Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested-the large rim of beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.     

The retinal pigment epithelium of Cr-deficient rats

The purpose of this study is to investigate the effect of Cr deficiency on the rat retina. Three-week-old Wistar Kyoto rats were divided into 2 groups. Cr-deficient rats were fed AIN-93G diet without Cr and deionized distilled water. Control rats were fed AIN-93G diet and deionized distilled water. The Cr and sugar concentrations in the whole blood and cholesterol concentration in the serum were measured. We observed the retina with an electron microscope, and counted phagocytized lamellar structures in the retinal pigment epithelium (RPE) before and after the start of light exposure on negative electron microscopic films. The whole blood Cr level of Cr-deficient rats was less than 0.2 microg/l. The blood sugar level of Cr-deficient rats was significantly higher than that of normal rats (p < 0.05). There were significantly more phagocytized lamellar structures in the RPE of Cr-deficient rats 1, 2, 7, 11 and 12 h after the start of light exposure than in that of normal rats (p < 0.05). However, no morphological abnormalities were found in the photoreceptor cells of Cr-deficient rats. Phagocytosis in the photoreceptor outer segment discs in the RPE was accelerated, but the pattern of the retinal circadian rhythm with maximum phagocytosis 2 h after exposure to light was unchanged. The Cr-deficient state may cause the membrane to degenerate, and phagocytosis of the photoreceptor outer segment discs in the RPE may be accelerated. This study provided an evidence of the nutritional importance of Cr in rat retina.     

CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

Suppressive effect of polysaccharides from the edible and medicinal mushrooms,Lentinus edodes and Agaricus blazei,on the expression of cytochrome P450s in mice

To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Balancing the central roles of the thylakoid proton gradient

The photosynthetic electron transfer chain generates proton motive force (pmf), composed of both electric field (Deltapsi) and concentration (DeltapH) gradients. Both components can drive ATP synthesis, whereas the DeltapH component alone can trigger feedback regulation of the antenna. It has often been suggested that a relatively large pmf is needed to sustain the energetic contributions of the ATP synthase reaction (DeltaG(ATP)), and that the Deltapsi component is dissipated during illumination, leading to an acidic lumen in the light. We suggest that this is incompatible with the stabilities of lumenal components and the observed activation of downregulation. Recent work on the chloroplast ATP synthase suggests that a more moderate pmf can sustain DeltaG(ATP). In addition, in vivo probes suggest that a substantial fraction of pmf can be stored as Deltapsi. Together, these factors should allow sufficient DeltaG(ATP) to maintain lumen pH in a range where lumenal enzyme activities are nearly optimal, and where the level of NPQ is regulated.     

Soybean germplasm pools in Asia revealed by nuclear SSRs

Soybean was domesticated in East Asia, where various kinds of landraces have been established as a result of adaptation to different environments and the diversification of food cultures. Asia is thus an important germplasm pool of soybean. In order to evaluate the genetic structure of the Asian soybean population, we analyzed allelic profiles at 20 simple-sequence repeat (SSR) loci of 131 accessions introduced from 14 Asian countries. The SSR loci produced an average of 11.9 alleles and a mean gene diversity of 0.782 in the accessions tested. Quantification theory III analysis and cluster analysis with the UPGMA method clearly separated the Japanese from the Chinese accessions, suggesting that the Japanese and Chinese populations formed different germplasm pools. The Korean accessions were involved in both germplasm pools, whereas most of the accessions from southeast and south/central Asia were derived from the Chinese pool. Relatively high genetic diversity and the absence of region-specific clusters in the southeast and south/central Asian populations suggest that soybean in these areas has been introduced repeatedly and independently from the diverse Chinese germplasm pool. The present study indicates that the two germplasm pools can be used as exotic genetic resources to enlarge the genetic bases of the respective Asian soybean populations.     

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.