Fatty acid derivatives and dammarane triterpenes from the glandular trichome exudates of Ibicella lutea and Proboscidea louisiana

Ibicella lutea and Proboscidea louisiana, both of the Martyniaceae family, are known for rich glandular trichomes on their leaves and stems. Chemical investigations of the glandular trichome exudates on leaves of the two plants furnished three types of secondary metabolites, glycosylated fatty acids, glycerides (2-O-(3,6-diacetyloxyfattyacyl)glycerols and 2-O-(3-acetyloxyfattyacyl)glycerols) and dammarane triterpenes. The glycosylated fatty acids from I. lutea were determined to be 6(S)-(6-O-acetyl-β-d-glucopyranosyloxy)-octadecanoic acid (1A), -eicosanoic acid (1B) and -docosanoic acid (1C), as well as their respective deacetyl congeners (2A, 2B and 2C), whereas P. louisiana furnished 8(S)-(6-O-acetyl-β-d-glucopyranosyloxy)-eicosanoic acid (3A) and -docosanoic acid (3B) and their respective deacetyl congeners (4A and 4B), together with 2B. Both plants contained 12 identical 2-O-[(3R,6S)-3,6-diacetyloxyfattyacyl]glycerols (5A-L), in which the fatty acyl moieties contained between 17 and 21 carbon atoms. The corresponding mono-acetyloxy compounds, 2-O-[(3R)-3-acetyloxyfattyacyl]glycerols (6A–L) were detected in both plants. Among these glycerides, ten compounds (5A, 5C, 5F, 5H, 5K, 6A, 6C, 6F, 6H and 6K) had iso-fattyacyl structures and four (5E, 5J, 6E and 6J) had anteiso-fattyacyl structures. A previously unknown dammarane triterpene, betulatriterpene C 3-acetate (7), was isolated together with three known dammarane triterpenes, 24-epi-polacandrin 1,3-diacetate (8), betulatriterpene C (9) and 24-epi-polacandrin 3-acetate (10) from I. lutea, whereas 12 dammarane triterpenes, named probosciderols A–L (12–23), and the known compound betulafolienetriol (11) were isolated from P. louisiana. The structures of these compounds were elucidated by spectroscopic analysis including 2D-NMR techniques and chemical transformations. The 6-O-acetylglucosyloxy-fatty acids 1A–C (42%) and the dammarane triterpenes 7–10 (31%) were the two most abundant constituents in the glandular trichome exudate of I. lutea, whereas the dammarane triterpenes 11–23 (47%) and the glucosyloxy-fatty acids (4A, 4B and 2B) (38%) were the most abundant constituents in the glandular trichome exudate of P. louisiana.     

Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.     

Development of an Ultra-Rapid Diagnostic Method Based on Heart-Type Fatty Acid Binding Protein Levels in the CSF of CJD Patients

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck^® H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck^® H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck^® H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck^® H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.     

Structure and Polymorphism Analysis of Transforming Growth Factor Beta Receptor 1 (TGFBR1) in Pigs

Many quantitative trait loci (QTL) for growth and reproductive traits have been detected on the porcine chromosome region 1qter (SSC1qter), making it one of the most important genomic regions for pig breeding. SSC1q corresponds to human chromosome 9, on which lies transforming growth factor beta receptor 1 (TGFBR1). We cloned the porcine TGFBR1 cDNA and gene (as a candidate for QTL) and analyzed the gene structure and polymorphism. Porcine TGFBR1 consists of 9 exons and 8 introns. Intron 2 is alternatively spliced at the acceptor site, resulting in two kinds of mRNA, with putative open reading frames of 1500 and 1512 bp in length. The shorter one encodes 499 amino acid residues. The amino acid sequence has 96.2 and 97.2% sequence similarity to those of human and bovine TGFBR1, respectively. The sequence similarity between porcine and murine TGFBR1 is 95.6%. We detected three single-nucleotide substitutions in exons 1, 2, and 7. Those in exons 1 and 7 are nonsynonymous substitutions resulting in Pro8Ser and Ile413Val substitutions, respectively.     

SHIP2 interaction with the cytoskeletal protein Vinexin

The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin alpha and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5)P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2-/- MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5)P3 5-phosphatase and as a modulator of focal contact formation.     

The retinol-retinoic acid metabolic pathway is impaired in the lumbar spine of a rat model of congenital kyphoscoliosis

Although congenital scoliosis is defined as a genetic disease characterized by a congenital and abnormal curvature of the spinal vertebrae, our knowledge of the genetic underpinnings of the disease is insufficient. We herein show that the downregulation of the retinol-retinoic acid metabolism pathway is involved in the pathogenesis of congenital scoliosis. By analyzing DNA microarray data, we found that the expression levels of genes associated with the retinol metabolism pathway were decreased in the lumbar spine of Ishibashi rats (IS), a rat model of congenital kyphoscoliosis. The expression of Adh1 and Aldh1a2 (alcohol dehydrogenase), two enzymes that convert retinol to retinoic acid in this pathway, were decreased at both the gene and protein levels. Rarα, a receptor of retinoic acid and bone morphogenetic protein 2, which play a central role in bone formation and are located downstream of this pathway, were also downregulated. Interestingly, the serum retinol levels of IS rats were higher than those of wild-type control rats. These results indicate that the adequate conversion from retinol to retinoic acid is extremely important in the regulation of normal bone formation and it may also be a key factor for understanding the pathogenesis of congenital scoliosis.     

Phylogeography of Pulsatilla cernua (Ranunculaceae), a grassland species,in Japan

The genetic diversity and structure of Pulsatilla cernua, a continental‐grassland relict, were investigated using variations in chloroplast DNA (cpDNA) and microsatellites of nuclear DNA. In the analyses of three cpDNA regions, 17 haplotypes were found in 24 populations of P. cernua from Japan, Korea, and Russia. Although the route and time of migration between the continent of Asia and Japan could not be well resolved, the cpDNA haplotype network suggests the existence of several ancient lineages in Japan and a recent secondary migration from Japan to the continent. Microsatellite analyses did not indicate genetic structure among the Japanese populations, indicating the existence of gene flow across the distribution area until recently. These results indicate that the present fragmentation of P. cernua in Japan may reflect a rapid, recent reduction from a previously large, continuous distribution.     

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles

Amino Acids - Carnosine (β-alanyl-l-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine...     

The interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of tight junctions and adherens junctions

The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the PM by using a Ca(2+)-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C) inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13 competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.