Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.     

Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells

Chromosome aberrations such as loss of chromosome 13 were frequently observed in human endothelial cells from umbilical cord veins (HUVEC). A recent study showed that the length of telomeric single-stranded 3'-overhangs (G-tails) is more important as an essential structure for chromosome maintenance than the net telomere length in telomere t-loop formation. Here, we have examined G-tail length using G-tail telomere HPA in normal and hTERT-transduced HUVECs. We found that forced expression of hTERT in HUVEC induced G-tail as well as total telomere length elongation. G-tail length was well correlated with total telomere length. However, hTERT introduction did not prevent chromosome aberrations such as loss of chromosome 13. Normal characteristics such as morphology, up-regulation of vWF, and tube formation were observed in hTERT-HUVEC as in young normal HUVEC. These results show that chromosome aberrations in HUVEC are independent of telomere G-tail and total telomere attrition.     

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.     

Role of aluminum-binding ligands in aluminum resistance of Eucalyptus camaldulensis and Melaleuca cajuputi

We investigated the roles of Al-binding ligands in Al exclusion from roots and in internal Al detoxification in roots as Al resistance mechanisms in two Al-resistant Myrtaceae trees, Eucalyptus camaldulensis Dehnh. and Melaleuca cajuputi Powell. The amounts of ligands secreted from roots and contained in root tips of these species were compared with those of an Al-sensitive species, Melaleuca bracteata F. Muell., after the roots were exposed to 0 or 1 mM AlCl₃ solution. Secretion of well-known ligands (citrate, oxalate, and malate) from roots under Al treatment was low in all species. However, in E. camaldulensis, the Al-binding capacity of root exudates under Al treatment was considerable and was higher than that in M. bracteata. Gel filtration chromatography revealed that a low-molecular-weight Al-binding ligand was secreted from roots in response to Al only in E. camaldulensis. On the other hand, the Al-binding capacity of cell sap in root tips under Al treatment was similar for the resistant and sensitive species. These results suggest that Al exclusion by secretion of the unknown low-molecular-weight Al-binding ligand from roots contributes to the Al resistance of E. camaldulensis, whereas M. cajuputi has developed Al-resistance mechanisms other than secretion of ligands from roots or concentration of internal ligands in root tips.     

Exploration of the correlation between solvation dynamics and internal dynamics of a protein

Protein function is intimately related to the dynamics of the protein as well as to the dynamics of the solvent shell around the protein. Although it has been argued extensively that protein dynamics is slaved to solvent dynamics, experimental support for this hypothesis is scanty. In this study, measurements of fluorescence anisotropy decay kinetics have been used to determine the motional dynamics of the fluorophore acrylodan linked to several locations in a small protein barstar in its various structural forms, including the native and unfolded states as well as the acid and protofibril forms. Fluorescence upconversion and streak camera measurements have been used to determine the solvation dynamics around the fluorophore. Both the motional dynamics and solvent dynamics were found to be dependent upon the location of the probe as well as on the structural form of the protein. While the (internal) motional dynamics of the fluorophore occur in the 0.1-3 ns time domain, the observed mean solvent relaxation times are in the range of 20-300 ps. A strong positive correlation between these two dynamical modes was found in spite of the significant difference in their time scales. This observed correlation is a strong indicator of the coupling between solvent dynamics and the dynamics in the protein.     

Positive narrowing pharyngoplasty with forearm flap for functional restoration after extensive soft palate resection

The restoration of velopharyngeal function after extensive soft palate resection to treat malignant oropharyngeal tumors is a major challenge to reconstructive surgeons. The authors had previously reconstructed soft palatal defects routinely with the folded forearm flap. A patient who had more than half of the soft palate excised experienced postoperative velopharyngeal dysfunction. To restore efficient velopharyngeal function, pharyngoplasty was additively applied where the folded ridge of the forearm flap was sutured to the posterior pharyngeal wall in an inverse manner of the pharyngeal flap technique. The essence of the procedure was positive narrowing of the nasopharyngeal space. Five patients who underwent this pharyngoplasty and another five who did not were evaluated for postoperative functions of speech intelligibility and of nasal regurgitation during oral feeding. The velopharyngeal movements of all patients were examined under a nasopharyngeal endoscope. The evaluations demonstrated that this surgical procedure afforded satisfactory results. This positive narrowing pharyngoplasty technique is simple, easy, and minimally invasive to the remaining healthy tissue, and it is the method of choice for the reconstruction of the soft palate after malignant tumor resection.     

Identification of Pip4k2β as a mechanical stimulus responsive gene and its expression during musculoskeletal tissue healing

To investigate the mechano-transduction system of cells, we identified genes responsive to a cyclic mechanical stimulus. MC3T3.E1 cells were cultured on a computer-controlled vacuum-pump-operated device designed to provide a cyclic mechanical stimulus. A maximum elongation of 15% of membrane at 10 cycles/min (3 s extension followed by 3 s relax per cycle) was repeated for 48 h. By means of a differential display, the gene expression pattern of cells exposed to the stimulus was compared with that of unexposed cells. As a result, a gene fragment that was exclusively expressed in mechanically stressed cells was identified. By using expressed sequence tag walking together with the oligo-capping method, this gene was identified as phosphatidylinositol 4-phosphate 5-kinase type II β (initially known as Pip5k2β but now reclassified as Pip4k2β). The specific up-regulation of Pip4k2β upon mechanical stimulus was also confirmed by using another apparatus, viz. a computer-controlled linearized-stepping motor system. To examine the involvement of the cyclic mechanical stimulus in the regulation of Pip4k2β expression in musculoskeletal tissue, we created an Achilles tendon transection model in rabbits. The temporal expression of Pip4k2β was assessed by means of a quantitative reverse-transcribed polymerase chain reaction. In the gastrocnemius muscle, expression of Pip4k2β rapidly decreased 1 week after transection but was restored to normal levels at 4 weeks. In the Achilles tendon, however, expression remained decreased until 4 weeks after transection. We suggest that the expression of Pip4k2β can be used as a marker for cells receiving a suitable mechanical stimulus. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.