Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC₅₀) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC₅₀ values in MKN-28 cells. For BVDU the EC₅₀ values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC₅₀ values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.     

Protein glutathionylation and oxidative stress

Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) demonstrated that glutathionyl hemoglobin (Hb) levels are increased in patients with diabetes, hyperlipidemia, uremia and Friedreich's ataxia. Glutathionylation of Hb is enhanced by oxidative stress. High performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) have also been developed for the quantification of glutathionyl Hb. Glutathionyl-lens proteins were detected in uremic patients and cataractous aged subjects. Glutathionylation of numerous enzymes is induced by oxidative stress, reduces their catalytic activities and may be involved in protection from the damaging effects of oxidative agents. Thioredoxin, glutaredoxin (thioltransferase) and protein disulfide isomerase are the key enzymes in controlling cellular oxidative stress that catalyze reduction of glutathionyl protein disulfide bonds. Thus, protein glutathionylation is closely associated with oxidative stress.     

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells

AimsMonocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1.Main methodsThe effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS).Key findingsDN + IS, DH, and DH + IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH + IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells.SignificanceIndoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.     

Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

Synchronization of spontaneous eyeblinks while viewing video stories

Blinks are generally suppressed during a task that requires visual attention and tend to occur immediately before or after the task when the timing of its onset and offset are explicitly given. During the viewing of video stories, blinks are expected to occur at explicit breaks such as scene changes. However, given that the scene length is unpredictable, there should also be appropriate timing for blinking within a scene to prevent temporal loss of critical visual information. Here, we show that spontaneous blinks were highly synchronized between and within subjects when they viewed the same short video stories, but were not explicitly tied to the scene breaks. Synchronized blinks occurred during scenes that required less attention such as at the conclusion of an action, during the absence of the main character, during a long shot and during repeated presentations of a similar scene. In contrast, blink synchronization was not observed when subjects viewed a background video or when they listened to a story read aloud. The results suggest that humans share a mechanism for controlling the timing of blinks that searches for an implicit timing that is appropriate to minimize the chance of losing critical information while viewing a stream of visual events.     

Metabolomic analysis of uremic toxins by liquid chromatography/electrospray ionization-tandem mass spectrometry

We applied the metabolomic analysis of comprehensive small-molecular metabolites using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis to identify uremic toxins accumulated in the serum of chronic renal failure (CRF) rats. CRF rats were produced by 5/6-nephrectomy. Indoxyl sulfate was demonstrated to be the first principal serum metabolite which differentiates CRF from normal, followed by phenyl sulfate, hippuric acid and p-cresyl sulfate. Then, we measured the serum levels of indoxyl sulfate, phenyl sulfate, hippuric acid and p-cresyl sulfate by the selected reaction monitoring (SRM) of LC/ESI-MS/MS, and demonstrated that these serum levels were markedly increased in CRF rats as compared with normal rats. As creatinine clearance decreased, the serum levels of the metabolites increased.     

The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.     

The carbohydrate-binding domain of Lafora disease protein targets Lafora polyglucosan bodies

Lafora's disease (LD) is an autosomal recessive and fatal form of epilepsy with onset in late childhood or adolescence. One of the characteristic features of LD pathology is the presence of periodic acid-Schiff (PAS) positive Lafora inclusion bodies. Lafora bodies are present primarily in neurons, but they have also been found in other organs. Histochemical and biochemical studies have indicated that Lafora bodies are composed mainly of polysaccharides. The LD gene, EPM2A, encodes a 331 amino acid long protein named laforin that contains an N-terminal carbohydrate-binding domain (CBD) and a C-terminal dual-specificity phosphatase domain (DSPD). Here we demonstrate that the CBD of laforin targets the protein to Lafora inclusion bodies and this property could be evolutionarily conserved. We also tested in vitro the effects of five LD missense mutations on laforin's affinity to Lafora body. While the missense mutant W32G failed to bind to purified Lafora body, four other mutants (S25P, E28L, F88L, and R108C) did not show any effect on the binding affinity. Based on these observations we propose the existence of a laforin-mediated glycogen metabolic pathway regulating the disposal of pathogenic polyglucosan inclusions. This is the first report demonstrating a direct association between the LD gene product and the disease-defining storage product, the Lafora bodies.