Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.     

Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).     

Terpenoids from Tripterygium doianum (Celastraceae)

The extract of Tripterygium doianum (Celastraceae) afforded three triterpenoids [3beta-acetoxy-11-ursen-13alpha,30-olide, 25-chloro-24-hydroxytirucall-7-en-3-one and tirucall-7-en-3,24-dione], two sesquiterpenoids [5alpha-acetoxy-1beta,8alpha-bis-cinnamoyl-4alpha-hydroxydihydroagarofuran and 5alpha-acetoxy-1beta-benzoyl-8alpha-cinnamoyl-4alpha-hydroxydihydroagarofuran] and nine known triterpenoids. Their structures were established based on spectroscopic studies.     

Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.)

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.     

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Identifying functional links between genes using conserved chromosomal proximity

Conservation of proximity of a pair of genes across multiple genomes generally indicates that their functions could be linked. Here, we present a systematic evaluation using 42 complete microbial genomes from 25 phylogenetic groups to test the reliability of this observation in predicting function for genes. We find a relationship between the number of phylogenetic groups in which a gene pair is proximate and the probability that the pair belongs to a common pathway. Our method produces 1586 links between ortholog families substantiated by observed proximity in genomes representing at least three phylogenetic groups. Of the pairs annotated in the KEGG database, 80% are in the same biological pathway in KEGG.     

Osteogenic growth peptide: from concept to drug design

Recently, the osteogenic growth peptide (OGP) and its C-terminal pentapeptide H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)] have attracted considerable clinical interest as bone anabolic agents and hematopoietic stimulators. They are present in mammalian serum in micromolar concentrations, increase bone formation and trabecular bone density, and stimulate fracture healing when administered to mice and rats. In cultures of osteoblastic and other bone marrow stromal cells, derived from human and other mammalian species, OGP regulates proliferation, alkaline phosphatase activity and matrix mineralization via an autocrine/paracrine mechanism. In vivo it also regulates the expression of type I collagen and the receptor for basic fibroblast growth factor. In addition, OGP and OGP(10-14) enhance hematopoiesis, including the stimulation of bone marrow transplant engraftment and hematopoietic regeneration after ablative chemotherapy. Apparently, the hematopoietic effects of these peptides are secondary to their effect on the bone marrow stroma. Detailed structure-activity relationship study identified the side chains of Tyr(10) and Phe(12) as the principal pharmacophores for OGP-like activity. Recently, it has been demonstrated that several cyclostereoisomers of OGP(10-14), including the analogue retro-inverso (Gly-Gly-D-Phe-Gly-D-Tyr), share the full spectrum of OGP-like bioactivities. Taken together, OGP represents an interesting case of a "housekeeping" peptide that plays an important role in osteogenesis and hematopoiesis, and interacts with its putative macromolecular target via distinct pharmacophores presented in a specific spatial organization.     

Thalictrum minus cell cultures and ABC-like transporter

Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.