Decrease in Serum Adiponectin Level Due to Obesity and Visceral Fat Accumulation in Children

Objective: To determine whether serum adiponectin is decreased in obesity and is restored toward normal level after treatment in children. Research Methods and Procedures: Subjects were 53 Japanese obese children, 33 boys and 20 girls (6 to 14 years old), and 30 age‐matched nonobese controls for measuring adiponectin (16 boys and 14 girls). Blood was drawn after an overnight fast, and the obese children were subjected to anthropometric measurements including waist and hip circumferences and skinfold thicknesses. Paired samples were obtained from 21 obese children who underwent psychoeducational therapy. Visceral adipose tissue area was measured by computed tomography. Adiponectin was assayed by an enzyme‐linked immunosorbent assay. Results: The serum levels of alanine aminotransferase, uric acid, triglyceride, total cholesterol, low‐density lipoprotein‐cholesterol, total cholesterol/high‐density lipoprotein‐cholesterol, apo B, apo B/apo A₁, and insulin in obese children were higher than the reference values. Serum adiponectin level was lower in the obese children than in the controls (6.4 ± 0.6 vs. 10.2 ± 0.8 mg/L, means ± SEM, p < 0.001). In 21 obese children whose percent overweight declined during therapy, the adiponectin level increased (p = 0.002). The adiponectin level was correlated inversely with visceral adipose tissue area in obese children (r = ?0.531, p < 0.001). The inverse correlations of adiponectin with alanine aminotransferase, uric acid, and insulin were significant after being adjusted for percentage overweight, percentage body fat, or sex. Discussion: Serum adiponectin level is decreased in obese children depending on the accumulation of visceral fat and is restored toward normal level by slimming.     

Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803

The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10^?5 and 8.2 × 10^?5, respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.     

Design of metalloporphyrin-carbohydrate conjugates for a new superoxide dismutase mimic with cellular recognition

Metalloporphyrin-carbohydrate conjugates have been synthesized as superoxide dismutase (SOD) mimics with cellular recognition. To synthesize the conjugates, aliphatic primary amino groups for conjugation were introduced, with the cationic pyridyl groups for the SOD activity of porphyrin preserved. The reductive amination between introduced amino groups and the reducing end of lactose was then carried out. The resulting conjugates consisting of manganese (Mn)-porphyrin surrounded by several lactose molecules possessed significant SOD activity and low cytotoxicity. Compared with metalloporphyrins having no lactose molecule, the recognition of the resulting conjugates by human hepatoma HepG2 cells increased. The cellular recognition was inhibited by competitors of beta-galactose. These results suggest that the Mn-porphyrin-lactose conjugates recognized the hepatic lectin on the cell surface.     

Comb-Type Copolymers for Controlled DNA Delivery

Abstract

Various comb-type copolymer containing a polycation as a main chain was design to construct delivery systems of DNAs. The comb-type copolymers having cell-specific polysaccharides were proved to be useful to deliver DNA to the target cells in vivo. Of interest, the copolymers with abundant side chains of hydrophilic polymers are capable of stabilizing DNA triplex. Further, injectable nanoparticles for controlled releases of DNAs were fabricated from the copolymer and a biodegradable polymer.     

Immunolocalization of cellular glutathione peroxidase in adult rat lungs and quantitative analysis after postembedding immunogold labeling

To determine the distribution of cellular glutathione peroxidase in rat lungs, the tissues were stained immunohistochemically. Quantitative analysis was performed in certain cell types of alveolar linings, after the ultrathin sections were stained by a postembedding immunogold technique. Immunoblot analysis revealed that homogenates of rat liver, heart, and lungs all gave a single band. Under the light microscope, the following tissues were stained intensely: epithelial cells, smooth muscle cells and glands of bronchi and bronchioles, type II alveolar cells, and alveolar macrophages. Under immunoelectron microscopy, type II alveolar cells and macrophages were abundant in mitochondria. The mitochondria, nucleus, and cytoplasm of macrophages were labeled almost twice as densely as the respective compartments of type II alveolar cells. Within cell types, the mitochondria were labeled twice as densely as the nuclei. The other particles were less than half as densely labeled as the nuclei. The labeling was slightly less dense in the cytoplasm than in the nucleus. The present study revealed that glutathione peroxidase occurred predominantly in the epithelial linings and metabolically active sites in rat lungs. The tissues that were previously found to be rich in superoxide dismutases were also rich in glutathione peroxidase.     

Postnatal development of galanin-like peptide mRNA expression in rat hypothalamus

We examined the developmental change of GALP mRNA in male and female rat hypothalamus during postnatal day 1 to 60, using in situ hybridization histochemistry. Neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA in the hypothalamus were also examined because they are important in the regulation of food intake. GALP mRNA was first detected in the arcuate nucleus (ARC) on day 8. GALP mRNA was gradually increased between day 8 and 14 and markedly increased between day 14 and 40, which is the weaning and pubertal period in rats. After day 40, there were no significant differences in GALP mRNA. In contrast to GALP, NPY and POMC mRNAs were detected in the ARC from day 1 and lasted to day 60. There was no sexual dimorphism in GALP, NPY and POMC mRNAs during postnatal development. Next, we examined the effect of the milk deprivation for 24 h on GALP, NPY and POMC mRNA in pups. GALP mRNA did not change by milk deprivation on day 9 and 15, while milk deprivation had a significant effect on NPY and POMC mRNA on day 15. These results suggest that the development of GALP may be associated with developmental changes such as weaning, feeding and maturation of reproductive functions. The regulatory mechanism of GALP mRNA is different from that of the NPY and POMC genes during postnatal development.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-alpha (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-kappaB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Involvement of α2‐antiplasmin in dendritic growth of hippocampal neurons

The α2‐Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule‐associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP^?/? mice than in the neurons from α2AP^+/+ mice. Exogenous treatment with α2AP enhanced the microtubule‐associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP‐induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen^?/? mice. The activation of p38 MAPK was involved in the α2AP‐induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.     

Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: quantitative analysis by postembedding method

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/m²) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.