Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons

We have reported previously that dopamine D₂ receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser³³² by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D₂ receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser³³² by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.     

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Background

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results

Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions

The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.     

Trace elements influenced by environmental changes in Lake Biwa: (I) Seasonal variations under suboxic hypolimnion conditions during 2007 and 2009

Dissolved oxygen (DO) in the Lake Biwa hypolimnion reached its lowest level of <1 mg kg^?1 in 2007. In this paper, we report the variations in the total dissolvable (TD), dissolved (D), and labile particulate (LP) fractions of Al, Si, P, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Mo, W, and U in Lake Biwa 2007 and 2009. Al and Fe species were predominantly in the form of LP-Al and LP-Fe and were strongly correlated with one another (r = 0.99), suggesting that the weathering of aluminous minerals and the supply of clay mineral particles are the main factors that influence the distributions of Al and Fe. Although D-Al increased in the summer epilimnion, D-Fe was relatively low, probably as a result of uptake by plants. Reductive release of Fe from the bottom was not seen. Mn was also dominated by LP-Mn, but this fraction showed a different distribution to those of LP-Al and LP-Fe. The D-Mn and LP-Mn concentrations varied by factors of 700–1000 and showed marked increases in the bottom water during stratification in 2007. We believe that Mn²⁺ was released from the sediments and oxidized by DO in the bottom water. Ni, Cu, Zn, and Cr, which exist as cationic species, had LP/TD ratios of 0.1–0.7 and relatively uniform distributions. Si, P, V, As, Mo, W, and U, which form oxoacid species, had LP/TD ratios of 0–0.8. Si, P, and As were characterized by nutrient-like profiles, V, W, and U showed summer maxima in the epilimnion, and Mo had a uniform distribution. TD-Mo increased in the bottom water along with TD-Mn, while TD-V and TD-W showed significant decreases. These results are likely attributable to differences in the adsorption of these elements onto manganese oxides and iron hydroxides.     

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

Antioxidant properties of aqueous extracts from red tide plankton cultures

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.     

Effect of histamine H1-receptor antagonist on the regulation of carbohydrate and lipid metabolism in rat liver

In order to elucidate the role of histamine in the liver, we studied the effect of a histamine H1-receptor antagonist on the carbohydrate and lipid metabolism in the rat liver. The administration of the H1-receptor antagonist decreased significantly the contents of glycogen and malonyl-CoA in the liver. However, it did not affect the levels of serum glucose and free fatty acid. These results suggest that histamine may play a part in the regulation of metabolism of carbohydrates and lipids in the liver.     

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.     

Indole-3-carbinol activates the cyclin-dependent kinase inhibitor p15(INK4b) gene

Indole-3-carbinol (I3C) is a naturally occurring compound found in vegetables such as broccoli and cauliflower, and has been shown to arrest human tumor cells in the G1 phase of the cell cycle. However, the molecular mechanism responsible for this effect has not been sufficiently elucidated. We report here that I3C activates the cyclin-dependent kinase (CDK) inhibitor p15INK4b gene through its promoter, accompanied by cell growth inhibition in HaCaT cells. Treatment with I3C almost did not affect the expressions of the other CDK inhibitors such as p19INK4d, p21WAF1 and p27Kip1. These results suggest that p15INK4b is an important molecular target of I3C among CDK inhibitors.     

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).