Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin.

In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (lambda greater than 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 leads to P380 and P380 leads to rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 leads to P380 and P380 leads to rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 leads to P380.     

Characteristics of phospholipids in microvillar membranes of octopus photoreceptor cells.

Characteristics of lipids in the microvillar membranes of octopus photoreceptor cells were studied in order to obtain some information on the membrane environment with rhodopsin in the invertebrate. (1) The membranes contain lipid and protein in almost equal proportion. The majority of lipids are phospholipids. Neutral lipids make up 16% of the total lipids, the major constituent of which is cholesterol. (2) Phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. Phosphatidylserine, ceramide 2-aminoethylphosphonate and sphingomyelin occur as minor components. An unidentified alkaline and acid stable phospholipid was found. (3) The predominant fatty acids of phosphatidylethanolamine and phosphatidylcholine are highly unsaturated such as 22 : 6, 20 : 5 and 20 : 4. The 22 : 6 and 20 : 5 are exclusively linked at the 2-position, but the 20 : 4 is linked significantly at the 1-position of the phospholipids. (4) Major molecular species are 16 : 0/22 : 6 (48.4%) and 16 : 0/20 : 4 (19.6%) in phosphatidylcholine, and 20 : 4/22 : 6 (50.7%) and 16 : 0/22 : 6 (25.6%) in phosphatidylethanolamine.     

Synthesis, properties and reactions of 3 beta-benzoyloxy-7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-ene.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (I) with gaseous HCl in chloroform at -40 degrees C gave, in 87% yield, 3 beta-benzoyloxy-7 alpha,15 beta-dichloro-5 alpha cholest-8(14)-ene (III). Reduction of the latter compound with lithium aluminum hydride in ether at room temperature for 20 min gave, in 86% yield, 7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-en-3 beta-ol (IV). The latter compound was fully characterized and assignments of the individual carbon peaks in the 13C nuclear magnetic resonance spectra of this sterol have been completed. Reduction of III with excess lithium aluminum hydride in refluxing ether for 4 days gave, in 74% yield, 5 alpha-cholesta-7,14-dien-3 beta-ol (VI). Reduction of the dichloro-steryl benzoate III with lithium triethylborohydride in tetrahydrofuran gave, in 88% yield, 5 alpha-cholest-8(14)-en-3 beta-ol (VII). A similar reduction using lithium triethylborodeuteride led to the formation of [7 beta, 15 xi-2 H2]-VIIa. Treatment of III with concentrated HCl in a mixture of chloroform and methanol gave, in 79% yield, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one (II) which was characterized as such and as the corresponding free sterol.     

Chromosomal aberrations and morphological transformation in hamster embryonic cells treated with potassium dichromate in vitro

The addition of K2Cr2O7, at concentrations ranging from 0.1 to 0.5 microng/ml, to hamster total embryonic cells for 24 h, resulted in consistent and drastic chromosomal aberrations including gaps, breaks and exchanges. The above effect, however, was reduced successfully by the addition of a reducing agent, Na2SO3. Among other chromium compounds examined, divalent and trivalent chromium salts were ineffective on chromosome morphology even at a concentration of 3.5 microng/ml as chromium, whereas a hexavalent compound, CrO3, was highly effective. K2Cr2O7 also enhanced the morphological transformation rate in a short-term colony assay, in whicy hamster embryonic cells (1x10(4) cells/60-mm dish) were treated and the morphology was observed 8 to 10 days after the treatment.     

Visualization of Ras-PI3K interaction in the endosome using BiFC

Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidylinositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome.     

High density lipoprotein binding protein of eel (Anguilla japonica) liver with specificity of binding to apoAI as a ligand

High-density lipoprotein (HDL) binding protein (HBP) was isolated from the microsomal fraction of eel liver homogenate by affinity chromatography with a HDL-column. After SDS-PAGE and blotting, HBP on the PVDF membrane was detected by FITC-labeled HDL and apolipoprotein AI (apoAI) as a ligand. HBP in the microsomal fraction was most abundant among microsomal, mitochondrial and cytosolic fractions. The HBP isolated by a HDL-column consisted of at least three proteins with low molecular weights of 18.5, 14.5 and 13.5 kDa; the main component was 14.5 kDa. These proteins are not products of protease digestion, as the procedure was carried out in the presence of protease inhibitors including (p-aminophenyl) methansulfonyl fluoride, 4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, aprotinin and EDTA. The HBP specifically bound to FITC-apoAI and faintly bound or did not bind to FITC-apoAII. Furthermore, binding of HDL labeled with lipophilic fluorescence to isolated eel hepatocytes was inhibited by the antibody to apoAI, but not inhibited by the antibody to apolipoprotein AII (apoAII). These results strongly suggest that the HBP isolated from the microsomal fraction is present on the plasma membrane of eel liver and plays important roles for the lipid transport through the interaction with HDL.     

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca²⁺-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 Å resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short β-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and β-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca²⁺-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.     

Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.     

6-Shogaol and 6-gingerol, the pungent of ginger, inhibit TNF-alpha mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes

In this study, we demonstrated that the two ginger-derived components have a potent and unique pharmacological function in 3T3-L1 adipocytes via different mechanisms. Both pretreatment of 6-shogaol (6S) and 6-gingerol (6G) significantly inhibited the tumor necrosis factor-α (TNF-α) mediated downregulation of the adiponectin expression in 3T3-L1 adipocytes. Our study demonstrate that (1) 6S functions as a PPARγ agonist with its inhibitory mechanism due to the PPARγ transactivation, and (2) 6G is not a PPARγ agonist, but it is an effective inhibitor of TNF-α induced c-Jun-NH₂-terminal kinase signaling activation and thus, its inhibitory mechanism is due to this inhibitory effect.