Cyclic peptide analysis of the biologically active loop region in the laminin alpha3 chain LG4 module demonstrates the importance of peptide conformation on biological activity

The laminin alpha3 chain LG4 module (alpha3LG4 module) has cell adhesion, heparin binding, migration, and neurite outgrowth activities. The LG4 module consists of a 14-stranded beta-sheet (A-N) sandwich structure. Previously, we identified the A3G756 sequence (KNSFMALYLSKGRLVFALG in the human laminin alpha3 chain 1411-1429) as a biologically active site in the alpha3LG4 module. The A3G756 sequence is located on the E and F strands based on a crystal structure-based sequence alignment. The Lys1421 and Arg1423 residues, critical amino acids for the biological activity of A3G756, are located on the E-F connecting loop region as a KGR sequence. In this study, we focused on the KGR sequence and investigated the structural requirements of the E-F connecting loop region in the alpha3LG4 module. We synthesized three linear peptides containing the KGR sequence at the middle and the N and C termini and also prepared three cyclic analogues corresponding to the linear peptides. cyclo-hEF3A (CLYLSKGRLVFAC), which is a cyclic peptide containing the KGR sequence at the middle, showed the strongest inhibitory effect on both the heparin binding and the cell attachment to the recombinant alpha3LG4 module protein. The cyclo-hEF3A peptide was more active for syndecan-4 binding and neurite outgrowth than the linear form. Furthermore, we found that the structure of cyclo-hEF3A is similar to that of the connecting E-F loop region in human laminin alpha3LG4 module by structural analysis using molecular dynamics simulations. These results suggest that the loop structure of the E-F connecting region of the alpha3LG4 module is important for its biological activities. The cyclo-hEF3A peptide may be useful for the development of therapeutic reagents especially for wound healing and nerve regeneration.     

Hypermetabolism of fat in V1a vasopressin receptor knockout mice

[Arg8]Vasopressin (AVP) has an antilipolytic action on adipocytes, but little is known about the mechanisms involved. Here, we examined the involvement of the V1a receptor in the antilipolytic effect of AVP using V1a receptor-deficient (V1aR-/-) mice. The levels of blood glycerol were increased in V1aR-/- mice. The levels of ketone bodies, such as acetoacetic acid and 3-hydroxybutyric acid, the products of the lipid metabolism, were increased in V1aR-/- mice under a fasting condition. Triacylglyceride and free fatty acid levels in blood were decreased in V1aR-/- mice. Furthermore, measurements with tandem mass spectrometry determined that carnitine and acylcarnitines in serum, the products of beta-oxidation, were increased in V1aR-/- mice. Most acylcarnitines were increased in V1aR-/- mice, especially in the case of 2-carbon (C2), C10:1, C10, C14:1, C16, C18:1, and hydroxy-18:1-carbon (OH-C18:1)-acylcarnitines under feeding rather than under fasting conditions. The analysis of tissue C2-acylcarnitine level showed that beta-oxidation was promoted in muscle under the feeding condition and in liver under the fasting condition. An in vitro assay using brown adipocytes showed that the cells of V1aR-/- mice were more sensitive to isoproterenol for lipolysis. These results suggest that the lipid metabolism is enhanced in V1aR-/- mice. The cAMP level was enhanced in V1aR-/- mice in response to isoproterenol. The phosphorylation of Akt by insulin stimulation was reduced in V1aR-/- mice. These results suggest that insulin signaling is suppressed in V1aR-/- mice. In addition, the total bile acid, taurine, and cholesterol levels in blood were increased, and an enlargement of the cholecyst was observed in V1aR-/- mice. These results indicated that the production of bile acid was enhanced by the increased level of cholesterol and taurine. Therefore, these results indicated that AVP could modulate the lipid metabolism by the antilipolytic action and the synthesis of bile acid via the V1a receptor.     

A Root Growth-promoting Factor,Capillarol, from Artemisia capillaris Thunb

A root growth-promoting factor was isolated from Artemisia capillaris Thunb. The chemical structure of this compound was elucidated as methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate (capillarol) on the basis of its spectroscopic analysis. At 5×10^–4m this compound promoted rice root growth to 180% of the control value.     

Real-Time Visualization of Neuronal Activity during Perception

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Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.     

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.     

FINE STRUCTURE OF THE OCTOPUS RETINA

The fine structure of the visual and the supporting cells and of the blood capillaries in the octopus retina is described. Lamellated structures contained in the proximal segment of the visual cell consist of compact arrays of dense membranes each of which is quintuple-layered and divides at its margins into two thinner sheets or membranes which are connected directly with the agranular or granular endoplasmic reticulum. Proximal to the deeper extremities of the rhabdomeres, the lateral plasma membranes of two adjoining visual cells contact each other forming a quintuple-layered compound membrane, which results in occlusion of the intercellular space. The central layer of the compound membrane is of high density, so that the membrane, as a whole, appears to be a single thick layer at low magnifications. The supporting cells are connected with the neighboring visual cells by two types of junctions. Long slender processes extend from the supporting cells to the surface of the retina through narrow spaces among the distal segments of the visual cells. The capillary endothelial cells are characterized by luminal surfaces irregularly contoured and by lateral surfaces elaborately interdigitated. The functional significance of the close contact between adjoining visual cells is discussed.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.