Comprehensive identification of terpene synthase genes and organ-dependent accumulation of terpenoid volatiles in a traditional medicinal plant Angelica archangelica L.

Angelica archangelica L. is a traditional medicinal plant of Nordic origin that produces an unusual amount and variety of terpenoids. The unique terpenoid composition of A. archangelica likely arises from the involvement of terpene synthases (TPSs) with different specificities, none of which has been identified. As the first step in identifying TPSs responsible for terpenoid chemodiversity in A. archangelica, we produced a transcriptome catalogue using the mRNAs extracted from the leaves, tap roots, and dry seeds of the plant; 11 putative TPS genes were identified (AaTPS1-AaTPS11). Phylogenetic analysis predicted that AaTPS1-AaTPS5, AaTPS6-AaTPS10, and AaTPS11 belong to the monoterpene synthase (monoTPS), sesquiterpene synthase (sesquiTPS), and diterpene synthase clusters, respectively. We then performed in vivo enzyme assays of the AaTPSs using recombinant Escherichia coli systems to examine their enzymatic activities and specificities. Nine recombinant enzymes (AaTPS2-AaTPS10) displayed TPS activities with specificities consistent with their phylogenetics; however, AaTPS5 exhibited a strong sesquiTPS activity along with a weak monoTPS activity. We also analyzed terpenoid volatiles in the flowers, immature and mature seeds, leaves, and tap roots of A. archangelica using gas chromatography-mass spectrometry; 14 monoterpenoids and 13 sesquiterpenoids were identified. The mature seeds accumulated the highest levels of monoterpenoids, with ß-phellandrene being the most prominent. α-Pinene and ß-myrcene were abundant in all organs examined. The in vivo assay results suggest that the AaTPSs functionally identified in this study are at least partly involved in the chemodiversity of terpenoid volatiles in A. archangelica.

Excessive gingival display treated with 2-piece segmental Le Fort I osteotomy: A case report.

BACKGROUND: The demand for orthognathic surgery has increased worldwide. Women with jaw deformity tend to have a worse quality of life than men owing to the deformity's negative effects on body image, low self-esteem, lack of self-confidence, and dissatisfaction with life. Therefore, they wish for more reliable treatment options. CASE DESCRIPTION: A woman aged 25 years and 9 months sought treatment for a convex profile and excessive gingival display caused by a skeletal Class II jaw-base relationship. Gingival exposure was up to 6.5 millimeters at full smile. She chose orthognathic surgery, and the authors performed a 2-piece segmental Le Fort I osteotomy and bilateral sagittal split ramus osteotomy. After active orthodontic treatment, the protrusive profile was improved, and an acceptable occlusion and an attractive smile were achieved. PRACTICAL IMPLICATIONS: It is hoped that 2-piece segmental Le Fort I osteotomy becomes a common treatment option for patients with protrusive profiles and excessive gingival displays.

A photoinduced growth system of peptide nanofibres addressed by DNA hybridization.

Spatiotemporal control of peptide nanofibre growth was achieved by photocleavage of a DNA-conjugated ß-sheet forming peptide that is linked through a photoresponsive amino acid residue. Peptide nanofibres were selectively formed by photocleaving the conjugate on a complementary DNA-immobilised glass substrate.

Adhesive Materials Utilizing a Thymine-Adenine Interaction and Thymine Photodimerization.

A cross-linking reagent having two adenine units was mixed with polymers bearing thymine units to form adhesive materials utilizing both intermolecular hydrogen bonding and in situ formation of covalent bonds. In the case of 58 mol % of adenine units relative to thymine units, formation of intermolecular hydrogen bonds because of a thymine-adenine interaction was observed at room temperature using FT-IR. The peel strength became weak with heating above 60 °C, indicating breakup of intermolecular bonds between thymine and adenine units. On the other hand, UV-vis spectral measurements showed that heating at 80 °C with 254 nm light irradiation facilitated the photodimerization reaction between thymine units even in the presence of adenine cross-linking reagents. This result was consistent with a large value for the peel strength (6.5 N/10 mm) after the dual treatment, heating at 80 °C with 400 mJ/cm2 of UV irradiation.

Imidazole Derivatives with an Intramolecular Hydrogen Bond as Thermal Latent Curing Agents for Thermosetting Resins.

Epoxy resins are important thermosetting resins widely used in industrial applications. Though imidazoles as curing agents have attracted particular attention because of their high reactivity in chain polymerizations with epoxides, polymerization of a liquid epoxy resin containing imidazoles proceeds gradually even at room temperature. This makes it difficult to use such mixtures as one-component materials for industrial applications. To improve the shelf life of the mixutures, we have developed a very simple and powerful thermal latent curing agent, a 2-(2-hydroxyphenyl)imidazole derivative (1), having an intramolecular hydrogen bond between the phenolic hydroxyl group and the nitrogen atom of the imidazole ring, leading to suppression of reactivity of 1 toward epoxy resins at room temperature. It was confirmed that high reactivity of 1 toward epoxy resins at 150 °C was based on breakage of the intramolecular hydrogen, whereas the epoxy resin composition showed long-term storage stability at room temperature.

Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco.

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kß1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.

Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins.

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.

Mandibular reconstruction using intraoral trifocal bone transport: report of a case.

On the mandibular reconstruction after tumor resection, it is easy to achieve esthetic and functional results when mandibular defect is relative small, however, it is difficult to reconstruct adequately larger defected mandible. Recently, with progress of devices, distraction osteogenesis that is the method of tissue regeneration is used as mandibular reconstruction. A 19-year-old male patient presented complaining of right lower jaw swelling. Biopsy suspected a multiple-cystic ameloblastoma in mandible. Under the general anesthesia, a mandibulectomy was performed from the right side ramus to the left side incisor. A mandibular reconstruction plate was attached to the proximal and distal bone segments. 2 types of intraoral distraction devices were placed inside the plate. These devices had 25 mm and 60 mm distraction length. After 9 days of latency, trifocal bone transport was started by 0.5 mm 2 times activation per day. After consolidation for 23 weeks, reconstruction plate and distraction devices were removed. 2.5 m x 2.0 cm iliac bone and cancellous bone were placed in the docking site with platelet rich plasma. The mandibular defect (85 mm) was reconstructed adequately using intraoral distraction osteogenesis trifocal bone transport technique. Symmetric facial balance was achieved. Now there is no recurrence and dental implants were placed on new bone.

Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides.

In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism.

Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides.

Phosphoinositides are believed to be involved in fundamental cellular events such as signal transduction and vesicular trafficking. Aberrant metabolisms of this lipid, caused by mutations in phosphoinositide kinases, phosphatases and lipases are known to be related to variety of human disorders such as diabetes and cancer. While the majority of such information is obtained by analyzing genetic and biochemical properties of phosphoinositide-metabolic enzymes, direct measurement of cellular content of the lipid is hindered by the lack of a simple method that is sensitive enough to measure phosphoinositides present in trace amounts in vivo. Here, we describe a novel, thin layer chromatography (TLC)-based method by which cellular phosphoinositides are separated, transferred and detected by specific phosphoinositide-binding domains. This method was applied to follow the generation of minor phosphoinositides, such as PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to insulin and to compare PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels in several cancer cell lines. The method has potential application not only in investigating the physiological roles of phosphoinositides, but also in diagnosing metabolic disease and cancer by directly assessing phosphoinositide levels in samples obtained from patients.

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis.

The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.

[A case of metastatic lung cancer responding well from tongue carcinoma using a combination of stereotactic radiation therapy and docetaxel/cisplatin/5-FU chemotherapy].

A 53-year-old man underwent partial glossectomy for tongue cancer on the right side (well-differentiated squamous cell carcinoma T 1 N 0 M 0). One and 9 months later, cervical lymph node metastasis was observed bilaterally. After bilateral radical neck dissection, 2 courses of NDP/5-FU combined chemotherapy were administered. However, 2 metastatic lung cancers were observed 14 months after initial treatment. Stereotactic radiation therapy of 50 Gy in 5 fractions and 6 courses of docetaxel/cisplatin/5-FU chemotherapy were administered. As a result, no recurrence was observed, and a complete response was obtained for 41 months after lung metastasis.

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool.

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.

Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4.

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, beta-, gamma-, delta-, epsilon-, and zeta-type. PLCdelta-type is reported to be composed of four isozymes, PLCdelta1-delta4. Here we report that a screening for mouse PLCdelta2 from a BAC library with primers that amplify a specific region of bovine PLCdelta2 resulted in isolation of one clone containing the mouse PLCdelta4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCdelta2 and PLCdelta4 in the mouse and human genomes, indicating that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4. However, PLCdelta2 Western blot analysis with a widely used commercial anti-PLCdelta2 antibody showed an expression pattern distinct from that of PLCdelta4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCdelta2, was smaller than an 85-kDa band detected by anti-PLCdelta4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCdelta4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCdelta2 antibody contained no PLC activity. From these data, we conclude that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4, and that three isozymes (delta1, delta3, and delta4) exist in the PLCdelta family.

Archaeal peptidyl prolyl cis-trans isomerases (PPIases) update 2004.

PPIases are ubiquitous in living organisms. While three families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases. Among 13 cyclophilins found in Archaea, only Halobacterium cyclophilin (HbsCyP19) has been characterized. This is a cyclosporin A (CsA) sensitive CyP with a molecular weight of 19.4 kDa. The PPIase activity and CsA sensitivity of HbsCyP19 is higher at higher salt concentration in the medium. No parvulin except a homolog in Cenarchaeum symbiosum has been found in Archaea. Two types of FKBPs, 26-30 kDa long-type and 17-18 kDa short-type FKBP, have been found in Archaea. Up to date, 12 short-type FKBPs and 18 long-type FKBPs have been known. The short-type FKBPs and N-terminal sequences of the long-type FKBPs are similar to each other and show homology to human FKBP12 (HsFKBP12). However, they have two insertion sequences in the regions corresponding to bulge and flap loops of HsFKBP12. The long-type archaeal FKBPs have additional ca. 100 amino-acid sequences at their C-terminal regions. A short-type archaeal FKBP from Methanothermococcus thermolithotrophicus has not only a PPIase activity but also a chaperone-like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates. Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the flap seemed to be important. Three-dimensional structure of this FKBP showed that the insertion in the flap makes a domain which has a hydrophobic surface. Coexpression of aggregation prone proteins with these archaeal FKBPs were shown to improve their expression in soluble fraction in Escherichia coli. Fusion protein of the archaeal FKBP and an aggregation prone protein also show improved expression of the latter in E. coli.

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities.

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.