zidovudine. Their transport in monolayers based on Caco-2 cells increase proportionally to concentration (in both directions) and zidovudine transport in Caco-2 cell monolayer does not show significant changes with lamivudine increasing concentrations. (C) 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4413-4419, 2009

Influence of Urea, Isopropanol, and Propylene Glycol on Rutin In Vitro Release from Cosmetic Semisolid Systems Estimated by Factorial Design

Rutin, one of the major flavonoids found in an assortment of plants, was reported to act as a sun protection factor booster with high anti-UVA defense, antioxidant, antiaging, and anticellulite, by improvement of the cutaneous microcirculation. This research work aimed at evaluating the rutin in vitro release from semisolid systems, in vertical diffusion cells, containing urea, isopropanol and propylene glycol, associated or not, according to the factorial design with two levels with center point. Urea (alone and in association with isopropanol and propylene glycol) and isopropanol (alone and in association with propylene glycol) influenced significant and negatively rutin liberation in diverse parameters: flux (g/cm2.h); apparent permeability coefficient (cm/h); rutin amount released (g/cm2); and liberation enhancement factor. In accordance with the results, the presence of propylene glycol 5.0% (wt/wt) presented statistically favorable to promote rutin release from this semisolid system with flux = 105.12 8.59 g/cm2.h; apparent permeability coefficient = 7.01 0.572 cm/h; rutin amount released = 648.80 53.01 g/cm2; and liberation enhancement factor = 1.21 0.07.

Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus

Aim of the study: Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. Materials and methods: LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 mu M CGA in presence of lipopolysaccharide from Escherichia coil (LPS, 5 mu g/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha [TNF-alpha; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, beta(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. Results: CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced beta(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression: inhibited neutrophil adhesion and neutrophil migration induced by fMLP. Conclusions: Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Application of hydrophobic resin adhesives to acid-etched dentin with an alternative wet bonding technique

Hydrophilic dentin adhesives are prone to water sorption that adversely affects the durability of resin-dentin bonds. This study examined the feasibility of bonding to dentin with hydrophobic resins via the adaptation of electron microscopy tissue processing techniques. Hydrophobic primers were prepared by diluting 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane/triethyleneglycol dimethacrylate resins with known ethanol concentrations. They were applied to acid-etched moist dentin using an ethanol wet bonding technique that involved: (1) stepwise replacement of water with a series of increasing ethanol concentrations to prevent the demineralized collagen matrix from collapsing; (2) stepwise replacement of the ethanol with different concentrations of hydrophobic primers and subsequently with neat hydrophobic resin. Using the ethanol wet bonding technique, the experimental primer versions with 40, 50, and 75% resin exhibited tensile strengths which were not significantly different from commercially available hydrophilic three-step adhesives that were bonded with water wet bonding technique. The concept of ethanol wet bonding may be explained in terms of solubility parameter theory. This technique is sensitive to water contamination, as depicted by the lower tensile strength results from partial dehydration protocols. The technique has to be further improved by incorporating elements of dentin permeability reduction to avoid water from dentinal tubules contaminating water-free resin blends during bonding. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res 84A: 19-29, 2008.

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria

We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extramitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extramitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.