Development of agmatine sensor using the combination of putrescine oxidase and agmatinase for squid freshness

Agmatine (Agm) is an indicator of squid freshness. The Agm sensor was developed using flow injection analysis (FIA) that consisted of the putrescine oxidase (PuOx) reactor, the agmatinase (AUH)-PuOx reactor and two oxygen electrodes. In the proposed sensor, the first step is that coexisting cadaverine (Cad) and putrescine (Put) are removed by passing through the PuOx reactor and the initial decomposition is determined by the amount of oxygen consumed, simultaneously. The second step is that the amount of Agm is determined by the amount of oxygen consumed in the AUH-PuOx reactor. The optimum conditions for the use of the Agm sensor were as follows: 50 mM HEPES containing MnSO4 at a final concentration of 5 mM, pH 8.0, flow rate of 0.6 mL min(-1) and injection volume of 50 microL. A single assay could be completed in approximately 3 min. A linear relationship was obtained between the output and the Agm concentration in the range of 0.01-1 mM Agm with a correlation coefficient of 0.999. The detection limit was 0.005 mM. The relative standard deviations (RSDs) were 3.14 and 1.19% (n = 20) for 0.1 and 0.3 mM Agm, respectively. The extracts of squid were injected into the proposed sensor and the results were compared with those obtained using the conventional high-performance liquid chromatography (HPLC) method. A correlation was observed between the results obtained by the proposed sensor and those obtained by the conventional method. The determination of squid freshness is one of the good uses of the proposed Agm sensor.     

4,5-Disubstituted-1,3-oxazolidin-2-imine derivatives: a new class of orally bioavailable nitric oxide synthase inhibitor

In our search for a novel class of inducible nitric oxide synthase (iNOS) inhibitors, 1,3-oxazolidin-2-imine was found to weakly inhibit iNOS. Further modifications of this compound resulted in a remarkable increase in both the in vivo and in vitro inhibitory activity and selectivity for iNOS.     

CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease

CD30, a TNFR family member, is expressed on activated CD4(+) and CD8(+) T cells and B cells and is a marker of Hodgkin's lymphoma; its ligand, CD30L (CD153) is expressed by activated CD4(+) and CD8(+) T cells, B cells, and macrophages. Signaling via CD30 can lead to proliferation or cell death. CD30-deficient (-/-) mice have impaired thymic negative selection and increased autoreactivity. Although human alloreactive T cells preferentially reside within the CD30(+) T cell subset, implicating CD30 as a regulator of T cell immune responses, the role of CD30/CD153 in regulating graft-vs-host disease (GVHD) has not been reported. We used a neutralizing anti-CD153 mAb, CD30(-/-) donor mice, and generated CD153(-/-) recipient mice to analyze the effect of CD30/CD153 interaction on GVHD induction. Our data indicate that the CD30/CD153 pathway is a potent regulator of CD4(+), but not CD8(+), T cell-mediated GVHD. Although blocking CD30/CD153 interactions in vivo did not affect alloreactive CD4(+) T cell proliferation or apoptosis, a substantial reduction in donor CD4(+) T cell migration into the gastrointestinal tract was readily observed with lesser effects in other GVHD target organs. Blockade of the CD30/CD153 pathway represents a new approach for preventing CD4(+) T cell-mediated GVHD.     

Critical role of OX40 in CD28 and CD154-independent rejection

Blocking both CD28 and CD154 costimulatory pathways can induce transplant tolerance in some, but not all, transplant models. Under stringent conditions, however, this protocol often completely fails to block allograft rejection. The precise nature of such CD28/CD154 blockade-resistant rejection is largely unknown. In the present study we developed a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (i.e., CD28/CD154 double-knockout (DKO) mice) and used this model to examine the role of novel costimulatory molecule-inducible costimulator (ICOS), OX40, 4-1BB, and CD27 in mediating CD28/CD154-independent rejection. We found that CD28/CD154 DKO mice vigorously rejected fully MHC-mismatched DBA/2 skin allografts (mean survival time, 12 days; n = 6) compared with the wild-type controls (mean survival time, 8 days; n = 7). OX40 costimulation is critically important in skin allograft rejection in this model, as blocking the OX40/OX40 ligand pathway, but not the ICOS/ICOS ligand, 4-1BB/4-1BBL, or CD27/CD70 pathway, markedly prolonged skin allograft survival in CD28/CD154 DKO mice. The critical role of OX40 costimulation in CD28/CD154-independent rejection is further confirmed in wild-type C57BL/6 mice, as blocking the OX40/OX40 ligand pathway in combination with CD28/CD154 blockade induced long term skin allograft survival (>100 days; n = 5). Our study revealed a key cellular mechanism of rejection and identified OX40 as a critical alternative costimulatory molecule in CD28/CD154-independent rejection.     

Regulation of postsurgical fibrosis by the programmed death-1 inhibitory pathway

Surgical adhesions are a common and often severe complication of abdominal or pelvic injury that cause pelvic pain, bowel obstruction, and infertility in women. Current treatments are of limited effectiveness because little is known about the cellular and subcellular processes underlying adhesiogenesis. Recently, we showed that Th1 alpha beta CD4(+) T cells mediate the pathogenesis of adhesion formation in a rodent model of this disease process. In this study, we demonstrate that in mice these T cells home directly to the site of surgically induced adhesions and control local chemokine production in a manner dependent on the CD28 T cell costimulatory pathway. Conversely, the inhibitory programmed death-1 pathway plays a central role in limiting adhesiogenesis, as programmed death-1 blockade was associated with increased T cell infiltration, chemokine production, and a concomitant exacerbation of disease. Our results reveal for the first time that the development of postsurgical fibrosis is under the tight control of positive and negative T cell costimulation, and suggest that targeting these pathways may provide promising therapies for the prevention of adhesion formation.     

Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (H_e) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (H_o) and expected heterozygosities (H_e) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( _IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (_IT ) was 0.98 and the genetic diversity indexes among populations, _ST and _ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system.