Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis

Phosphatidylinositol-3,4,5-trisphosphate (PtdInsP(3)) regulates diverse cellular functions, including cell proliferation and apoptosis, and has roles in the progression of diabetes and cancer. However, little is known about its production. Here, we describe fluorescent indicators for PtdInsP(3) that allow a spatio-temporal examination of PtdInsP(3) production in single living cells. After ligand stimulation, PtdInsP(3) levels increased to a larger extent at the endomembranes (that is, the endoplasmic reticulum and the Golgi) than at the plasma membrane. This increase was found to originate from in situ production at the endomembranes, a process stimulated directly by receptor tyrosine kinases endocytosed from the plasma membrane to the endomembranes. The demonstration of PtdInsP(3) production through receptor endocytosis addresses a long-standing question about how signalling pathways downstream of PtdInsP(3) are activated at intracellular compartments remote from the plasma membrane.     

Synthesis of fluorescent-labeled aeruginosin derivatives for high-throughput fluorescence correlation spectroscopy assays

The design and solid-phase synthesis of effective fluorescent-labeled aeruginosin derivatives and their application to the fluorescence correlation spectroscopy (FCS)-based competitive binding assay of an aeruginosin library are described. The phenolic hydroxyl group on the (R)-3-(4-hydroxyphenyl)lactic acid (d-Hpla) residue was observed to be suitable for connecting Rhodamine green derivative with minimum loss of biological activity. In addition, the FCS-based binding assay of the library using fluorescent-labeled chemical probes was also achieved.     

Insulin-like growth factor-I stimulates cell proliferation in the outer layer of Hertwig’s epithelial root sheath and elongation of the tooth root in mouse molars in vitro

To elucidate the mechanism of root formation in tooth development, we examined the role of insulin-like growth factor I (IGF-I) on early root formation in mandibular first molar teeth from 5-day-old mice. Immunohistochemistry revealed the specific localization of the IGF-I receptor in Hertwigs epithelial root sheath (HERS) in the tooth root. The effect of IGF-I on root development, especially on HERS, was subsequently examined in vitro. The control culture showed normal development of HERS and the periodontium, resembling that in vivo. However, the presence of 100 ng/ml IGF-I resulted in elongation of HERS and increased cell proliferation in its outer layer. These effects were negated by the addition of antibodies specific for IGF-I. Thus, we propose that IGF-I is involved in early root formation by regulating the mitotic activity in the outer layer of HERS.This study was partly supported by grants to N.F. from KAKENHI (no. 13671909), to N.F. and M.J.T. from the Promotion of 2001-Multidisciplinary Research Projects in 2001–2005, and to N.F. and K.I. from Iwate Medical University—Keiryokai Research Foundation (no. 64).     

The Endemic Fritillaria Species of Japan

Summary.  This paper reviews and illustrates the seven known species of Fritillaria subgenus Fritillaria that are endemic to Japan and describes a new, eighth species, Fritillaria tokushimensis Akasawa, Katayama et Naito. A key for their identification is provided.     

Photoswelling and light-inactivation of isolated chloroplasts I. Change in lipid content in light-aged chloroplasts

The formation of lipid peroxide and changes in the lipid compositionof isolated chloroplasts aged in the light or dark, were investigatedin more detail. Lipid peroxide formation was observed in thethylakoid membrane as well as in the supernatant from dark-agedchloroplasts. Light was necessary for its formation in bothsystems. We confirmed that the peroxidation of lipids formedduring aging did not induce the inhibition of photochemicalactivities in chloroplasts. Aged chloroplasts underwent decompositionof their endogeneous monogalactolipid and phosphatidylcholine(lecithin) resulting in free fatty acids and lysophosphatidylcholine(lysolecithin). Decomposition of monogalactolipid occurred inboth the light- and dark-aged chloroplasts. The change of lecithinto lysolecithin was stimulated by illumination. This suggeststhat the peroxidation of lipids occurs as a result of the illuminationof free fatty acids released from monogalactolipid and lecithinin the thylakoid membranes, and that the change of lecithinto lysolecithin is related to the inactivation of photochemicalactivities and to swelling in light-aged chloroplasts. ¹ Present address: Department of Microbiology, Ishikawa ResearchLaboratory for Public Health and Environment, Minma, Kanazawa,Japan. (Received August 15, 1974; )     

Determination of Biotinylated Proteins as an Index for Purification of Plasma Membrane using Surface Plasmon Resonance-based Optical Biosensor

Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Synthesis and structure–activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.