Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl^-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm^-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of Sweet Gem were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter--glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl^-1 BA and 200 M -hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl^-1 BA 250 mgl^-1 carbenicillin, and 100 mgl^-1 kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl^-1 BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.     

Kruppel-like factor 4 attenuates osteoblast formation,function, and cross talk with osteoclasts

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)₂D₃-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)₂D₃-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.     

An in situ calibration of an ultrasound transducer: a potential application for an ultrasonic indentation test of articular cartilage

A change in mechanical properties of articular cartilage would be considered one of the most reliable signs of cartilage degeneration. While an indentation method has the potential to measure the cartilage properties in vivo, an accurate measurement of cartilage thickness in situ is technically difficult. An ultrasound transducer has often been used to measure the cartilage thickness. However, its accuracy is limited by the lack of an accurate measurement of the ultrasound speed of cartilage, for the ultrasound speed varies according to the pathological conditions of the tissue. Therefore, the objective of this study is to develop an in situ calibration method of predicting the true ultrasound speed of cartilage and thus allow the ultrasound transducer to measure the thickness of the tissue with great accuracy. By simultaneously implementing an indentation testing protocol using the ultrasound transducer as an indenter, this method can also provide an indentation stiffness measurement of cartilage.The feasibility of the proposed method was examined using normal and proteoglycan-depleted cartilage specimens. It was found that the true ultrasound speed measured by the in situ calibration method was sensitive to the proteoglycan depletion (1735+/-35 m/s for normal, and 1598+/-28 m/s for proteoglycan-depleted cartilage), and that the measured cartilage thickness was consistently accurate regardless of the tissue condition. The measured indentation stiffness of articular cartilage was also sensitive to the tissue condition. Thus, this study demonstrates that the proposed ultrasonic indentation technique can be used to accurately identify the abnormality of articular cartilage in situ.     

Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1

Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG(2k)-N(ter)-GLP-1 and (ii) isomers of Lys(26), Lys(34) modified PEG(2k)-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG(2k)-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P = 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG(2k)-N(ter)-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG(2k)-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG(2k)-Lys-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the half-life, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically mono-PEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.     

Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.     

Endogenous N-cadherin in a subpopulation of MDCK cells: distribution and catenin complex composition

Epithelial (E)-cadherin plays a critical role in developing a normal epithelial phenotype but neural (N)-cadherin can disrupt epithelial shape, at least in carcinoma-derived cells. Here the normal epithelial cell line MDCK was used to select for a trypsin-sensitive (TS-MDCK) subpopulation that expresses low levels of endogenous N-cadherin. Similar amounts of E-cadherin and all catenins are found in both TS-MDCK and trypsin-resistant cells (TR-MDCK), but TS-MDCK are less phenotypically epithelioid and more motile, and junctional proteins are more detergent soluble. In TS-MDCK, N-cadherin is largely nonjunctional; a similar N-cadherin distribution and mesenchymal phenotype are found in TR-MDCK transfected to express low levels of exogenous N-cadherin. Little N-cadherin was attracted to junctions between TS-MDCK and hTERT-RPE1 cells, a retinal pigment epithelium-derived line that expresses dominantly N-cadherin. No differences were seen in E-cadherin-catenin complexes in TS- and TR-MDCK, but N-cadherin-catenin complexes in TS-MDCK have more abundant p120 catenin. Overall, the results indicate that E- and N-cadherin assemble stoichiometrically different complexes with p120 in the same cells. Further, N-cadherin does not participate with E-cadherin in a zonular epithelial junction in normal MDCK epithelial cells. Rather, even low levels of endogenous N-cadherin contribute to a disrupted epithelial phenotype, resembling the effect of N-cadherin on carcinoma-derived epithelial cells.