Effects of length and position of an extended linker on sequence-selective DNA recognition of zinc finger peptides

Engineered zinc finger proteins revealed that a linker sequence connecting zinc finger units has a significant effect on the DNA binding property of the protein. The recognition for a noncontiguous DNA target beyond the current recognition code of zinc finger proteins has never been determined because of the limitation of a zinc finger framework. DNA recognition of zinc finger proteins is limited only to a contiguous subset of three base pairs. We propose the recognition for a noncontiguous DNA target by inserting amino acids into the canonical linker between zinc finger units. The sequence selectivity of the new zinc finger peptides was evaluated by gel mobility shift assays. DNase I footprinting analyses clearly showed different DNA binding of various linker-extended zinc finger peptides. The application of a SPR measurement also revealed a DNA sequence selectivity of peptides. Insertion of three amino acids is enough for recognition of a noncontiguous DNA target with sequence selectivity. An extended linker will be useful for expansion of the recognition code of zinc finger proteins and for development of a new role for linker sequences in DNA binding of zinc finger proteins.     

Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.     

Salivary cystatins influence ingestion of capsaicin-containing diets in the rat

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.