Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2

The signaling mechanisms responsible for bone morphogenetic protein (BMP) induced osteoblast differentiation remains poorly understood. Previous research demonstrated that Smad proteins are the substrates and the mediators of BMP bound serine/threonine receptor kinase. In the present study, we examined the possible involvement of extracellular signal-regulated kinase (Erk) in the BMP induced osteoblast differentiation of mesenchymal progenitor cell C3H10T1/2. Our results indicate that BMP-2 inducement increased MAP kinase activity in mesenchymal progenitor cell line C3H10T1/2. Contrary to previous reports, this increased MAP kinase activity showed a latent but sustained pattern. Elevation of Erk1 and Erk2 protein levels was observed simultaneously. RT-PCR results demonstrated that the elevation of Erk protein level in BMP-2 induced cells was from the upregulation of mRNA expression. Furthermore, upregulated Erk proteins present enhanced phosphorylation. By using a dominant-negative Erk2 cell line, we demonstrated that nonfunctional Erk2 partially eliminated BMP-2 induced cell proliferation and ALP activity in the C3H10T1/2 cell. These results indicate that Erk is involved in BMP-2 induced osteoblast differentiation. The results also demonstrate that a latent and sustained signaling pattern exists in BMP induced signaling cascade.     

Solution structure of an N-capping peptide from the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The RNA genome of HDV encodes two proteins, the small delta antigen and the large delta antigen, which differ only with the latter having an additional 19 amino acids at the C-terminus. Previously, we have shown that dAg24-50, a synthetic peptide corresponding to residues 24-50 of the N-terminal leucine-repeat region of hepatitis delta antigen, binds to the viral RNA and forms an alpha-helical conformation in TFE-containing solution. However, it exhibited low alpha-helicity (less than 5%) in the absence of TFE. In order to obtain biologically active delta antigen peptides with higher structural stability in solution, an N-capping 21-residue polypeptide corresponding to residues 24-38 of hepatitis delta antigen (dAg(Cap24-38am)) was synthesized and, surprisingly, its solution structure was found to be a stable alpha-helix (64%) by circular dichroism and 1H NMR techniques. Moreover, the structure of the capping box shows the characteristic L-shaped bend perpendicular to the helix axis. This structural knowledge provides a molecular basis for understanding the role of the N-terminal leucine-repeat region of hepatitis delta antigen and has a significant potential for the development of diagnostic and therapeutic methods for HDV.     

Lack of obvious 50 kilobase pair DNA fragments in DNA fragmentation factor 45-deficient thymocytes upon activation of apoptosis

The DNA fragmentation factor 45 (DFF45/ICAD) is a key subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation during apoptosis in vivo. To further assess the importance of DFF45 in chromosomal DNA degradation, we induced apoptosis in wild-type control and DFF45 deficient thymocytes and compared the cleavage of chromosomal DNA to 50 kilobase pair size fragments. We found that there is a lack of obvious large chromosomal DNA fragments upon treatments by various apoptotic agents in DFF45 deficient thymocytes. The major organ systems in the DFF45 mutant mice either two months or fifteen months of age appear normal. These results suggest that functional DFF45 is required for cleavage of DNA into both large size and oligonucleosomal size fragments in thymocytes during apoptosis. However, deficiency in DFF45 apparently does not significantly affect normal mouse development and tissue homeostasis.     

铜锌超氧物歧化酶与过氧化氢反应中羟自由基的形成

应用脱氧核糖降解法研究了离体条件下Cu,Zn-SOD与H2O2反应产生·OH,并对其机理进行了探讨。H2O2可使Cu,Zn-SOD失活,在失活过程中有·OH产生,甲酸钠和苯甲酸钠均能不同程度地保护Cu,Zn-SOD和降低H2O2与Cu,Zn-SOD反应中·OH的产额;热失活SOD也可和H2O2反应生成·OH,且效能高于活性Cu,Zn-SOD;用螫合剂脱去Cu,Zn-SOD的金属辅基后,脱辅基的SOD蛋白不能和H2O2反应产生·OH;Cu2＋和H2O2反应产生·OH的效率很高,而Zn2＋产生·OH的效率很低。实验结果提示Cu,Zn－SOD与H2O2反应产生的·OH可能是SOD活性中心的Cu2＋与H2O2发生Fenton反应的结果．     

Dietary Response of Chimpanzees and Cercopithecines to Seasonal Variation in Fruit Abundance. II. Macronutrients

In a continuation of our study of dietary differentiation among frugivorous primates with simple stomachs, we present the first comparison of differences in dietary macronutrient content between chimpanzees and cercopithecine monkeys. Previously we have shown that chimpanzee and monkey diets differ markedly in plant part and species content. We now examine whether this diet diversity is reflected in markedly different dietary macronutrient levels or the different feeding strategies yield the same macronutrient levels in their diets. For each primate group we calculated the total weighted mean dietary content of 4 macronutrients: crude lipid (lipid), crude protein (CP), water-soluble carbohydrates (WSC), and total nonstructural carbohydrates (TNC). We also calculated 4 fiber fractions: neutral-detergent fiber (NDF), which includes the subfractions hemicellulose (HC), cellulose (Cs), and sulfuric acid lignin (Ls). The HC and Cs are potentially fermentable fibers and would contribute to the energy provided by plant food, depending on the hind gut fermenting capacity of the individual primate species. The chimpanzee diet contained higher levels of WSC and TNC because during times of fruit abundance the chimpanzees took special advantage of ripe fruit, while the monkeys did not. The monkey diets contained higher levels of CP because the monkeys consumed a constant amount of leaf throughout the year. All four primate species consumed diets with similar NDF levels. However, the chimpanzees also took advantage of periods of ripe fruit abundance to decrease their Ls levels and to increase their HC levels. Conversely, the monkey diets maintained constant levels of the different fiber fractions thoughout the year. Nevertheless, despite these differences, the diets of the 4 frugivores were surprisingly similar, considering the substantial differences in body size. We conclude that the chimpanzee diet is of higher quality, particularly of lower fiber content, than expected on the basis of their body size.     

Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.