Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.     

Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Liver X receptor alpha (LXRalpha) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRalpha also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRalpha/RXRalpha (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRalpha might modulate cholesterol metabolism.     

A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)

The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa (  J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca^{2 +} . Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca^{2 +} , and a 40-kDa Ca^{2 +} -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca^{2 +} -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48   /   80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca^{2 +} receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of ¹²⁵I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two type of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Quantitation of the glycation intermediate 3-deoxyglucosone by oxidation with rabbit liver oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography

A simple and sensitive method for the detection of 3-deoxyglucosone was developed using oxidation with crude oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography (HPLC). Oxoaldehyde dehydrogenase was prepared from rabbit liver and partially characterized. 2-Keto-3-deoxygluconic acid produced from 3-deoxyglucosone by oxoaldehyde dehydrogenase was derivatized with 1,2-diamino-4,5-methylenedioxybenzene, and the fluorescent products were detected and quantitated by HPLC using a solvent containing borate. In the presence of borate, 2-keto-3-deoxygluconic acid was completely separated from N-acetylneuraminic acid. The detection limit of 3-deoxyglucosone was 2.5 pmol/injection (10 μl) at a signal-to-noise ratio of 3. This method was used to confirm the inhibitory effect of aminoguanidine on glycation.     

Consistency index of daily activity pattern and its correlations with subjective ratings of QOL

Sleep and Biological Rhythms - Since sleep is under circadian and homeostatic regulation, the status of regulatory mechanisms can be known from daily activity patterns, including both sleep and...     

Vesicle transport and processing of the precursor to 2S albumin in pumpkin

Cell fractionation of pulse-chase-labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.
The primary structure of the precursor (16 578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N-terminus indicates that the precursor is a preproprotein that is converted into pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off co-translationally on the C-terminal side of alanine residue 22 of prepro2S albumin. By contrast, post-translational cleavages occur on the C-terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.     

Heterologous Expression and Subcellular Localization of Pumpkin Seed Tonoplast Intrinsic Proteins (TIP) in Yeast Cells

Two tonoplast intrinsic proteins (TIP) of pumpkin seeds, pMP23and MP28, were expressed in yeast cells under control of theGAL1 promoter, and the subcellular localization of the proteinswas analyzed. The pMP23 and MP28 stably accumulated in the yeastvacuolar membrane when the proteins were expressed in the proteinaseA-deficient strain (pep4), which lacks the activities of vacuolarproteases. However, pMP23 and MP28 did not accumulate in thewild-type strain; the expressed pMP23 and MP28 were degradedin a proteinase A-dependent manner. These results indicate thatpMP23 and MP28 are transported to the vacuolar membrane whenexpressed in yeast. ⁵Present address: Department of Biochemistry and Molecular Biology,The Pennsylvania State University, University Park, PA16802,U.S.A.     

Dose–response relationship from longitudinal data with response‐dependent dose modification using likelihood methods

In some clinical trials or clinical practice, the therapeutic agent is administered repeatedly, and doses are adjusted in each patient based on repeatedly measured continuous responses, to maintain the response levels in a target range. Because a lower dose tends to be selected for patients with a better outcome, simple summarizations may wrongly show a better outcome for the lower dose, producing an incorrect dose–response relationship. In this study, we consider the dose–response relationship under these situations. We show that maximum‐likelihood estimates are consistent without modeling the dose‐modification mechanisms when the selection of the dose as a time‐dependent covariate is based only on observed, but not on unobserved, responses, and measurements are generated based on administered doses. We confirmed this property by performing simulation studies under several dose‐modification mechanisms. We examined an autoregressive linear mixed effects model. The model represents profiles approaching each patient's asymptote when identical doses are repeatedly administered. The model takes into account the previous dose history and provides a dose–response relationship of the asymptote as a summary measure. We also examined a linear mixed effects model assuming all responses are measured at steady state. In the simulation studies, the estimates of both the models were unbiased under the dose modification based on observed responses, but biased under the dose modification based on unobserved responses. In conclusion, the maximum‐likelihood estimates of the dose–response relationship are consistent under the dose modification based only on observed responses.