Developmental and Visual Input-Dependent Regulation of the CB1 Cannabinoid Receptor in the Mouse Visual Cortex

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.     

Induction of intratumoral tumor necrosis factor by a synthetic lipid A analog, ONO-4007, with less tolerance in repeated administration and its implication in potent antitumor effects with low toxicity

To evaluate the anti-tumor characteristics of ONO-4007, a synthetic analog of lipid A, the authors examined its acute toxicity and anti-tumor activity in a mouse MM46 mammary tumor system in comparison with LA-15-PP, an E. coli-type synthetic lipid A and LPS. Systemic and local (tumor site) induction of tumor necrosis factor (TNF) by a single i.v. shot of ONO-4007 and LA-15-PP correlated with manifestation of their toxicity, showing that ONO-4007 is 100-fold less effective than LA-15-PP. However, a protocol of repeated administration (3 shots twice a week) exhibited about 10 times more therapeutic potency of ONO-4007 for cancer therapy than expected in the above experiments. In a dose inducing submaximal systemic and intratumoral TNF production, repeated injections (twice a week) of ONO-4007 (10 mg/kg), LA-15-PP (0.1 mg/kg) and LPS (0.1 mg/kg) commonly generated a tolerant state in the systemic response (serum and liver) to subsequent stimulation. The intratumoral response was retained with this repeated administration of ONO-4007, but was not with LA-15-PP or LPS. TIM (tumor-infiltrating macrophages) isolated from mice pre-injected with ONO-4007 and LA-15-PP were found to lose their response to both substances, but the response was rapidly recovered until 72 h after injection and virtually no difference was observed in their response to either drug. The in vitro treatment of naive TIM with ONO-4007 or LA-15-PP for 2 h depressed the response to both substances and the depression continued for 72 h even in culture with fresh medium. The relatively high efficacy of ONO-4007 in cancer therapy likely depends on the retraction of the tolerant state, especially at the tumor site where the response to ONO-4007 is recovered much more efficiently than that to lipid A. While constant recruitment of macrophages to tumor tissue might be involved in the difference of tolerance recovery between this region and others, selective response to ONO-4007 may not be explained simply by the sensitivity of recruited TIM. Pharmacokinetical experiments revealed that repeated injections of LA-15-PP enhanced its clearance from blood circulation, while the clearance of ONO-4007 was stable after repeated injections. Thus, pharmacokinetical properties of ONO-4007 may also possibly be implicated in this event.     

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.     

Mechanism of rapid-phase insulin response to elevation of portal glucose concentration

The hepatoportal region is important for glucose sensing; however, the relationship between the hepatoportal glucose-sensing system and the postprandial rapid phase of the insulin response has been unclear. We examined whether a rapid-phase insulin response to low amounts of intraportal glucose infusion would occur, compared that with the response to intrajugular glucose infusion in conscious rats, and assessed whether this sensing system was associated with autonomic nerve activity. The increases in plasma glucose concentration did not differ between the two infusions at 3 min, but the rapid-phase insulin response was detected only in the intraportal infusion. A sharp and rapid insulin response was observed at 3 min after intraportal infusion of a small amount of glucose but not after intrajugular infusion. Furthermore, this insulin response was also induced by intraportal fructose infusion but not by nonmetabolizable sugars. The rapid-phase insulin response at 3 min during intraportal infusion did not differ between rats that had undergone hepatic vagotomy or chemical sympathectomy with 6-hydroxydopamine compared with control rats, but this response disappeared in rats that had undergone chemical vagotomy with atropine. We conclude that the elevation of glucose concentration in the hepatoportal region induced afferent signals from undetectable sensors and that these signals stimulate pancreas to induce the rapid-phase insulin response via cholinergic nerve action.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

Preclinical Validation of Talaporfin Sodium-Mediated Photodynamic Therapy for Esophageal Squamous Cell Carcinoma

Photodynamic therapy (PDT) kills cancer cells via a photochemical reaction mediated by an oncotropic photosensitizer. Herein, we performed an experimental preclinical study to validate the anti-tumour effect of talaporfin sodium-mediated PDT (t-PDT) for esophageal squamous cell carcinoma (ESCC) cells. We used human ESCC cells derived from various differentiation grades or resistant to 5-fluorouracil (5-FU). The cytotoxic effect of t-PDT was determined by evaluating cell viability, apoptosis and generation of reactive oxygen species (ROS) and DNA double-strand breaks. Furthermore, the anti-tumour effect of t-PDT was assessed using an anchorage-independent cell-growth assay and xenograft transplantation models. t-PDT induced potent cytotoxicity in ESCC cells independent of their differentiation grade or 5-FU resistance. Moreover, t-PDT induced robust apoptosis, as indicated by cell shrinkage, perinuclear vacuolization, nuclear fragmentation and induction of annexin V-positive cells. This apoptotic response was accompanied by concurrent activation of ROS, and induction of DNA double-strand breakage. Importantly, t-PDT suppressed efficiently anchorage-independent cell growth as well as ESCC-xenografted tumor formation. In aggregate, t-PDT showed anti-tumor potential for ESCC cells with various histological grades or chemoresistance, providing a novel translational rationale of t-PDT for the treatment of ESCC.     

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.     

Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.