Versican modulates embryonic chondrocyte morphology via the epidermal growth factor-like motifs in G3

This investigation was designed to characterize the effect of the extracellular matrix molecule versican on chondrocyte morphology, using the well-studied chondrocyte cell culture system. When cultured chondrocytes reverted or "dedifferentiated" to a fibroblast-like morphology, we found that versican expression was significantly enhanced. Transfection of chondrocytes, isolated from embryonic chicken sterna, with a chicken miniversican construct accelerated the reversion process, while expression of an antisense construct inhibited it. A mutant miniversican lacking two epidermal growth factor-like motifs (versicanDeltaEGF) promoted differentiation, as shown by morphological changes and changes in the expression of other extracellular matrix molecules. A truncated versican mutant, the G3DeltaEGF, a G3 domain lacking its two epidermal growth factor-like motifs, also enhanced differentiation. This effect is related to G3DeltaEGF-induced change in cytoskeleton, since transfected cells exhibited misassembly of actin filaments. This article thus provides the first evidence that versican modulates chondrocyte morphology via changes in cytoskeletal structure, and may imply that extracellular matrix molecules play an important role in cell differentiation.     

The involvement of TNF-alpha-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-beta-stimulated human dendritic cells to tumor cells

TNF-alpha-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34(+) stem cell-derived DCs (CD34DCs) and human CD14(+) monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-beta stimulation. Moreover, IFN-beta could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-beta-stimulated DCs partially contributed to the increased cytotoxicity of DCS: Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-beta-stimulated DCS: In contrast, NF-kappaB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCS: Our studies demonstrate that IFN-beta-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8(+) CTL cells.     

SeO(2) induces apoptosis with down-regulation of Bcl-2 and up-regulation of P53 expression in both immortal human hepatic cell line and hepatoma cell line

An immortal human hepatic cell line HL-7702 and human hepatoma cell line SMMC-7721 were treated with 3-30 microM SeO(2). SeO(2) at 30 microM markedly inhibited cell proliferation and viability, and prompted apoptosis of both normal hepatic and hepatoma cells after 48h treatment. SeO(2) could also down-regulate the Bcl-2 level, greatly in HL-7702 and slightly in SMMC-7721 cells, but up-regulate wild type P53 level a little in HL-7702 and significantly in SMMC-7721 cells. The Bcl-2/P53 value was closely correlated with the apoptotic rate as well as SeO(2) concentrations.     

Factors influencing the levels of fatty acid synthase complex activity in fowl

There is a notable discrepancy between the FAS (fatty acid synthase) activity of four types of fowl (egg chicken, meat chicken, egg duck, and meat duck) with distinctively different body fat levels. There is a 14.8 fold difference per unit body weight between the maximum and minimum FAS activities. The three major factors affecting this discrepancy are liver weight per unit body weight, which is 2.3 times greater in meat ducks than in egg chickens, the amount of FAS protein per gram of liver, which is 1.85 times greater in meat ducks than in egg chickens, and the FAS specific activity in meat ducks, which is 3.5 times greater in meat ducks than in egg chickens. Within the same species of egg chickens, the abdomen fat per kg of body weight at 470 days after egg production is 66 times greater than 90 days before egg production and the liver FAS activity is increased 9.6 fold. The 9.6 fold FAS activity increase resulted from an increase in the specific activity, since the liver weight per kilogram of body weight remained constant at approx. 20 grams and the FAS weight per gram of liver also remained constant at approx. 4.5 mg. This shows that the control of the basic FAS activity level which is closely related to the level of body fat does not mainly arise from genetic control. For the same kind of fowl, the control of the basic FAS activity level occurs after gene expression. It is suggested that control may be imposed in the folding phase when new peptides give rise to functional proteins.     

Maskin is a CPEB-associated factor that transiently interacts with elF-4E

In Xenopus, the CPE is a bifunctional 3' UTR sequence that maintains maternal mRNA in a dormant state in oocytes and activates polyadenylation-induced translation during oocyte maturation. Here, we report that CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor we term maskin. Maskin contains a peptide sequence that is conserved among elF-4E-binding proteins. Affinity chromatography demonstrates that CPEB, maskin, and elF-4E reside in a complex in oocytes, and yeast two-hybrid analyses indicate that CPEB and maskin bind directly, as do maskin and elF-4E. While CPEB and maskin remain together during oocyte maturation, the maskin-elF-4E interaction is substantially reduced. The dissolution of this complex may result in the binding of elF-4E to elF-4G and the translational activation of CPE-containing mRNAs.     

Model dependence of the phylogenetic inference: relationship among carnivores, Perissodactyls and cetartiodactyls as inferred from mitochondrial genome sequences

Some previous analysis of mitochondrial proteins strongly support the Carnivora/Perissodactyla grouping excluding Cetartiodactyla (Artiodactyla + Cetacea) as an outgroup, but the support of the hypothesis remains equivocal from the analysis of several nuclear-encoded proteins. In order to evaluate the strength of the support by mitochondrial proteins, phylogenetic relationship among Carnivora, Perissodactyla, and Cetartiodactyla was estimated with the ML method by using the updated data set of the 12 mitochondrial proteins with several alternative models. The analyses demonstrate that the phylogenetic inference depends on the model used in the ML analysis; i.e., whether the site-heterogeneity is taken into account and whether the rate parameters are estimated for each individual proteins or for the concatenated sequences. Although the analysis of concatenated sequences strongly supports the Carnivora/Perissodactyla grouping, the total evaluation of the separate analyses of individual proteins, which approximates the data better than the concatenated analysis, gives only ambiguous results, and therefore it is concluded that more data are needed to resolve this trichotomy.     

The involvement of singlet oxygen in copper-phenanthroline/H2O2-induced DNA base damage: a chemiluminescent study

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and H2O2 causes DNA base damage as well as strand breakage. We have reported in previous work that a strong chemiluminescence was followed by DNA base damage in this system, which is characteristic of guanine. In the present work, the mechanism of the chemiluminescence was studied. Results show that the luminescence was inhibited by all three classes of reactive oxygen species (*OH, O2-, (1)O2) scavengers to different degrees. Singlet oxygen scavengers showed the most powerful inhibition while the other two classes of scavengers were relatively weaker. The emission intensity in D2O was 3-fold that in H2O. Comparing the effect of scavengers on the luminescence of DNA with that of dGMP, the ratio of inhibition was similar. On the other hand, DNA breakage analysis showed that inhibition by the singlet oxygen scavenger NaN3 of strand breakage was strong and comparable to that of the scavengers of the two oxygen radicals. The results suggest that singlet oxygen may be a major factor for the chemiluminescence of guanine, while DNA strand breakage may be caused by many active species.     

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 receptor (IL-2R) subunits. Mussel IL-2R alpha and IL-2R beta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL-2R gamma is smaller than that in vertebrates. Both lipopolysaccharide and IL-2 induce IL-2R alpha expression, and such induction depends on the concentration of both agonists.     

The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.     

Systemic gene delivery expands the repertoire of effective antiangiogenic agents

Cationic liposome-DNA complex (CLDC)-based intravenous gene delivery targets gene expression to vascular endothelial cells, macrophages and tumor cells. We used systemic gene delivery to identify anti-angiogenic gene products effective against metastatic spread in tumor-bearing mice. Specifically, CLDC-based intravenous delivery of the p53 and GM-CSF genes were each as effective as the potent antiangiogenic gene, angiostatin, in reducing both tumor metastasis and tumor angiogenesis. Combined delivery of these genes did not increase anti-tumor activity, further suggesting that each gene appeared to produce its antimetastatic activity through a common antiangiogenic pathway. CLDC-based intravenous delivery of the human wild type p53 gene transfected up to 80% of tumor cells metastatic to lung. Furthermore, it specifically induced the expression of the potent antiangiogenic gene, thrombospondin-1, indicating that p53 gene delivery in vivo may inhibit angiogenesis by inducing endogenous thrombospondin-1 expression. CLDC-based delivery also identified a novel anti-tumor activity for the metastasis suppressor gene CC3. Thus, CLDC-based intravenous gene delivery can produce systemic antiangiogenic gene therapy using a variety of different genes and may be used to assess potential synergy of combined anti-tumor gene delivery and to identify novel activities for existing anti-tumor genes.