Apoptotic neurodegeneration induced by influenza A virus infection in the mouse brain

Neurodegeneration in the brain induced by the WSN strain of influenza A virus was investigated after stereotaxic introduction into the olfactory bulb of C57BL/6 mice. Immunohistochemistry detected WSN virus-infected neurons in the anterior olfactory nucleus as early as day 3 postinfection. Thereafter, they became shrunken and showed loss of neurite-immunolabeling and chromatin condensation. Infected neurons died by day 12, degenerating into multiple small granular bodies. The terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling method demonstrated DNA fragmentation in infected neurons at day 7 and also in such granular bodies at day 12. In perforin-deficient mice, the appearance of virally induced apoptotic neurodegeneration was delayed and virus infection continued for a longer period of 35 days postinfection. These findings indicate that perforin-mediated neuroapoptosis appears significant in exterminating the intracellular pathogen at an early stage of infection.     

A case of maxillary sarcoma in a chimpanzee (Pan troglodytes)

Oral malignancy is rare in chimpanzees. A 34‐year‐old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.     

A simple set-and-mix assay for screening of protein kinase inhibitors in cell lysates

Here we developed a simple set-and-mix assay to perform high-throughput screening of protein kinase A (PKA) inhibitors from the LOPAC 1280 compound library. This assay is based on the color change of gold nanoparticles on aggregation induced by a cationic substrate peptide as coagulant. In spite of the simplicity of this assay system, this assay can be applied to drug screening based on cellular kinases. We successfully found several highly active inhibitors, including compounds that have not been reported before.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.     

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer’s disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC₅₀ = 6.1 μM) and cyclophilin, another type of PPIase, (IC₅₀ = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.     

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Obesity-related metabolic abnormalities, including chronic inflammation and oxidative stress, increase the risk of colorectal cancer. Dysregulation of the renin–angiotensin system (RAS) also plays a critical role in obesity-related metabolic disorders and in several types of carcinogenesis. In the present study, we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and angiotensin-II type 1 receptor blocker (ARB), both of which inhibit the RAS, on the development of azoxymethane (AOM)-initiated colonic premalignant lesions in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given 4 weekly subcutaneous injections of AOM (15 mg/kg body weight), and then, they received drinking water containing captopril (ACE inhibitor, 5 mg/kg/day) or telmisartan (ARB, 5 mg/kg/day) for 7 weeks. At sacrifice, administration of either captopril or telmisartan significantly reduced the total number of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, compared to that observed in the control group. The expression levels of TNF-α mRNA in the colonic mucosa of AOM-treated db/db mice were decreased by captopril and telmisartan. Captopril lowered the expression levels of TNF-α, IL-1β, IL-6, and PAI-1 mRNAs, while telmisartan lowered the expression levels of COX-2, IL-1β, IL-6, and PAI-1 mRNAs in the white adipose tissues of these mice. In addition, these agents significantly reduced the levels of urinary 8-OHdG, a surrogate marker of oxidative damage to DNA, in the experimental mice. These findings suggested that both ACE inhibitor and ARB suppress chemically-induced colon carcinogenesis by attenuating chronic inflammation and reducing oxidative stress in obese mice. Therefore, targeting dysregulation of the RAS might be an effective strategy for chemoprevention of colorectal carcinogenesis in obese individuals.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.     

The Arabidopsis calcium-dependent protein kinase, CPK6, functions as a positive regulator of methyl jasmonate signaling in guard cells

Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca(2+) signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca(2+) signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca(2+)-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.