Ken Oofusa

Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone‐induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana. Anti‐tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis, and adult newts, Cynopus pyrrhogaster, and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone‐treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.

Approach for in Vivo Protein Binding of 5-n-Butyl-pyrazolo[1,5-a]pyrimidine Bioactivated in Chimeric Mice with Humanized Liver by Two-Dimensional Electrophoresis with Accelerator Mass Spectrometry

Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.

Visualization of the initiation and sequential expansion of the metamorphic conversion of anuran larval skin into the precursor of adult type

A tadpole of bullfrog, Rana catesbeiana, is originally covered with the larval skin over its entire body. Drastic changes arise in both the epidermis and the subcutaneous connective tissue at an early developmental stage, producing the precursor of adult type skin (pre‐adult skin). It was found that calcium is a useful probe to detect the region where the precursor formation has occurred because its deposition in the upper part of subcutaneous collagen bundles coincides with the appearance of the pre‐adult skin. Whole‐mount in situ staining of tadpoles with alizarin red S revealed the initiation site of the premetamorphic transformation of the larval skin into the adult precursor and its ensuing region‐dependent expansion. The pre‐adult skin first emerged at TK II to III (TK, Taylor and Kollros staging) t lateral sides of the body, which led us to postulate that ‘the center for premetamorphic skin transformation’ is formed at the specific site in this region. This center moved dorsally and then ventrally, then reached to the most proximal region of the tail, yielding a unique sequential conversion pattern by around TK V when the conversion was completed in the trunk. The present study also visualized the process of the hindlimb skin transformation.

1‐Benzyl‐1,2,3,4‐tetrahydroisoquinoline binds with tubulin β, a substrate of parkin, and reduces its polyubiquitination

J. Neurochem. (2010) 114, 1291–1301.

Cloning and characterization of the full length cDNA encoding α2 type I collagen of bullfrog Rana catesbeiana

Abstract The present study determined nucleotide sequences of the full-length cDNA of α2 chain of bullfrog type I collagen. Hybridization of a bullfrog cDNA library with human α1 type I collagen cDNA yielded a clone named 6A-1 which was 3449 bp long and lacked a 5′ region of the gene. A 5′ region containing the translation initiation site was amplified by the reverse transcription polymerase chain reaction using poly(A)+RNA from tadpole tail tissues as template, and oligonucleotides encoding the translation initiation region of mammalian fibrillar collagens and the Gly-X-Y repeat region of clone 6A-1 as primers. As a result we obtained a 1518 bp long clone Y31. A 355 bp long clone Y31-9 was produced by extending clone Y31 from its ATG codon to a 127 bp upstream region. Combining these three clones, the complete nucleotide sequence of the full-length cDNA was determined which contained 4692 bp as a whole and 4065 bp in the open reading frame. The comparison of its structure with known collagen cDNAs of various vertebrates showed that the cDNA obtained codes for α2(I) chain of bullfrog. Its deduced amino acid sequence revealed the complete conservation of seven cysteine residues in the C-propeptide and three lysine residues in the N-telopeptide through the helical domain. Northern blot analysis revealed that the thyroid hormone regulated the expression of α2(I) collagen in an organ-dependent manner: intense up-regulation in the back skin and intestine, weak and transient up-regulation in the liver, and initial down-regulation, but later up-regulation in the tail. Prolactin increased its expression in both the back skin and tail. These results suggested that the expression of bullfrog α2(I) collagen is cooperatively regulated by these two metamorphosis-regulating hormones.

Chimeric Mice with Hepatocyte-humanized Liver as an Appropriate Model to Study Human Peroxisome Proliferator–activated Receptor-α

Peroxisome proliferator (PP)–activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of β- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

Thyroid hormone-dependent expression of bullfrog tadpole collagenase gene

Collagenase has been suggested to play a pivotal role in tissue remodeling during anuran metamorphosis. RNA blot analysis and nuclear run-on assay showed that collagenase genes of caudal cells of tadpoles of the bullfrog, Rana catesbeiana, are up-regulated by thyroid hormone after only 24 h. The gene for collagenase was cloned from genome DNA of the bullfrog and its structure, including the 5′-upstream region with-1 kbp, was determined. The gene contained 4 exons with sequences of the enzyme active centre in exon 2. Cis-acting regulatory sequences were recognized in the 5′-upstream region as AP-1 and Sp1, which had also been found in the mammalian collagenase gene. A special sequence was noticed near −870 bp, 5′-AG-GTAAGAACAGGATA-3′, that satisfies a general criterion for the identification of the thyroid hormone responsive element (TRE) proposed by Umesono et al. (1991) and is homologous to the element reported in genes of thyroid-hormone responsive proteins such as thyroid hormone receptor (TR) β, growth hormone, malic enzyme and myosin heavy chain. Results of the gel mobility shift assay supported the idea that the sequence functions as a TRE. The supershift procedure demonstrated that the complex formed in the gel mobility shift assay contains TR. The present study revealed the uniqueness of bullfrog collagenase as a metamorphosis-associated enzyme, because, to our knowledge, known mammalian collagenase genes do not contain THE in their transcription regulatory regions.

Identification and characterization of nucleoplasmin 3 as a histone-binding protein in embryonic stem cells.

Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self‐renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull‐down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification‐related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)‐induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA‐induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells.

Characterization of a collagenolytic enzyme released from wounded planarians Dugesia japonica.

Planarians (Dugesia japonica) were cultured on gels of type I bovine collagen under various conditions that damaged the worms to test the possibility that the animals secrete a collagenolytic enzyme when they are wounded. The planarians were shown to release a potent collagenolytic enzyme around their body when the animals received damage‐inducing treatments such as heating at 37 °C, freezing and thawing, and amputation. This release of the enzyme did not require the synthesis of RNA or protein. Isolated planarian cells did not release the enzyme even when cultured at 37 °C. The collagenolytic activity was found in the supernatants, but not in the insoluble fraction prepared from disintegrated tissues of the wounded animals. These results indicated that the enzyme was stored extracellularly in the normal planarian body. Partial purification of the enzyme and the action spectra of various protease inhibitors on the enzyme showed that the enzyme was a neutral type I collagen‐degrading 40 kDa metalloproteinase. We named this enzyme the planarian collagenase.

Zone analysis by two-dimensional electrophoresis with accelerator mass spectrometry of in vivo protein bindings of idiosyncratic hepatotoxicants troglitazone and flutamide bioactivated in chimeric mice with humanized liver

Analyses using electrophoresis with accelerator mass spectrometry revealed that in vivo bioactivated radiolabeled troglitazone and flutamide, both known to be hepatotoxic in humans, bound nonspecifically to a variety of microsomal and cytosolic proteins in livers from chimeric mice with humanized liver. Unlike those of radiolabeled diazepam (rarely hepatotoxic) and previously reported 5-n-butyl-pyrazolo[1,5-a]pyrimidine (limited hepatotoxicity), some troglitazone and flutamide binding proteins were located in the top right area in a zone analysis, representing high covalent binding contents and high target protein concentrations. Among a variety of liver microsomal proteins bound, the binding target proteins of troglitazone and flutamide with the highest covalent binding contents (in terms of pmol equivalent per μg target protein) were 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase, respectively. Troglitazone and flutamide were activated to reactive metabolites and apparently bound to different target proteins in livers from chimeric mice with humanized liver. The highest covalent binding contents for troglitazone were higher than that for flutamide under the present conditions. These results indicate that the drug metabolism mediated by humanized livers (leading to binding in vivo) in combination with a zone analysis of covalent binding contents/target protein concentration data could be a good tool for evaluating the relationship between the nonspecific protein binding behavior of medicines and potential hepatotoxicity in humans. Thus, testing whether protein binding data of new medicines are unbalanced with respect to deviation from an inverse relationship or the presence of data points in the high covalent binding/high protein concentration zone might be an important concept in evaluating hepatotoxic potential.