Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 parts per thousand salinity), as well as potential routes of Na(+) uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell inviginations, respectively. These findings ire discussed regarding the putative movement of Na(+) across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations. (C) 2008 Elsevier Inc. All rights reserved.

Differential adjustment in gill Na(+)/K(+)- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase alpha-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25 parts per thousand S. During a 10-day acclimation period to 25 parts per thousand S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase alpha-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25 parts per thousand S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21 parts per thousand S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Association of the MCP-1-2518 A/G Polymorphism and No Association of Its Receptor CCR2-64 V/I Polymorphism with Lupus Nephritis

Objective. To evaluate whether the A/G polymorphism at position 2518 in the regulatory region of the monocyte chemoattractant protein-1 (MCP-1) or the V/I polymorphism at position 64 of the receptor. CCR2, are associated with lupus nephritis (LN) or any clinical characteristics of the disease or with renal survival in a patient population. Methods. We selected 197 patients with lupus nephritis and 220 matched healthy controls for study. MCP-1 and CCR2 genotyping was performed by polymerase chain reaction. Clinical and laboratory data were compiled from patients` charts over followup that ranged from 6 months to 10 years. Results. The GIG genotype of MCP-1 was more common in LN patients (p = 0.019), while the A allele was associated with healthy controls (p = 0.007) as was the V allele of CCR2 (p = 0.046) compared to LN patients. Clinical index measures [SLE Disease Activity Index (SLEDAI)], immunological markers, renal histology, renal function at enrollment, and renal survival were not influenced by these polymorphisms. A less aggressive renal disease, measured by renal SLEDAI index, was associated with the V allele of the CCR2 gene polymorphism. Conclusion. These findings support that MCP-1 2518 GIG is associated with LN but there was no association of this genotype with renal function or renal survival. When studying CCR2 64 V/I polymorphism we showed a positive association of the V allele with healthy controls but no association of the genotype with LN patients. (First Release March 152010; J Rheumatol 2010;37:776-82; doi:10.3899/jrheum.090681)

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Endomyocardial fibrosis: pathological and molecular findings of surgically resected ventricular endomyocardium

Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions affecting the inflow tract and apex of one or both ventricles, which show fibrous thickening of the endocardium and adjacent myocardium. Surgical treatment is recommended for patients in functional classes III or IV (New York Heart Association). The gross and histological features of the heart have been comprehensively studied in autopsies, but studies in surgical samples are still lacking. Histological and immunohistochemical features of EMF in surgical samples collected from 32 patients were described and correlated with clinical data. Polymerase chain reaction (PCR) and reverse transcription-PCR, performed on formalin fixed endomyocardial samples, were used retrospectively to detect genomes of certain cardiotropic viruses and Toxoplasma gondii. Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III, with predominance of the former type. Besides fibrosis, a chronic inflammatory process and an anomalous lymphatic rich vascular pattern were observed in the deep endocardium, connected to the terminal coronary circulation of the myocardium, which might be an important pathological finding concerning EMF pathogenesis. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents (6/12, 50%); however, their role in the disease pathogenesis is still controversial.

Dengue and dengue hemorrhagic fever among adults: Clinical outcomes related to viremia, serotypes, and antibody response

Background. Clinical manifestations of dengue vary in different areas of endemicity and between specific age groups, whereas predictors of outcome have remained controversial. In Brazil, the disease burden predominantly affects adults, with an increasing trend toward progression to dengue hemorrhagic fever (DHF) noted. Methods. A cohort of adults with confirmed cases of dengue was recruited in central Brazil in 2005. Patients were classified according to the severity of their disease. Associations of antibody responses, viremia levels (as determined by real-time polymerase chain reaction [PCR]), and serotypes (as determined by multiplex PCR) with disease severity were evaluated. Results. Of the 185 symptomatic patients > 14 years of age who had a confirmed case of dengue, 26.5% and 23.2% were classified as having intermediate dengue fever (DF)/ DHF (defined as internal hemorrhage, plasma leakage, manifested signs of shock, and/ or thrombocytopenia [platelet count, <= 50,000 platelets/mm(3)]) and DHF, respectively. The onset of intermediate DF/ DHF and DHF occurred at a late stage of disease, around the period of defervescence. Patients with DHF had abnormal liver enzyme levels, with a > 3-fold increase in aspartate aminotransferase level, compared with the range of values considered to be normal. Overall, 65% of patients presented with secondary infections with dengue virus, with such infection occurring in similar proportions of patients in each of the 3 disease category groups. Dengue virus serotype 3 (DV3) was the predominant serotype, and viremia was detected during and after defervescence among patients with DHF or intermediate DF/ DHF. Conclusions. Viremia was detected after defervescence in adult patients classified as having DHF or intermediate DF/ DHF. Secondary infection was not a predictor of severe clinical manifestation in adults with infected with the DV3 serotype.

Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Characterization of dimethacrylate polymeric networks: A study of the crosslinked structure formed by monomers used in dental composites

The resin phase of dental composites is mainly composed of combinations of dimethacrylate comonomers, with final polymeric network structure defined by monomer type/reactivity and degree of conversion. This fundamental study evaluates how increasing concentrations of the flexible triethylene glycol dimethacrylate (TEGDMA) influences void formation in bisphenol A diglycidyl dimethacrylate (BisGMA) co-polymerizations and correlates this aspect of network structure with reaction kinetic parameters and macroscopic volumetric shrinkage. Photopolymerization kinetics was followed in real-time by a near-infrared (NIR) spectroscopic technique, viscosity was assessed with a viscometer, volumetric shrinkage was followed with a linometer, free volume formation was determined by positron annihilation lifetime spectroscopy (PALS) and the sol-gel composition was determined by extraction with dichloromethane followed by (1)H NMR analysis. Results show that, as expected, volumetric shrinkage increases with TEGDMA concentration and monomer conversion. Extraction/(1)H NMR studies show increasing participation of the more flexible TEGDMA towards the limiting stages of conversion/crosslinking development. As the conversion progresses, either based on longer irradiation times or greater TEGDMA concentrations, the network becomes more dense, which is evidenced by the decrease in free volume and weight loss after extraction in these situations. For the same composition (BisGMA/TEGDMA 60-40 mol%) light-cured for increasing periods of time (from 10 to 600 s), free volume decreased and volumetric shrinkage increased, in a linear relationship with conversion. However, the correlation between free volume and macroscopic volumetric shrinkage was shown to be rather complex for variable compositions exposed for the same time (600 s). The addition of TEGDMA decreases free-volume up to 40 mol% (due to increased conversion), but above that concentration, in spite of the increase in conversion/crosslinking, free volume pore size increases due to the high concentration of the more flexible monomer. In those cases, the increase in volumetric shrinkage was due to higher functional group concentration, in spite of the greater free volume. Therefore, through the application of the PALS model, this study elucidates the network formation in dimethacrylates commonly used in dental materials. (C) 2010 Elsevier Ltd. All rights reserved.

Influence of matrix composition on polymerization stress development of experimental composites

Objective. Stress development at the tooth/restoration interface is one of the most important reasons for failure of adhesive restorations. The aim of this study was to evaluate the influence of BisGMA/TEGDMA (B/T) and UDMA/TEGDMA (U/T) ratios on polymerization stress (PS) and on the variables related to its development: degree of conversion (DC), polymerization maximum rate (Rp(max)), volumetric shrinkage (VS), elastic modulus (E), stress relaxation (SR) and viscosity of experimental composites. Method. Composites were formulated containing B/T or U/T in mol% ratios of 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3 and 8: 2, and 15 wt% of fumed silica. PS was determined with a universal testing machine. VS was measured with a linometer. E and SR were obtained in three-point bending. DC and Rp(max) were determined by real time NIR spectroscopy and viscosity was measured in viscometer. Data were submitted to one-way ANOVA, Tukey test (alpha = 0.05%) and regression analyses. Results. PS, VS, E and DC decreased and viscosity and Rp(max) increased with base monomer content in both series. PS showed strong correlation with VS, DC and viscosity. PS, VS and DC were higher and viscosity was lower for UDMA-based materials. Significance. Reduced viscosity, kinetics parameters and molecular characteristics led UDMA-based composites to elevated conversion and relatively lower PS at lower TEGDMA contents, compared to B/T composites. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Contraction stress determinants in dimethacrylate composites

The influence of composite organic content on polymerization stress development remains unclear. It was hypothesized that stress was directly related to differences in degree of conversion, volumetric shrinkage, elastic modulus, and maximum rate of polymerization encountered in composites containing different BisGMA (bisphenylglycidyl dimethacrylate) concentrations and TEGDMA ( triethylene glycol dimethacrylate) and/or BisEMA ( ethoxylated bisphenol-A dimethacrylate) as co-monomers. Stress was determined in a tensilometer. Volumetric shrinkage was measured with a mercury dilatometer. Elastic modulus was obtained by flexural test. We used fragments of flexural specimens to determine degree of conversion by FT-Raman spectroscopy. Reaction rate was determined by differential scanning calorimetry. Composites with lower BisGMA content and those containing TEGDMA showed higher stress, conversion, shrinkage, and elastic modulus. Polymerization rate did not vary significantly, except for the lower value of the 66% TEGDMA composite. We used linear regressions to evaluate the association between polymerization stress and conversion (R-2 = 0.905), shrinkage ( R-2 = 0.825), and modulus ( R-2 = 0.623).

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Persistent Impairment of Testicular Histology and Sperm Motility in Adult Rats Treated with Cisplatin at Peri-Puberty

Cisplatin is one of the most widely used and effective chemotherapeutic agents for the treatment of several human malignancies. This study evaluated the effects of peri-pubertal cisplatin administration on several reproductive end-points and the reversibility of these effects in adulthood. Peri-pubertal Wistar male rats (45 days old) were divided into two groups: control (saline 0.9%) and cisplatin (1 mg/kg/day, 5 days/week, for 3 weeks, i.p.). The study was conducted in two steps and evaluations were performed at ages of 66 (post-pubertal age) and 140 (adult age) days on: (i) organ weights, serum gonadotropins and testosterone levels, sperm counts, motility and morphology, testicular histomorphometry, spermatogenesis kinetics, Sertoli cell number and in situ detection of apoptotic germ cells and (ii) sexual behaviour, fertility and intratesticular testosterone. At the end of cisplatin therapy, rats showed reductions in sperm production and reserves, sperm with progressive movement, tubular diameter, intratesticular testosterone and fertility potential, but increased numbers of TUNEL-positive seminiferous tubules, immotile sperm and pre-implantation losses compared with control. Moreover, cisplatin-treated post-pubertal rats displayed impaired testicular histopathology and sexual behaviour. Serum gonadotropins and testosterone levels, sperm morphology, spermatogenesis kinetics and Sertoli cell number were comparable between experimental groups at both ages. Alterations found in post-puberty were recovered at adulthood, except for sperm motility and damage to testicular histology. The persistence of these cisplatin effects, despite the unaltered fertility after natural mating in rats, may have implications for reproductive function of young boys undergoing cancer therapy, given the lower reproductive efficiency in human beings compared with rats.

GHASP: an H alpha kinematic survey of spiral and irregular galaxies - IX. The near-infrared, stellar and baryonic Tully-Fisher relations

We studied, for the first time, the near-infrared, stellar and baryonic Tully-Fisher relations for a sample of field galaxies taken from a homogeneous Fabry-Perot sample of galaxies [the Gassendi HAlpha survey of SPirals (GHASP) survey]. The main advantage of GHASP over other samples is that the maximum rotational velocities were estimated from 2D velocity fields, avoiding assumptions about the inclination and position angle of the galaxies. By combining these data with 2MASS photometry, optical colours, HI masses and different mass-to-light ratio estimators, we found a slope of 4.48 +/- 0.38 and 3.64 +/- 0.28 for the stellar and baryonic Tully-Fisher relation, respectively. We found that these values do not change significantly when different mass-to-light ratio recipes were used. We also point out, for the first time, that the rising rotation curves as well as asymmetric rotation curves show a larger dispersion in the Tully-Fisher relation than the flat ones or the symmetric ones. Using the baryonic mass and the optical radius of galaxies, we found that the surface baryonic mass density is almost constant for all the galaxies of this sample. In this study we also emphasize the presence of a break in the NIR Tully-Fisher relation at M(H,K) similar to -20 and we confirm that late-type galaxies present higher total-to-baryonic mass ratios than early-type spirals, suggesting that supernova feedback is actually an important issue in late-type spirals. Due to the well-defined sample selection criteria and the homogeneity of the data analysis, the Tully-Fisher relation for GHASP galaxies can be used as a reference for the study of this relation in other environments and at higher redshifts.

Measurement of the epicycle frequency in the Galactic disc and initial velocities of open clusters

A new method to measure the epicycle frequency kappa in the Galactic disc is presented. We make use of the large data base on open clusters completed by our group to derive the observed velocity vector (amplitude and direction) of the clusters in the Galactic plane. In the epicycle approximation, this velocity is equal to the circular velocity given by the rotation curve, plus a residual or perturbation velocity, of which the direction rotates as a function of time with the frequency kappa. Due to the non-random direction of the perturbation velocity at the birth time of the clusters, a plot of the present-day direction angle of this velocity as a function of the age of the clusters reveals systematic trends from which the epicycle frequency can be obtained. Our analysis considers that the Galactic potential is mainly axis-symmetric, or in other words, that the effect of the spiral arms on the Galactic orbits is small; in this sense, our results do not depend on any specific model of the spiral structure. The values of kappa that we obtain provide constraints on the rotation velocity of the in particular, V(0) is found to be 230 +/- 15 km s(-1) even if the scale (R(0) = 7.5 kpc) of the Galaxy is adopted. The measured kappa at the solar radius is 43 +/- 5 km s(-1) kpc(-1). The distribution of initial velocities of open clusters is discussed.

HI aperture synthesis and optical observations of the pair of galaxies NGC 6907 and 6908

NGC 6908, an S0 galaxy situated in the direction of NGC 6907, was only recently recognized as a distinct galaxy, instead of only a part of NGC 6907. We present 21-cm radio synthesis observations obtained with the Giant Metrewave Radio Telescope (GMRT) and optical images and spectroscopy obtained with the Gemini-North telescope of this pair of interacting galaxies. From the radio observations, we obtained the velocity field and the H I column density map of the whole region containing the NGC 6907/8 pair, and by means of the Gemini multi-object spectroscopy we obtained high-quality photometric images and 5 angstrom resolution spectra sampling the two galaxies. By comparing the rotation curve of NGC 6907 obtained from the two opposite sides around the main kinematic axis, we were able to distinguish the normal rotational velocity field from the velocity components produced by the interaction between the two galaxies. Taking into account the rotational velocity of NGC 6907 and the velocity derived from the absorption lines for NGC 6908, we verified that the relative velocity between these systems is lower than 60 km s(-1). The emission lines observed in the direction of NGC 6908, not typical of S0 galaxies, have the same velocity expected for the NGC 6907 rotation curve. Some emission lines are superimposed on a broader absorption profile, which suggests that they were not formed in NGC 6908. Finally, the H I profile exhibits details of the interaction, showing three components: one for NGC 6908, another for the excited gas in the NGC 6907 disc and a last one for the gas with higher relative velocities left behind NGC 6908 by dynamical friction, used to estimate the time when the interaction started in (3.4 +/- 0.6) x 10(7) yr ago.