Lipopolysaccharide-induced Overproduction of Nitric Oxide and Overexpression of iNOS and Interleukin-1β Proteins in Zinc-deficient Rats

Zinc deficiency leads to decreased cellular immune responses. The overproduction of nitrogen species derived from inducible nitric oxide synthase (iNOS), its enzyme, and interleukine-1 beta (IL-1β), and inflammatory cytokine have been implicated in immune responses. The goal of this study was to investigate the effects of lipopolysaccharide (LPS)-induced changes in NO metabolites, iNOS, and IL-1β protein expression in the lungs of zinc-deficient rats. Male Sprague–Dawley rats (body weight, 100 g) were divided into two groups and were fed either a zinc-deficient diet (ZnD) or a zinc-containing diet (Cont). After 4 weeks on these diets, rats received a 10-mg/kg dose of LPS injected via the tail vein and were then maintained for an additional 72 h. To determine total NO concentrations in the blood, serum zinc concentration, iNOS protein expression, IL-1β, and iNOS immunohistochemistry, blood and lung samples were obtained at pre-LPS injection, 5, 24, and 72 h after injection. Total NO levels were significantly increased at 5, at 24, and at 72 h after LPS injection compared with pre-LPS injection level in ZnD group; significant changes in total NO levels was elevated at 5 h from at pre-LPS level but not significant changes from basal level at 24 and 72 h in the control group. Based on western blot analyses and immunohistochemistry, clear bands indicating iNOS and IL-1β protein expression and iNOS antibody-stained inflammatory cells were detected at 5 and 24 h in the ZnD group and 5 h in the Cont group, not observed at 24 and 72 h in the control group. These results suggest that zinc deficiency induces overexpression of iNOS and IL-1β proteins from inflammatory cells around the alveolar blood vessels, resulting in overproduction of total NO and persisted inflammatory response in the zinc-deficient rat lung. Taken together, overexpression of LPS-induced iNOS, overproduction of iNOS-derived NO, and overexpression of IL-1β may induce nitrosative and oxidative stresses in the lung, and these stresses may be involved low immunity of zinc deficiency states.     

Essential structure of opioid κ receptor agonist nalfurafine for binding to the κ receptor 3: Synthesis of decahydro(iminoethano)phenanthrene derivatives with an oxygen functionality at the 3-position and their pharmacologies

To clarify the essential structures of an opioid κ receptor selective agonist, nalfurafine, for binding to the κ receptor, we designed and synthesized the decahydro(iminoethano)phenanthrene derivatives with an oxygen functionality at the 3-position. The introduction of a hydroxy group to the derivatives increased the affinity and selectivity to the κ receptor regardless of the configuration at the 3-position. However, their affinities were lower than those of nalfurafine with the phenolic hydroxy group. The results suggested that the acidity of the hydroxy group would play an important role in the interaction with the opioid receptor. The low affinities of the 3-keto derivatives indicated that the 3-hydroxy group may participate in the hydrogen bonding with the receptor site not as a hydrogen acceptor but as a hydrogen donor. This is the first experimental evidence for a role as a hydrogen donor for the 3-hydroxy group in morphinans. Furthermore, the κ selectivities in these derivatives with the 6α-amide side chain were affected by the the 3-hydroxy group. The obtained structure–activity relationship information is expected to be useful for the design of more selective ligands for the κ receptor.     

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor

Background

The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).

Methods

Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer''s (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).

Results

Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.

Conclusions

Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.     

Effect of <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> on TIMP-1 secretion from mouse melanoma cell

Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.     

Brief measure for screening complicated grief: reliability and discriminant validity

Background

Complicated grief, which is often under-recognized and under-treated, can lead to substantial impairment in functioning. The Brief Grief Questionnaire (BGQ) is a 5-item self-report or interview instrument for screening complicated grief. Although investigations with help-seeking samples suggest that the BGQ is valid and reliable, it has not been validated in a broader population.

Methodology/Principal Findings

A questionnaire was mailed to a randomly selected sample (n = 5000) residing in one of 4 areas of Japan. The BCQ was examined for responders who were bereaved more than 6 months and less than 10 years (n = 915). Non-specific psychological distress was assessed with the K6 screening scale. Multiple group confirmatory factor analysis supported a uni-dimensional factor structure and the invariance of parameters across gender and age. Cronbach''s alpha was sufficiently high (alpha = .75) to confirm internal consistency. Average Variance Extracted (0.39) was higher than the shared covariance (0.14) between BGQ and K6, suggesting discriminant validity.

Conclusions

The results of this study support the reliability and validity of the BGQ in the Japanese population. Future studies should examine predictive validity by using structured interviews or more detailed scales for complicated grief.     

Cell surface biotinylation by azaelectrocyclization: easy-handling and versatile approach for living cell labeling

Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Detection of Legionella pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test

The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella®) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.