Fbxw7 acts as an E3 ubiquitin ligase that targets c-Myb for nemo-like kinase (NLK)-induced degradation

The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-beta-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7alpha directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7alpha, suggesting that the c-Myb/Fbxw7alpha interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7alpha and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7alpha-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7.     

Liposome-mediated gene therapy in the kidney

Gene therapy directed to the kidney has been attempted to improve renal disorders such as inherited kidney diseases and common renal diseases that cause interstitial fibrosis, tubular atrophy, and glomerulosclerosis. Viral and non-viral vectors have been tried and been modulated to obtain sufficient transgene expression. However, gene delivery to the kidney is usually difficult because of characteristics of renal cell biology. Among non-viral vectors, the liposome system is a promising procedure for kidney-targeted gene therapy. Using cationic liposome, tubular cells were effectively transduced by retrograde injection of liposome/cDNA complex. Although transgene expression was reportedly modest using cationic liposomes, this method improved renal disease models such as carbonic anhydrase II deficiency and unilateral ureteral obstruction. In contrast, HVJ-liposome system is an effective transfection method to glomerular cells using intra-renal arterial infusion and improved glomerular disease models such as glomerulonephritis and glomerulosclerosis. In addition, intra-renal pelvic injection of DNA by HVJ-liposome system showed transgene expression in interstitial fibroblasts. In kidney-targeted gene therapy, liposome-mediated gene transfer is an attractive method because of its simplicity and reduced toxicity. In spite of modest transgene expression, several renal disease models were successfully modulated by liposome system. Although one limitation of liposome-mediated gene delivery is the duration of transgene expression, the liposome/cDNA complex can be repeatedly administered due to the absence of an immune response.     

Cleavage of the xylosyl serine linkage between a core peptide and a glycosaminoglycan chain by cellulases

We previously found that endo-beta-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-beta-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-beta-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-beta-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides.     

Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and the N-glycosidase activities of their recombinant proteins on ribosomes and DNA in comparison with other isoforms

Pokeweed antiviral proteins (PAPs) are single-chain ribosome-inactivating proteins (RIPs) isolated from several organs of Phytolacca americana (Pokeweed) that are characterized by their ability to depurinate not only ribosomes but also various nucleic acids. PAP-S is one of the isoforms found in seeds. In this study, we obtained three different genomic clones encoding two forms of PAP-S (here designated as PAP-S1 and PAP-S2) and alpha-PAP after PCR using a pair of degenerated primers based on the known N- and C-terminal amino acid sequences of PAP-S. The nucleotide sequences of the genomic clones contained no introns. The deduced amino acid sequences of PAP-S1 and PAP-S2, which showed 83% identity to each other, were found to correspond to sequences reported independently for PAP-S protein and cDNA, respectively, demonstrating that at least two forms of PAP-S actually exist in seeds of the same plant. The recombinant PAP-S1, PAP-S2, alpha-PAP, and PAP I (a form appearing in spring leaves) exhibit the same level of depurinating activity on rat ribosomes, while their efficiencies on Escherichia coli ribosomes and salmon sperm DNA differ substantially from one another in the order of PAP I > alpha-PAP > PAP-S1 > PAP-S2 and alpha-PAP > PAP I > PAP-S1 > PAP-S2. Structural comparisons suggest that the large difference in ribosome recognition between PAP-S1 (or S2) and PAP I is caused by the alteration of residues adjacent to the adenine-binding site.     

The structure of rooster-comb dermatan sulfate. Fragmentation of the polysaccharide chains by chondroitinase AC-II digestion,and by periodate oxidation,followed by alkali cleavage

The polysaccharide-chain fragments of rooster-comb dermatan sulfates (RC-20 and RC-30) were obtained by chondroitinase AC-II digestion and by periodate oxidation, followed by alkaline cleavage, and their structures analyzed both quantitatively and qualitatively. RC-20 having a lower d-glucuronic acid content (22.6%) is composed preponderantly of large clusters of N-acetyldermosine sulfate (M_r～17 600–41 000) at the nonreducing terminal, whereas RC-30, having a higher d-glucuronic acid content, (41.4%) is poor in this cluster. Both RC-20 and RC-30 have an N-acetyldermosine sulfate cluster (M_r 6500–7300) within the polysaccharide chains. Most N-acetylchondrosine sulfate units of RC-20 and RC-30 exist as clusters, the large clusters (M_r～17 600) being preponderant in RC-30; both RC-20 and RC-30 contain a large proportion of N-acetylchondrosine sulfate clusters (M_r 3500 and 9000) that corresponds to the uronic acid content. In RC-30, most N-acetyldermosine disulfate units (13.4%) are linked to N-acetylchondrosine sulfate units or clusters.     

Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.     

Comparative characterization of the oah2 gene homologous to the oah1 of Thermus thermophilus HB8

The oah2 gene homologous to the oah1 of Thermus thermophilus HB8 was cloned and sequenced. It comprised 1,236 bp encoding a protein of 412 amino acid residues and was overexpressed. The gene product, also having O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity, was purified to homogeneity and characterized comparatively with the oah1 product. The two proteins shared many characteristics.