Involvement of ceramide in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation in primary cultured cortical neurons of rats.

The complex molecular cascades of ischemic tolerance in brain cells remain unclear. Recently, sphingolipid-related metabolite ceramide has been implicated as a second messenger in many biological functions, including neuronal survival and death. The present study, therefore, examined the roles of ceramide (Cer) in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) using primary cultured cortical neurons of rats. Preconditioning of the neurons with sublethal 1-h OGD produced robust neuroprotection against cell death induced by lethal 3-h OGD imposed 12 h after preconditioning when measured by the MTT assay. Analysis of sphingolipids using LC-MS/MS showed that the ischemic preconditioning resulted in significant increases in the levels of C(16 : 0) Cer, C(18 : 0) Cer, C(20 : 0) Cer, C(24 : 0) Cer, C(24 : 1) Cer and the total ceramide contents compared with the sham-washed control group. However, sphingomyelin contents were not significantly changed by the ischemic preconditioning, suggesting that ceramides were increased through the de novo synthetic pathway. In the case of severe OGD paradigm, levels of ceramide and sphingomyelin in the lethal OGD group were not significantly different from those of the control group or the lethal OGD group with preconditioning at any time points studied. Treatment with an inhibitor of de novo ceramide synthesis, fumonisin B(1), during the ischemic preconditioning period completely blocked preconditioning-induced ischemic tolerance. Moreover, application of a non-cytotoxic concentration of exogenous cell-permeable ceramide produced neuroprotection against lethal OGD. The results suggest that ceramides increased by sublethal OGD preconditioning play an important role in induction of ischemic tolerance.

Screening and identification of virus-encoded RNA silencing suppressors.

RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities.

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase.

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).

A crown reconstructing method for resin-embedded transparent teeth.

OBJECTIVE: Resin-embedded transparent teeth are devoid of the original crown morphology, because enamel is lost by demineralization. This study was designed to reproduce artificial enamel and to reconstruct the original crown morphology for resin-embedded transparent teeth. METHOD AND MATERIALS: The impression of the coronal portion of human permanent teeth was taken with a silicone impression material. After demineralization, drawing ink was injected into the pulp cavities. The ink-infiltrated teeth were made transparent with methyl salicylate and embedded with polyester resin. Urethane prepolymer was injected into the impression, and the resin-embedded teeth were reinserted into the impression. After polymerization of the urethane resin, the specimens and the urethane resin were removed from the impression. RESULTS: The original crown morphology of the resin-embedded transparent teeth could be precisely reconstructed with artificial and removable enamel. The resin-embedded teeth showed morphologic details of the black-stained pulp cavities through the transparent dentin and cementum. CONCLUSION: This study established a crown reconstructing method for resin-embedded transparent teeth. The specimens will be useful for demonstration of morphology of teeth and pulp cavities.