Molecular cloning and characterization of a RING-H2 finger protein, ANAPC11, the human homolog of yeast Apc11p.

Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases. The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed. This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids. The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes. In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte. The ANAPC11 gene is located at the human chromosome 17q25. ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected. Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment.

Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization.

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.

Morin hydrate: a potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs.

The co-incubation of morin hydrate with either doxorubicin or mitomycin C could minimize the toxicity of these anti-tumor drugs on cardiovascular cells, such as red blood cells, human umbilical vein endothelial cells (ECV304) and primary mouse cardiomyocytes, whereas morin hydrate did not lower the cytotoxicity of the drugs on human hepatocellular carcinoma cells (HepG2). Morin hydrate may not exert its antioxidant effect by enhancing the antioxidant enzymatic activity because it did not cause any induction on the mRNA levels of manganese superoxide dismutase expression in ECV304 cells and HepG2 cells.

Cloning and characterization of a cDNA encoding a novel fibroblast growth factor preferentially expressed in human heart.

A novel human fibroblast growth factor (hFGF), which shows 75% sequence homology with fibroblast growth factor-9, was isolated in random sequencing of a human heart cDNA library. The full-length sequence is 928 bp, the encoded protein is composed of 168 amino acid residues, and its pI value and molecular weight were estimated to be 8.13 and 19.1 kDa, respectively. RT-PCR using Marathon human heart cDNA shows that the coding region is approximately 507 bp. Southern hybridization showed a single band which indicates that this is a single copy gene. Northern hybridization done on a human multiple tissues blot showed that the gene is preferentially expressed in human heart, very weakly detectable in human brain and not detectable in 18 other different human tissues.

Activation of the anti-tumor effector cells by Radix bupleuri.

Radix bupleuri, the root of Bupleuri spp., Chinese medicinal herbs used for the treatment of influenza, malaria and menstrual disorders, were extracted with hot water and separated into five different fractions (RB, RBI, RBII, RBIII and RBIV) by stepwise alcohol precipitation. One of these fractions, RBI, was then fractionated into RBIa and RBIb by gel filtration using G-100 Sephadex. These two fractions were further purified into RBIai, RBIaii and RBIbi, RBIbii fractions respectively by ion-exchange chromatography using DEAE-Sephadex. Each of these fractions is a heteropolymer consisting mainly of carbohydrate and varying proportions of protein and uronic acid. RBIaii was found to show strong anti-tumor activities in sarcoma-bearing mice. Mechanistic studies showed that RBIaii exhibited a potent activating effect on the cytotoxic activity of macrophages, NK and LAK cells against tumor cells. In addition, RBIaii could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Furthermore, RBIaii could induce the release of interferon-gamma by lymphocytes in vitro.