Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

Lilliputians get into the limelight: Novel class of small peptide genes in morphogenesis

Generally, bioactive small peptides are derived from precursors with signal sequences at their N-terminal ends, which undergo modification and proteolysis through a secretory pathway. By contrast, small peptides encoded in short open reading frames (sORF) lack signaling sequences and therefore are released into the cytoplasm, which may result in their having functions distinct from those of secreted peptides. Several small peptides encoded by sORF are involved in the morphogenesis of multicellular organisms. POLARIS, ROTUNDIFOLIA4, and Enod40 are plant peptides that are involved, respectively, in root formation, leaf shape control, and cortical cell division during nodule formation. Brick1 / HSPC300 is an evolutionarily conserved component of the actin reorganization complex. polished rice / tarsal-less and mille-pattes encode related small peptides that are required for epithelial morphogenesis in Drosophila and segmentation in Tribolium . There are only a few known examples of small peptides encoded by sORF, and their molecular functions are still largely obscure. Nevertheless, an increasing number of sORF genes is being identified, and further research should reveal their roles in novel molecular mechanisms underlying developmental events.     

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.     

Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4(+) T cell lines generated in vitro were adoptively transferred into Rag-2(-/-) mice with primed B cells derived from Dsg3-immunized Dsg3(-/-) mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Ralpha in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.     

MDA-MB-231 produces ATP-mediated ICAM-1-dependent facilitation of the attachment of carcinoma cells to human lymphatic endothelial cells

We examined the effects of supernatants of culture media of MDA-MB-231 and MCF-7 cells on the expression of adhesion molecules on human lymphatic endothelial cells (LECs) and evaluated whether the overexpression of adhesion molecules facilitated the attachment of carcinoma cells to LECs. The 48-h stimulation of MDA-MB-231, but not MCF-7, supernatant produced a significant expression of ICAM-1 on human LECs but little or no expression of E-selectin. Chemical treatment with dialyzed substances of <1,000 molecular weight (MW) caused a complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating, digestion with protease, or chemical treatment with dialyzed substances of <500 MW produced no significant effect on the supernatant-mediated response. ATP (10(-7) M) caused overexpression of ICAM-1 on human LECs similar to that produced by the supernatant of MDA-MB-231. The ATP- and MDA-MB-231 supernatant-mediated responses were significantly reduced by treatment with 10(-6) M suramin (a purinergic P2X and P2Y receptor antagonist). In attachment assays, 10(-7) M ATP or MDA-MB-231 supernatant produced a significant increase in the attachment of carcinoma cells to human LECs. The treatment with 10(-6) M suramin caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. Additional treatment with anti-ICAM-1 antibody also caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. The experimental findings suggest that MDA-MB-231 may release or leak ATP, which produces the overexpression of ICAM-1 on human LECs through activation of purinergic P2X and/or P2Y receptors and then facilitates ICAM-1-mediated attachment of carcinoma cells to LECs.     

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.     

The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2

PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.     

Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.     

Synthesis of Methylenecyclobutyl- and Cyclobutenyl Adenine,Potent Antiviral Carbocyclic Analogues of Oxetanocin A

Abstract

9-Cyclobutyladenines bearing both methylene and hydroxymethyl groups, 3 and 4, were prepared by dehydration of carbocyclic oxetanocin A (1a). Introduction of a double bond into cyclobutane ring was achieved by allylic oxidation of N ⁶-benzoyl-9-[3-methylenecyclobutyl]adenine (12), which after several steps, afforded 9-[3-(hydroxy-methyl)-2-cyclobutenyl)adenine (5).