Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the beta-lactam producer Penicillium chrysogenum

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of g-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. (C) 2011 Elsevier Inc. All rights reserved.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF - 1 mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL`s and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p < 0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL. (C) 2009 Elsevier Ltd and ISBI. All rights reserved.

Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector. Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs). Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

THE SUMMATION OF VOLUNTARY AND ELECTRICAL ELICITED STIMULUS INCREASES JOINT TORQUE?

The aim of this study is to analyze the effect of neuromuscular electrical stimulation (NMES) on myoelectrical activity and on joint torque during isometric plantar flexion contraction. Ten healthy young adult subjects participate in this study. The electrodes for NMES are placed along posterior thigh along ciatic nerve trajectory. It is measured the myoelectrical activity and the isometric torque generated by ankle plantar flexion with an isokinetic dynamometer. The conditions of isometric contractions are maximum isometric voluntary contraction (MIVC), NMES, and association of both (MIVC+NMES). The results show lower torque during NMES and larger SOL activity compare to the others. Besides, in order to keep the same objective task (to produce the same level of torque), neuromuscular adaptations are necessary on the common drive.

Time course of strength and power recovery after resistance training with different movement velocities

Ide, BN, Leme, TCF, Lopes, CR, Moreira, A, Dechechi, CJ, Sarraipa, MF, da Mota, GR, Brenzikofer, R, and Macedo, DV. Time course of strength and power recovery after resistance training with different movement velocities. J Strength Cond Res 25(7): 2025-2033, 2011-The purpose of this study was to evaluate the time course of strength and power recovery after a single bout of strength training designed with fast and slow contraction velocities. Nineteen male subjects were randomly divided into 2 groups: the slow-velocity contraction (SV) group and the fast velocity contraction (FV) group. Resistance training protocols consisted of 5 sets of 12 repetition maximum (5 x 12RM) with 50 seconds of rest between sets and 2 minutes between exercises. Contraction velocity was controlled by the execution time for each repetition (SV-6 seconds to complete concentric and eccentric phases and for FV-1.5 seconds). Leg Press 45 degrees 1RM (LP 1RM), horizontal countermovement jump (HCMJ), and right thigh circumference (TC) were accessed in 6 distinct moments: base (1 week before exercise), 0 (immediately after exercises), 24, 48, 72, and 96 hours after exercise protocol. The SV and FV presented significant LP 1RM decrements at 0, and these were still evident 24-48 hours postexercise. The magnitude of decline was significantly (p<0.05) higher for FV. The SV and FV presented significant HCMJ decrements at 0, but only for FV were these still evident 24-72 hours postexercise. The SV and FV presented significant TC increments at 0, and these were still evident 24-48 hours postexercise for SV but for FV it continued up to 96 hours. The magnitude of increase was significantly (p<0.05) higher for FV. In conclusion, the fast contraction velocity protocol resulted in greater decreases in LP 1RM and HCMJ performance, when compared with slow velocity. The results lead us to interpret that this variable may exert direct influence on acute muscle strength and power generation capacity.

Comparative analysis of the gas-phase reactions of cylindrospermopsin and the difference in the alkali metal cation mobility

Cylindrospermopsin (CYN) belongs to a group of toxins produced by several strains of freshwater cyanobacteria. It is a compact zwitterionic molecule composed of a uracil section and a tricyclic guanidinium portion with a primarily hepatotoxic effect. Using low multi-stage and high-resolution mass spectrometry, the gas-phase reactions of this toxin have been investigated. Our data show that collision-induced dissociation (CID) spectra of CYN are dominated by neutral losses, and three major initial fragmentation pathways are clearly distinguishable. Interestingly, comparative analysis of protonated and cationizated molecules showed a significant difference in the balance of the SO(3) and terminal ring elimination. These data indicate that the differential ion mobility of H(+), Li(+), Na(+) and K(+) leads to different fragmentation pathways, giving rise to mass spectra with different profiles. Copyright (C) 2008 John Wiley & Sons, Ltd.

Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca2+ influx. To this end, the effect of PA on phenylephrine- and KCI-induced increases in cytosolic calcium concentration ([Ca2+](c)) measured by the variation in the ratio of fluorescence intensities (R340/ 380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 mu mol L-1) and KCI (30 or 90 mmol L-1) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (110, 100, 200 mu mol L-) attenuated the contraction induced by CaCl2 (0.5 nmol L(-)1 or 2.5 mmol L-1) in denuded rat aorta exposed to Ca2+- free medium containing phenylephrine (0.1 mu mol L-1) or KCI (30 mmol L-1). Interestingly, the inhibitory effect displayed by PA on CaCl2-induced contraction was more pronounced when KCI was used as the stimulant. Phenylephrine- and KCI-induced increases in (Ca2+,](c) were inhibited by PA. Similarly, verapamil, a Ca2+-channel blocker, also inhibited the increase in [Ca2+](c) induced by either phenylephrine or KCI. Finally, bolus injection of PA (1-15 mg kg(-1)) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca2+ influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

The effects of pH and ionic strength on topical delivery of a negatively charged porphyrin (TPPS(4))

Meso-tetra-[4-sulfonatophenyl]-porphyrin (TPPS(4)) is a charged porphyrin derivate used in photodynamic therapy (PDT) by parenteral administration. This study means to investigate potential enhancement for its topical delivery by determining the TPPS(4) dependence on the environmental characteristics and applying iontophoresis. In order to accomplish this task, cathodal and anodal iontophoresis as well as passive delivery of the drug were studied in vitro and in vivo in function of its concentration, pH and ionic strength. A reduction in drug concentration as well as the NaCl elimination from donor formulation at pH 2.0 increased TPPS(4) passive permeation through the skin in vitro. Iontophoresis improved TPPS(4) delivery across the skin when applied in solutions containing NaCl at pH 2.0, regardless electrode polarity. However, at pH 7.4, the amount of TPPS(4) permeated by iontophoresis was not different from that one permeated after passive experiments from a solution containing NaCl. Despite the fact that iontophoresis did not improve TPPS(4) transdermal delivery at this specific condition, in vivo experiments showed that 10 min of iontophoresis quickly and homogeneously delivered TPPS(4) to deeper skin layers when compared to passive administration, which is an important condition for topical treatment of skin tumors with PDT. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association.

Vascular biology of magnesium and its transporters in hypertension

Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on myriad biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoconstrictor agents. Mammalian cells regulate Mg(2+) concentration through special transport systems that have only recently been characterized. Magnesium efflux occurs via Na(2+)-dependent and Na(2+)-independent pathways. Mg(2+) influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A2, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg(2+) deficiency in hypertension and other cardiovascular pathologies. In particular, increased Mg(2+) efflux through dysregulation of the vascular Na(+)/Mg(2+) exchanger and decreased Mg(2+) influx due to defective vascular and renal TRPM6/7 expression/activity may be important in altered vasomotor tone and consequently in blood pressure regulation. The present review discusses the role of Mg(2+) in vascular biology and implications in hypertension and focuses on the putative transport systems that control magnesium homeostasis in the vascular system. Much research is still needed to clarify the exact mechanisms of cardiovascular Mg(2+) regulation and the implications of aberrant cellular Mg(2+) transport and altered cation status in the pathogenesis of hypertension and other cardiovascular diseases.

Degree of conversion and mechanical properties of a BisGMA : TEGDMA composite as a function of the applied radiant exposure

Objective: Verify the influence of radiant exposure (H) on composite degree of conversion (DC) and mechanical properties. Methods: Composite was photoactivated with 3, 6, 12, 24, or 48 J/cm(2). Properties were measured after 48-h dry storage at room temperature. DC was determined on the flat surfaces of 6 mm x 2 mm disk-shaped specimens using FTIR. Flexural strength (FS) and modulus (FM) were accessed by three-point bending. Knoop microhardness number (KHN) was measured on fragments of FS specimens. Data were analyzed by one-way ANOVA/Tukey test, Student`s t-test, and regression analysis. Results: DC/top between 6 and 12 J/cm(2) and between 24 and 48 J/cm(2) were not statistically different. No differences between DC/top and bottom were detected. DC/bottom, FM, and KHN/top showed significant differences among all H levels. FS did not vary between 12 and 24 J/cm(2) and between 24 and 48 J/cm(2). KHN/bottom at 3 and 6 J/cm(2) was similar. KHN between top and bottom was different up to 12 J/cm(2). Regression analyses having H as independent variable showed a plateau region above 24 J/cm(2). KHN increased exponentially (top) or linearly (bottom) with DC. FS and FM increased almost linearly with DC/bottom up to 55% conversion. Conclusions: DC and mechanical properties increased with radiant exposure. Variables leveled off at high H levels. (C) 2007 Wiley Periodicals, Inc.

Effect of Conventional and Resin-modified Glass-Ionomer Liner on Dentin Adhesive Interface of Class I Cavity Walls After Thermocycling

Objective: The aim of this in vitro study was to analyze the effect of glass-ionomer cement as a liner on the dentin/resin adhesive interface of lateral walls of occlusal restorations after thermocycling. Materials and Methods: Occlusal cavities were prepared in 60 human molars, divided into six groups: no liner (1 and 4); glass-ionomer cement (GIC, Ketac Molar Easymix, 3M ESPE) (2 and 5); and resin-modified glass-ionomer cement (RMGIC, Vitrebond, 3M ESPE) (3 and 6). Resin composite (Filtek Z250, 3M ESPE) was placed after application of an adhesive system (Adper Single Bond 2, 3M ESPE) that was mixed with a fluorescent reagent (Rhodamine B) to allow confocal microscopy analysis. Specimens of groups 4, 5 and 6 were thermocycled (5 degrees C-55 degrees C) with a dwell time of 30 seconds for 5000 cycles. After this period, teeth were sectioned in approximately 0.8-mm slices. One slice of each tooth was randomly selected for confocal microscopy analysis. The other slices were sectioned into 0.8 nun x 0.8 mm beams, which were submitted to microtensile testing (MPa). Data were analyzed using two-way ANOVA and Tukey test (p < 0.05). Results: There was no detectedstatistical difference on bond strength among groups (alpha < 0.05). Confocal microscopy analysis showed a higher mean gap size in group 4(12.5 mu m) and a higher percentage of marginal gaps in the thermocycled groups. The RNIGIC liner groups showed the lowest percentage of marginal gaps. Conclusions: Lining with RMGIC resulted in less gap formation at the dentin/resin adhesive interface after artificial aging. RMGIC or GIC liners did not alter the microtensile bond strength of adhesive system/resin composite to dentin on the lateral walls of Class I restorations.

Influence of curing rate on softening in ethanol, degree of conversion, and wear of resin composite

Purpose: To investigate the effect of curing rate on softening in ethanol, degree of conversion, and wear of resin composites. Methods: With a given energy density and for each of two different light-curing units (QTH or LED), the curing rate was reduced by modulating the curing mode. Thus, the irradiation of resin composite specimens (Filtek Z250, Tetric Ceram, Esthet-X) was performed in a continuous curing mode and in a pulse-delay curing mode. Wallace hardness was used to determine the softening of resin composite after storage in ethanol. Degree of conversion was determined by infrared spectroscopy (FTIR). Wear was assessed by a three-body test. Data were submitted to Levene`s test, one and three-way ANOVA, and Tukey HSD test (alpha= 0.05). Results: Immersion in ethanol, curing mode, and material all had significant effects on Wallace hardness. After ethanol storage, resin composites exposed to the pulse-delay curing mode were softer than resin composites exposed to continuous cure (P< 0.0001). Tetric Ceram was the softest material followed by Esthet-X and Filtek Z250 (P< 0.001). Only the restorative material had a significant effect on degree of conversion (P< 0.001): Esthet-X had the lowest degree of conversion followed by Filtek Z250 and Tetric Ceram. Curing mode (P= 0.007) and material (P< 0.001) had significant effect on wear. Higher wear resulted from the pulse-delay curing mode when compared to continuous curing, and Filtek Z250 showed the lowest wear followed by Esthet-X and Tetric Ceram. (Am J Dent 2011;24:115-118).

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Influence of different light sources and photo-activation methods on degree of conversion and polymerization shrinkage of a nanocomposite resin

The purpose of this study was to evaluate the influence of different light sources and photo-activation methods on degree of conversion (DC%) and polymerization shrinkage (PS) of a nanocomposite resin (Filtek (TM) Supreme XT, 3M/ESPE). Two light-curing units (LCUs), one halogen-lamp (QTH) and one light-emitting-diode (LED), and two different photo-activation methods (continuous and gradual) were investigated in this study. The specimens were divided in four groups: group 1-power density (PD) of 570 mW/cm(2) for 20 s (QTH); group 2-PD 0 at 570 mW/cm(2) for 10 s + 10 s at 570 mW/cm(2) (QTH); group 3-PD 860 mW/cm(2) for 20 s (LED), and group 4-PD 125 mW/cm(2) for 10 s + 10 s at 860 mW/cm(2) (LED). A testing machine EMIC with rectangular steel bases (6 x 1 x 2 mm) was used to record the polymerization shrinkage forces (MPa) for a period that started with the photo-activation and ended after two minutes of measurement. For each group, ten repetitions (n = 40) were performed. For DC% measurements, five specimens (n = 20) for each group were made in a metallic mold (2 mm thickness and 4 mm diameter, ISO 4049) and them pulverized, pressed with bromide potassium (KBr) and analyzed with FT-IR spectroscopy. The data of PS were analyzed by Analysis of Variance (ANOVA) with Welch`s correction and Tamhane`s test. The PS means (MPa) were: 0.60 (G1); 0.47 (G2); 0.52 (G3) and 0.45 (G4), showing significant differences between two photo-activation methods, regardless of the light source used. The continuous method provided the highest values for PS. The data of DC% were analyzed by Analysis of Variance (ANOVA) and shows significant differences for QTH LCUs, regardless of the photo-activation method used. The QTH provided the lowest values for DC%. The gradual method provides lower polymerization contraction, either with halogen lamp or LED. Degree of conversion (%) for continuous or gradual photo-activation method was influenced by the LCUs. Thus, the presented results suggest that gradual method photo-activation with LED LCU would suffice to ensure adequate degree of conversion and minimum polymerization shrinkage.

Continuous and gradual photo-activation methods: influence on degree of conversion and crosslink density of composite resins

Thermal properties and degree of conversion (DC%) of two composite resins (microhybrid and nanocomposite) and two photo-activation methods (continuous and gradual) displayed by the light-emitting diode (LED) light-curing units (LCUs) were investigated in this study. Differential scanning calorimetry (DSC) thermal analysis technique was used to investigate the glass transition temperature (T(g)) and degradation temperature. The DC% was determined by Fourier transform infrared spectroscopy (FT-IR). The results showed that the microhybrid composite resin presented the highest T(g) and degradation temperature values, i.e., the best thermal stability. Gradual photo-activation methods showed higher or similar T(g) and degradation temperature values when compared to continuous method. The Elipar Freelight 2 (TM) LCU showed the lowest T(g) values. With respect to the DC%, the photo-activation method did not influence the final conversion of composite resins. However, Elipar Freelight 2 (TM) LCU and microhybrid resin showed the lowest DC% values. Thus, the presented results suggest that gradual method photo-activation with LED LCUs provides adequate degree of conversion without promoting changes in the polymer chain of composite resins. However, the thermal properties and final conversion of composite resins can be influenced by the kind of composite resin and LCU.