Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Decrease in polyamines with aging and their ingestion from food and drink

Changes in polyamine levels during aging were measured in 3-, 10- and 26-week-old female mice. The level of polyamines in pancreas, brain, and uterus was maintained during these periods. The level of spermidine slightly decreased in intestine, and decreased significantly in thymus, spleen, ovary, liver, stomach, lung, kidney, heart and muscle during these periods. In skin, the level of spermidine was maximal in 10-week-old mice and markedly reduced in 26-week-old mice. The results suggest that maintenance of polyamine levels may play important roles in the function of the pancreas, brain and uterus in 3- to 26-week-old mice. We next looked for polyamine-rich food materials as a dietary source of polyamines. Foods found to be rich in polyamines included wheat germ, rice bran, black rice, Philippine mango, green pepper, Japanese pumpkin, nuts, fermented pickles, pond smelt, turban shell viscera, whelk viscera, salted salmon roe, salted cod roe, beef intestine (boiled) and liver of eel, beef, pork and chicken; and, as previously reported, soybean, fermented soybean (natto), mushrooms, orange and green tea leaf. These results offer useful information when it becomes necessary to ingest polyamines from food.     

Decoding Reveals Plasticity in V3A as a Result of Motion Perceptual Learning

Visual perceptual learning (VPL) is defined as visual performance improvement after visual experiences. VPL is often highly specific for a visual feature presented during training. Such specificity is observed in behavioral tuning function changes with the highest improvement centered on the trained feature and was originally thought to be evidence for changes in the early visual system associated with VPL. However, results of neurophysiological studies have been highly controversial concerning whether the plasticity underlying VPL occurs within the visual cortex. The controversy may be partially due to the lack of observation of neural tuning function changes in multiple visual areas in association with VPL. Here using human subjects we systematically compared behavioral tuning function changes after global motion detection training with decoded tuning function changes for 8 visual areas using pattern classification analysis on functional magnetic resonance imaging (fMRI) signals. We found that the behavioral tuning function changes were extremely highly correlated to decoded tuning function changes only in V3A, which is known to be highly responsive to global motion with human subjects. We conclude that VPL of a global motion detection task involves plasticity in a specific visual cortical area.     

Systematic SAR study of the side chain of nonsecosteroidal vitamin D₃ analogs

A series of nonsecosteroidal vitamin D(3) analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal which was bound by (E)-olefin linker to the bisphenyl core. Calculating the volume of the side chain showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coefficient based on the 55% rule, and that this resulted in the most potent in vitro activity.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Use of flowering gene <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in the phylogenetic analysis of bambusoid and early diverging grasses

A nuclear gene, FLOWERING LOCUS T (FT) homolog, was cloned from Phyllostachys meyeri as PmFT. Its putative copy number was estimated as four by Southern blot analysis, and the two copies were completely sequenced. Twenty-seven FT homolog sequences of bambusoid and early diverging grasses comprised 172-bp exons, and 357- to 785-bp introns exhibited 0-58.9% pairwise divergence with six modal levels. Parsimony analyses of the FT homologs rooted at Pharus virescens produced six equally parsimonious trees. In the strict consensus tree, five clades were resolved; they were affected by divergence of the intron region rather than exon region. The basal clade was Puelioideae, followed by Olyreae clade including Oryza sativa. Streptogyneae clade combined the Olyreae clade with terminal sister clades of the Bambuseae, i.e., pantropical bamboos and East Asiatic temperate bamboos. The global topology suggested that FT homologs are significant for resolving the tribe level. However, the phylogeny of FT homologs does not resolve monophyly in Bambusoideae because of intercalary positioning by Streptogyneae clade. We discussed the role of FT homologs in controlling the inflorescence architecture and position of Streptogyneae in the bamboo phylogeny.     

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca²⁺ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca²⁺ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus. This revised version was published online in August 2006 with corrections to the Cover Date.