Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one‐step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole‐cell Candida antarctica lipase B‐expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase‐expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.     

Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.     

Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): insights from plastid matK and nuclear ribosomal ITS sequences

We conducted phylogenetic analyses using two DNA sequence data sets derived from matK, the maturase-coding gene located in an intron of the plastid gene trnK, and the internal transcribed spacer region of 18S–26S nuclear ribosomal DNA to examine relationships in subtribe Aeridinae (Orchidaceae). Specifically, we investigated (1) phylogenetic relationships among genera in the subtribe, (2) the congruence between previous classifications of the subtribe and the phylogenetic relationships inferred from the molecular data, and (3) evolutionary trends of taxonomically important characters of the subtribe, such as pollinia, a spurred lip, and a column foot. In all, 75 species representing 62 genera in subtribe Aeridinae were examined. Our analyses provided the following insights: (1) monophyly of subtribe Aeridinae was tentatively supported in which 14 subclades reflecting phylogenetic relationships can be recognized, (2) results are inconsistent with previous classifications of the subtribe, and (3) repeated evolution of previously emphasized characters such as pollinia number and apertures, length of spur, and column foot was confirmed. It was found that the inconsistencies are mainly caused by homoplasy of these characters. At the genus level, Phalaenopsis, Cleisostoma, and Sarcochilus are shown to be non-monophyletic.     

A role of EphB4 receptor and its ligand,ephrin-B2, in erythropoiesis

Erythropoiesis is regulated not only by erythropoietin but also by microenvironments which are composed of transmembrane molecules. We have previously shown that a receptor tyrosine kinase EphB4 is predominantly expressed on human erythroid progenitors in bone marrow. EphB4 is expressed in approximately 45% of hematopoietic progenitor cells, which are CD34-positive and c-Kit-positive in human umbilical cord blood (hUCB). The transmembrane ligand for EphB4 or ephrin-B2 is expressed on bone marrow stromal cells and arterial endothelial cells. When such EphB4-positive hematopoietic progenitor cells were co-cultured with stromal cells which express ephrin-B2, they were immediately detached from stromal cells and differentiated to mature erythroid cells. At that time, expression of EphB4 immediately down-regulated. In contrast, on ephrin-B2 non-expressing stromal cells, they remained EphB4-positive cells and the generated number of mature erythroid cells was less than that on ephrin-B2 expressing stromal cells. Additionally, ephrin-B2 expression on endothelial cells up-regulated under hypoxic condition. Taken together, we propose that one of the molecular cues that regulate erythropoiesis is ephrin-B2 on stromal cells.     

Inhibition by nitric oxide of Ca(2+) responses in rat pancreatic alpha-cells

This study examined the effect of nitric oxide (NO) on the cytosolic free Ca(2+) concentration ([Ca(2+)](c)) of alpha-cells isolated from rat pancreatic islets. When extracellular glucose was reduced from 7 to 0 mM, about half of the alpha-cells displayed [Ca(2+)](c) oscillations. Nicardipine, a Ca(2+) channel blocker, terminated the oscillations, while thapsigargine, an inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, did not affect them, suggesting that the [Ca(2+)](c) oscillations were produced by periodic Ca(2+) influx via L-type voltage-operated Ca(2+) channels. NOC 7, an NO donor, did not cause any changes in [Ca(2+)](c) at 7 mM glucose, but reduced [Ca(2+)](c) or terminated [Ca(2+)](c) oscillations at 0 or 2.8 mM glucose. A similar inhibitory effect on [Ca(2+)](c) of alpha-cells was caused by 8-bromo-cGMP. When the [Ca(2+)](c) of alpha-cells was elevated by L-arginine in the presence of N(omega)-nitro-L-arginine, an NO synthase inhibitor, the subsequent application of NOC 7 and 8-bromo-cGMP reduced [Ca(2+)](c). As there is a direct relationship between [Ca(2+)](c) and glucagon release, these results suggest that the NO-cGMP system in rat pancreatic islets reduces glucagon release by suppressing [Ca(2+)](c) responses in alpha-cells.     

Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic,terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.     

Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart

The serine-threonine kinase, Akt, inhibits cardiomyocyte apoptosis acutely both in vitro and in vivo. However, the effects of chronic Akt activation in the heart are unknown. To address this issue, we generated transgenic mice (TG+) with cardiac-specific expression of a constitutively active mutant of Akt (myr-Akt) driven by the myosin heavy chain-alpha promoter. Three TG+ founders (9-19 weeks) died suddenly with massive cardiac dilatation. Two viable TG+ lines (TG564 and TG20) derived from independent founders demonstrated cardiac-specific transgene expression as well as activation of Akt and p70S6 kinase. TG564 (n = 19) showed cardiac hypertrophy with a heart/body weight ratio 2.3-fold greater than littermates (n = 17, p < 0.005). TG20 (n = 18) had less marked cardiac hypertrophy with a heart/body weight ratio 1.6-fold greater than littermates (n = 17, p < 0.005). Isolated TG564 myocytes were also hypertrophic with surface areas 1.7-fold greater than littermates (p < 0.000001). Echocardiograms in both lines demonstrated concentric hypertrophy and preserved systolic function. After ischemia-reperfusion, TG+ had a 50% reduction in infarct size versus TG- (17 +/- 3% versus 34 +/- 4%, p < 0.001). Thus, chronic Akt activation is sufficient to cause a spectrum of phenotypes from moderate cardiac hypertrophy with preserved systolic function and cardioprotection to massive cardiac dilatation and sudden death.     

Changes in the proliferative activity of epidermal melanocytes in serum-free primary culture during the development of ultraviolet radiation B-induced pigmented spots in hairless mice

Long-term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR-1 X HR/De)F1. To clarify the cellular mechanism for the development of these UVB-induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation. Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum-free medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation). Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small-sized pigmented spots) and 37 (the stage of development of medium-sized pigmented spots) weeks after the cessation of 8-week UVB exposure. At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed. With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes. These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB-irradiated skin increases with the development of pigmented spots.