Constant variation in structure and function of geometrical isomers of acitretin under natural light

Acitretin, a beneficial retinoid, was shown to undergo constant structural interconversions among its geometrical isomers (all-trans-acitretin, 9-cis-acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin, etc.) by photoisomerization under natural light. The photoisomerization was zero order reaction with an apparent velocity of 4x107 M/min under illumination by white fluorescent lamps (1, 200 lx). An equilibrium mixture of the geometrical isomers (all-trans-acitretin 20%, 9-cis-acitretin 15%, 13-cis-acitretin 30%, 9, 13-di-cis-acitretin 15%, and unidentified compounds 20%) was formed at around 30 min. Equilibrium mixtures with similar composition were obtained by photoisomerization reactions starting from other geometrical isomers. Geometrical isomers of acitretin thus formed, showed different effects to induce differentiation of human acute promyelocytic leukemia cells (HL-60 cells): activity of all-trans-acitretin (ED50, 3.2 x 10(-6) M), 9-cis-acitretin (ED50, 2.3 x 10(-5)M), 13-cis-acitretin (ED50, 1.1 x 10(-5)M), 9, 13-di-cis-acitretin (ED50, 2.6 x 10(-6)M). 9-cis-Acitretin acted synergistically with all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin on HL-60 cells. On the other side, all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin acted additively. Geometrical isomers of acitretin showed different effects on differentiation of human epidermal keratinocytes; expression of keratinocyte differentiation markers, keratin 1 and keratin 10, were suppressed more strongly by 9-cis-acitretin and 13-cis-acitretin as compared to all-trans-acitretin or 9, 13-di-cis-acitretin.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

Design and functional analysis of actomyosin motor domain chimera proteins

To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia,Oreochromis niloticus

Piscine DAX1 and SHP cDNAs with an open reading frame encoding 296 and 258 amino acid residues, respectively, as well as SHP partial gene fragment, were cloned from Nile tilapia. Phylogenetic analyses of DAX1s, SHPs, and homologous EST fragments indicate that DAX1 and SHP are conserved in gene structure and are present throughout vertebrates. A single band of approximately 1.4kb for DAX1 and of approximately 1.2kb for SHP was detected in the Northern blot analysis. Tissue distribution analysis by RT-PCR showed that fish DAX1 and SHP mRNAs are widely expressed in adult tissues, with the most abundant expression in gonads and liver, respectively. DAX1 and SHP were also detected in gonads of both sexes at 5-90 days after hatching (dah). However, the expression of DAX1 is weak at 5 and 10dah and then significantly up-regulated between 10 and 15dah, whereas the expression of SHP is moderate and consistent during the ontogeny.     

Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation

Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton.     

Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.     

Inducible brain COX-2 facilitates the recurrence of hippocampal seizures in mouse rapid kindling

Brain cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, is rapidly and transiently induced by convulsions in hippocampal and cortical neurons. Therefore, we examined the effects of COX-2 on the 'rapid kindling' development in COX-2 knockout mice and in mice treated with nimesulide, a COX-2-selective inhibitor. Rapid kindling development was examined based on the incidence of hippocampal EEG seizures and behavioral seizures following repetitive electrical stimulation of the perforant path at an interval of 40 s, and on the total afterdischarge (AD) duration induced by 50 stimulations. In addition, we measured COX-2 mRNA expression by in situ hybridization and PGE2 concentration using enzyme immunoassay following rapid kindling stimulation. The results suggested that brain COX-2 mRNA levels were markedly increased in the hippocampal neurons and the concentration of PGE2 was elevated significantly, and that the incidence of AD and seizure behavior induction and the total AD duration were significantly decreased under conditions of COX-2 deficiency. Therefore, we concluded that inducible COX-2 facilitates the recurrence of hippocampal seizures.     

Overproduction of highly active trypanosome alternative oxidase in Escherichia coli heme-deficient mutant

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in humans and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs, because it does not exist in the host. In this study, we established a system for overproduction of highly active TAO in Eschericia coli heme-deficient mutant. Kinetic analysis of recombinant enzyme and TAO in Trypanosoma brucei brucei mitochondria revealed that recombinant TAO retains the properties of native enzyme, indicating that recombinant TAO is quite valuable for further biochemical study of TAO.