Stimulation of Prostaglandin EP2 Receptors on RGC-5 Cells in Culture Blunts the Negative Effect of Serum Withdrawal

Reduced neurotrophic support is one possible cause for retinal ganglion cells dying in glaucoma. Experiments were designed to investigate the effect of EP2 receptor agonist butaprost on transformed retinal ganglion (RGC-5) cells where reduced neurotrophic support was simulated by serum withdrawal. Cultures were analysed for cell viability, flow cytometry, reactive oxygen species and apoptosis. Western blot and immunohistochemistry were used to provide information for the occurrence of PGE₂ receptor-types. We demonstrated the existence of all four types of PGE₂ receptors in RGC-5 cells and exposure of cultures to butaprost resulted in an elevation of cAMP. Serum deprivation induced RGC-5 cell death was significantly attenuated by butaprost as well as by rolipram and forskolin where intracellular cAMP levels were increased. These data are of value in relation to the possible use of EP2 receptor agonists to reduce both elevated intraocular pressure and retinal ganglion cell death as occurs in glaucoma.     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

Protective effect of ginseng sapogenins against 2,2'-azobis (1-aminopropane) dihydrochloride (AAPH)-induced LLC-PK₁ cell damage

The effect of ginseng sapogenins, aglycone parts of ginsenosides, against oxidative damage by radical generator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), in renal epithelial LLC-PK(1) cells was investigated to identify the structural characteristics of sapogenins to have renoprotective effects. Of the tested sapogenins, Δ(20(21))-protopanaxatriol showed the strongest protective effect against the AAPH-induced LLC-PK(1) cell damage. Based on the structure and stronger activity of Δ(20(21))-protopanaxatriol than the other sapogenins, the hydroxyl group in C-6 and double bond in C-20(21) position were important for renoprotective effect of sapogenin against oxidative stress.     

BRCA2 fine-tunes the spindle assembly checkpoint through reinforcement of BubR1 acetylation

Germline mutations that inactivate BRCA2 promote early-onset cancer with chromosome instability. Here, we report that BRCA2 regulates the spindle assembly checkpoint (SAC). Previously, we reported that BubR1 acetylation is essential for SAC activity. In this study we show that BRCA2 recruits the PCAF acetyltransferase and aids in BubR1 acetylation during mitosis. In the absence of BRCA2, BubR1 acetylation is abolished, and the level of BubR1 decreases during mitosis. Similarly, Brca2-deficient mouse embryonic fibroblasts exhibited weak SAC activity. Transgenic mice that were engineered to have interruptions in the BRCA2-BubR1 association exhibited marked decrease of BubR1 acetylation, weakened SAC activity, and aneuploidy. These transgenic mice developed spontaneous tumors at 40% penetrance. Moreover, immunohistochemical analyses of human breast cancer specimens suggested that BRCA2 mutation and BubR1 status is closely linked. Our results provide an explanation for how mutation of BRCA2 can lead to chromosome instability without apparent mutations in SAC components.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

Predominant Clonal Accumulation of CD8+ T Cells with Moderate Avidity in the Central Nervous Systems of Theiler's Virus-Infected C57BL/6 Mice

Induction of antigen-specific CD8⁺ T cells bearing a high-avidity T-cell receptor (TCR) is thought to be an important factor in antiviral and antitumor immune responses. However, the relationship between TCR diversity and functional avidity of epitope-specific CD8⁺ T cells accumulating in the central nervous system (CNS) during viral infection is unknown. Hence, analysis of T-cell diversity at the clonal level is important to understand the fate and function of virus-specific CD8⁺ T cells. In this study, we examined the Vβ diversity and avidity of CD8⁺ T cells specific to the predominant epitope (VP2_121-130) of Theiler''s murine encephalomyelitis virus. We found that Vβ6⁺ CD8⁺ T cells, associated with epitope specificity, predominantly expanded in the CNS during viral infection. Further investigations of antigen-specific Vβ6⁺ CD8⁺ T cells by CDR3 spectratyping and sequencing indicated that distinct T-cell clonotypes are preferentially increased in the CNS compared to the periphery. Among the epitope-specific Vβ6⁺ CD8⁺ T cells, MGX-Jβ1.1 motif-bearing cells, which could be found at a high precursor frequency in naïve mice, were expanded in the CNS and tightly associated with gamma interferon production. These T cells displayed moderate avidity for the cognate epitope rather than the high avidity normally observed in memory/effector T cells. Therefore, our findings provide new insights into the CD8⁺ T-cell repertoire during immune responses to viral infection in the CNS.Theiler''s murine encephalomyelitis virus (TMEV) is a member of the Cardiovirus genus within the Picornaviridae family (43). This virus is a common enteric pathogen among wild mice but rarely causes neurological disease (57). However, when it infects susceptible mice (e.g., the SJL/J [SJL] strain) intracerebrally, it reproducibly induces a chronic immune-mediated demyelinating disease that has been studied as an infectious model of human multiple sclerosis (MS) (10, 30). In contrast, infection of resistant mice like those of the C57BL/6 (B6) strain results in strong antiviral immune responses that clear the virus effectively and prevent disease development (24, 31). Therefore, immune responses in B6 mice have been often compared to those in susceptible SJL mice to understand the nature of protective versus pathogenic immunity in these mice.It has been shown that the major histocompatibility complex (MHC) H-2D locus is a critical genetic factor for resistance to TMEV-induced demyelinating disease (9, 49). For example, expression of the H-2D^b transgene makes susceptible FVB mice resistant by inducing strong H-2D^b-restricted VP2_121-130-specific CD8⁺ T-cell responses (36). This acquired resistance is abolished when VP2_121-130-specific T cells are tolerized by introducing the VP2 transgene (45). These results strongly suggest that CD8⁺ T cells generated in the presence of H-2D^b are critical for viral clearance from the central nervous system (CNS). Since the cardinal difference between the resistant B6 and susceptible SJL strains is the quantity, not the quality, of virus-specific CD8⁺ T cells (23, 32), strong CD8⁺ T-cell responses are probably required to prevent viral persistence and the consequent development of demyelinating disease. More than threefold more virus-specific CD8⁺ T cells were found in the CNSs of resistant B6 mice than in those of susceptible SJL mice at the acute phase of infection. Thus, the level of virus-specific CD8⁺ T cells at an early phase of the immune response may be a critical factor in resistance to the disease.Many recent investigations indicate that oligoclonal CD8⁺ T cells accumulate in the CNSs of MS patients (4, 38, 51). In addition, CD8⁺ T cells may also induce the development of experimental autoimmune encephalomyelitis (EAE) (54). Therefore, clonal expansion of certain CD8⁺ T cells may be associated with the pathogenesis of demyelinating diseases. However, B6 mice, which are resistant to TMEV-induced demyelinating disease, induce strong CD8⁺ T-cell responses to a single predominant epitope (VP2_121-130), i.e., ≥70% of CNS-infiltrating CD8⁺ T cells (41, 42). These CD8⁺ T cells result in effective viral clearance yet remain at a low level in the CNS more than 120 days postinfection (dpi) without detectable pathology (42). This inconsistency led us to investigate the shape and quality of the T-cell receptor (TCR) repertoire accumulating in the CNSs of B6 mice.The CD8⁺ T-cell responses induced after viral infection have previously been investigated with other animal viruses, including influenza virus, lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), and Borna disease virus (11, 14, 35, 47, 58). Among these models, the detailed T-cell Vβ repertoire in the CNS was described only in the MHV model (46). CD8⁺ T-cell responses against TMEV in B6 mice are primarily against a single predominant epitope (22, 36, 41). However, virtually no study of the TCR Vβ repertoires of virus-specific CD8⁺ T cells has been reported. Furthermore, it is not yet known whether a particular TCR Vβ repertoire is associated with the avidity and/or function of CD8⁺ T cells in the CNS. Since protective versus pathogenic CD8⁺ T cells may correlate with their Vβ repertoire and T-cell function, these studies may help to elucidate the underlying mechanisms of protection versus pathogenesis of CD8⁺ T cells in the CNS.In this study, we have addressed several important questions about the CD8⁺ T-cell repertoire in the CNS. First, what is the pattern of Vβ usage in TMEV-infected B6 mice? Second, are there differences in the antigen-specific CD8⁺ T-cell clonotypes between the CNS and periphery? Third, are the T-cell clonotypes maintained in the CNS during the viral infection? Fourth, what is the functional avidity of T cells accumulating in the CNS during this virus infection? Last, what possible factors are associated with repertoire selection and expansion in the CNS? Our results show that Vβ6⁺ CD8⁺ T cells preferentially expand in the CNS during viral infection. Further analyses of the CDR3 region of antigen-specific Vβ6⁺ CD8⁺ T cells by spectratyping and sequencing indicate that distinct T-cell clonotypes are expanded in the CNS compared to those in the periphery. T cells expressing a particular Vβ6-CDR3-Jβ1.1 sequence are preferentially retained in the CNS during the course of viral infection. Interestingly, these T cells are capable of producing gamma interferon (IFN-γ) upon stimulation and display moderate avidity for the cognate epitope. We believe that our findings will provide important information regarding the CD8⁺ T-cell repertoire during viral infection and that these results may help to provide a better understanding of antiviral CD8⁺ T-cell immunity in the CNS.     

ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled

Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/β-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with β-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/β-catenin signaling pathway by phosphorylating Dsh.     

Molecular mechanisms governing plant responses to high temperatures

The increased prevalence of high temperatures(HTs) around the world is a major global concern, as they dramatically affect agronomic productivity. Upon HT exposure, plants sense the temperature change and initiate cellular and metabolic responses that enable them to adapt to their new environmental conditions.Decoding the mechanisms by which plants cope with HT will facilitate the development of molecular markers to enable the production of plants with improved thermotolerance. In recent decades, genetic, physiological, molecular, and biochemical studies have revealed a number of vital cellular components and processes involved in thermoresponsive growth and the acquisition of thermotolerance in plants. This review summarizes the major mechanisms involved in plant HT responses, with a special focus on recent discoveries related to plant thermosensing, heat stress signaling, and HT-regulated gene expression networks that promote plant adaptation to elevated environmental temperatures.     

Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai

Antimicrobial activity of the 18 prenylated flavonoids, which were purified from five different medicinal plants, was evaluated by determination of MIC using the broth microdilution methods against four bacterial and two fungal microorganisms (Candida albicans, Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis and S. aureus). Papyriflavonol A, kuraridin, sophoraflavanone D and sophoraisoflavanone A exhibited a good antifungal activity with strong antibacterial activity. Kuwanon C, mulberrofuran G, albanol B, kenusanone A and sophoraflavanone G showed strong antibacterial activity with 5–30 μg/ml of MICs. Morusin, sanggenon B and D, kazinol B, kurarinone, kenusanone C and isosophoranone were effective to only gram positive bacteria, and broussochalcone A was effective to C. albicans. IC₅₀ values of papyriflavonol A, kuraridin, sophoraflavanone D, sophoraisoflavanone A and broussochalcone A in HepG2 cells were 20.9, 37.8, 39.1, 22.1, and 22.0 μg/ml, respectively. These results support the use of prenylated flavonoids in Asian traditional medicine to treat microbial infection and indicate a high potential for prenylated flavonoids as antimicrobial agents as well as anti-inflammatory agents.