Cell surface changes accompanying aging in human diploid fibroblasts : III. Division age and senescence revealed by concanavalin A-mediated red blood cell adsorption

The relationship between cell size, [³H]thymidine incorporation capacity, and cell surface property of human diploid fibroblasts was investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. Small cells in late passage populations adsorbed RBCs well with the RBC coating method (in which ConA-coated RBCs are adsorbed to fibroblasts) as did large cells of this population, while small cells in early passage populations did not. The RBC adsorption capacity of rapidly dividing cells with this method differed among young, middle-aged and old cell populations. The results suggest that temporal cell size and [³H]thymidine incorporating capacity is not a measure of the division age of human diploid cells at the individual cell level. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to ConA-coated fibroblasts) occurred to non-dividing cells of the populations. Thus, the increase in RBC adsorption with this method is considered to be a reflection of the increase in non-dividing cells at phase III. Our results support the hypothesis that RBC adsorption with the RBC and fibroblast-coating methods represents a cell surface marker for division age and senescence of human diploid cells, respectively, at the individual cell level.     

The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.     

Wasabi leaf extracts attenuate adipocyte hypertrophy through PPARγ and AMPK

Hypertrophy of adipocytes in obese adipose tissues causes metabolic abnormality by adipocytokine dysregulation, which promotes type 2 diabetes mellitus, hypertension, and dyslipidemia. We investigated the effects of wasabi (Wasabia japonica Matsum) leaf extracts on metabolic abnormalities in SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF), which are a model of metabolic syndrome. Male SHRSP/ZF rats aged 7 weeks were divided into two groups: control and wasabi leaf extract (WLE) groups, which received water or oral treatment with 4 g/kg/day WLE for 6 weeks. WLE improved the body weight gain and high blood pressure in SHRSP/ZF rats, and the plasma triglyceride levels were significantly lower in the WLE group. Adipocyte hypertrophy was markedly prevented in adipose tissue. The expression of PPARγ and subsequent downstream genes was suppressed in the WLE group adipose tissues. Our data suggest that WLE inhibits adipose hypertrophy by suppressing PPARγ expression in adipose tissue and stimulating the AMPK activity by increased adiponectin.     

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-gamma, the production of proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (< 5 kDa) and high (> 30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.     

Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism

We have previously shown that royal jelly (RJ) promoted collagen production by skin fibroblasts in the presence of ascorbic acid-2-O-alpha-glucoside (AA-2G). In this study, we purified the honeybee RJ-derived collagen production-promoting factor (HBRJ-CPF) from an alkali-solubilized fraction of RJ by C18 reverse-phase column chromatography. The elution profile by the C18 column chromatography and the molecular mass of the purified HBRJ-CPF material coincided with those of 10-hydroxy-2-decenoic acid (10H2DA). We then examined the collagen production-promoting activities of several commercially available fatty acids contained in RJ. We found that 10H2DA and 10-hydroxydecanoic acid increased the collagen production in a dose-dependent manner. Furthermore, 10H2DA induced the fibroblast cell line, NHDF, to produce transforming growth factor-beta 1 (TGF-beta 1) which is an important factor for collagen production. As expected, the collagen production-promoting activity of 10H2DA was neutralized by the anti-TGF-beta 1 antibody. These result suggest that HBRJ-CPF identified as 10H2DA promoted the collagen production of AA-2G-treated fibroblasts by inducing TGF-beta 1 production.     

Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).     

Network structures and thermal properties of polyurethane films prepared from liquefied wood

Polyurethane (PU) films were prepared by solution-casting after co-polymerization of liquefied woods (LWs) and polymeric methylene diphenylene diisocyanate (PMDI). The resulting PU films had various [NCO]/[OH] ratios ranging from 0.6 to 1.4 and contained 5.0-16.8% dissolved woody components at the [NCO]/[OH] ratio of 1.0. The crosslink densities of the films with [NCO]/[OH] ratios of 0.6-1.4 increased remarkably with increasing [NCO]/[OH] ratio. Similarly, there were large increases in glass transition temperatures (Tg). These characteristics could be attributed to effective incorporation of PMDI into the LW. The crosslink densities and Tg of the PU films prepared at the [NCO]/[OH] ratio of 1.0 increased because the amounts of dissolved woody components in the films increased. It is concluded that the dissolved woody components acted as crosslinking points in PU network formations. The thermal degradation of the PU films at an [NCO]/[OH] ratio of more than 0.8 or with more than 10.6% dissolved wood started above 262 degrees C under an N2 atmosphere. The thermostability was lost at low crosslink density or with large amount of co-polymerized glycerol structures in the PU networks.     

Trehalose-producing operon treYZ from Arthrobacter ramosus S34

Arthrobacter ramosus S34, which produces trehalose from maltooligosaccharide, was isolated. A trehalose-producing operon, treYZ, was cloned from the genome. Expression experiments with treY and treZ confirmed that they coded malto-oligosyltrehalose synthase and malto-oligosyltrehalose trehalohydrolase, respectively. The amino acid sequence of TreY from A. ramosus S34 and that from Arthrobacter sp. Q36 did not show high identity, nor did those of TreZ.