Heritability of Eating Behavior Assessed Using the DEBQ (Dutch Eating Behavior Questionnaire) and Weight‐related Traits: The Healthy Twin Study

The heritability of eating behavior and body weight–related traits in Asian populations has not been reported. The purpose of this study was to estimate the heritability of eating behavior and the body weight–related traits of current weight and self‐reported past weight among twins and their families. Study subjects were 2,144 Korean, adult, same‐sex twins and their families at the ages between 20 and 65 years (443 monozygotic (MZ) and 124 dizygotic (DZ) twin pairs, and 1,010 individuals of their family). The Dutch Eating Behavior Questionnaire (DEBQ) was used to assess three eating behavior subscales measuring restraint, emotional eating, and external eating. A variance component approach was used to estimate heritability. After consideration of shared environmental effects and adjustment for age and sex effects, the heritability estimates ± s.e. among twins and their family members were 0.31 ± 0.036 for restraint, 0.25 ± 0.098 for emotional eating, 0.25 ± 0.060 for external eating, 0.77 ± 0.032 for measured current body weight, and 0.70 ± 0.051 for self‐reported weight at 20 years old. The three DEBQ subscales were associated with all weight related traits after adjustment for age and sex. These results suggest eating behaviors and weight‐related traits have a genetic influence, and eating behaviors are associated with obesity indexes. Our findings from Korean twin family were similar to those reported in Western populations.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver

In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.