Sesquiterpene constituents from the liverwort Bazzania japonica

The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC-MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (-)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni²⁺ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more -helix and less -structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.     

Age-related changes of element contents in human tendon of the iliopsoas muscle and the relationships among elements

To elucidate compositional changes of the tendons with aging, the authors investigated age-related changes of element contents in the tendon of the iliopsoas muscle by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 14 men and 14 women, ranging in age from 4 to 102 yr. The insertion tendons of the iliopsoas were removed from the cadavers after ordinary dissection and also surgically from patients with congenital dislocation of the hip. It was found that both Mg and P in the tendon of the iliopsoas muscle increased significantly with aging, but the other elements, such as S, Ca, Na, Zn, Fe, and Si, hardly changed with aging. With regard to the relationship among element contents, extremely significant correlations were found between P and S contents, between Mg and Na contents, and between S and Zn contents. In addition, very significant correlations were found between Si and either S or Zn contents. However, there was no significant correlation between Ca and P contents. These results revealed that with regard to age-related changes of element contents and the relationships among element contents, the tendon of the iliopsoas muscle was different from the Achilles tendon, in which both Mg and P decreased significantly with aging and there were significant correlations each other among the contents of S, Mg, and P.     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.     

A missense mutation (GGC[435Gly]-->AGC[Ser]) in exon 8 of the CYP11B2 gene inherited in Japanese patients with congenital hypoaldosteronism

OBJECTIVES: To clarify the underlying molecular mechanism of corticosterone methyl oxidase type II (CMO II) deficiency, Japanese patients newly diagnosed with CMO II deficiency were investigated. METHODS: We analyzed the patients' genomic DNA sequence on all 9 exons of the CYP11B2 gene. In addition, restriction fragment length polymorphism (RFLP) analysis and expression studies were performed. RESULTS: The analysis showed that the patients homozygously retained a missense mutation, Gumacr;GC[435Gly]-->Aumacr;GC[Ser], in the CYP11B2 gene. Expression studies indicated that the steroid 18-hydroxylase/oxidase activities of the mutant enzyme were substantially reduced. CONCLUSION: These results support the hypothesis that this mutation causes CMO II deficiency in the patients, and are in accordance with our theory that the partial loss of P-450(C18) activities causes CMO II deficiency.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Spatial differences and seasonal changes of net carbonate accumulation on some coral reefs of the Ryukyu Islands, Japan

This study sought to understand short-term spatial changes in accretion and erosion on (experimental) carbonate blocks on three coral reefs of the Ryukyu Islands, Japan. The principal objectives were to differentiate net accretion/erosion according to season, location, depth and substrate-type. At all locations the summer season showed more positive net weight changes and higher coralline algal coverage than the winter season. Windward reefs revealed higher net accretion and higher coralline algal coverage than leeward reefs. Massive (Holocene) Porites blocks showed highest net loss, followed by Pleistocene carbonate and (Holocene) Acropora blocks. High population densities of Echinometra mathaei (de Blainville) were recorded on reefs adjacent to large human populations and overall net carbonate loss significantly correlated to densities of E. mathaei type A.