Tatsuo Shimada

YB-1 is important for an early stage embryonic development: neural tube formation and cell proliferation.

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. β-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1-/- mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1-/- embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1-/- mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.

Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro

When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibitory effect of anti-FGF antibody was dose-dependent and maximal inhibition was observed at 2.0 micrograms/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor.

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III1, III13, and III14), with III13 and III14 assumed to be the major sites. The interaction between III2–3 and III12–14 was inhibited by DP, whereas the interaction between I1–5 and III12–14 was specifically and strongly enhanced by DP. Because the interaction between III2–3 and III12–14 is involved in forming a globular conformation of fibronectin, and that between I1–5 and III12–14 is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

The endocrine pancreas of spontaneously diabetic db db mice: microangiopathy as revealed by transmission electron microscopy

Abnormalities in ultrastructures of islet capillaries were detected in db/db mice, with the visual inspection and morphometry of electron micrographs. The observed changes are: (1) capillary scarcity; (2) increase in the mean and diversity of capillary size; (3) pericapillary edema and fibrosis; (4) hypertrophy of the pericyte and abundance therein of actin-like microfilaments; and (5) luminal irregularity. Changes (2), (3) and (4) are conceived to indicate hyperperfusion, capillary hypertension and secondary vascular response. In particular, such pericyte changes were found to be shared by other organs whose capillaries are susceptible to diabetic complications.

Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy

 The developmental mechanism of tubulointerstitial fibrosis in diabetic nephropathy (DN) has not been elucidated. Tubulointerstitial fibrosis, as well as glomerulosclerosis, occurs in DN. Myofibroblasts which overproduce extracellular matrix are present in the renal interstitium in diabetics, although they are almost never seen in normal kidneys. The myofibroblasts appear to originate from interstitial fibroblasts. In addition, transforming growth factor-β1 (TGF-β1), which can evoke myofibroblast transformation, is detected in interstitial cells in the diabetic kidney, but not in the normal kidney. Taken together, these findings led us to speculate that TGF-β1 induces the transformation of interstitial fibroblasts into myofibroblasts, followed by tubulointerstitial fibrosis. Based on this speculation, we discuss the developmental mechanism of tubulointerstitial fibrosis in this review.

A procedural method for modeling the purkinje fibers of the heart.

The Purkinje fibers are located in the ventricular walls of the heart, just beneath the endocardium and conduct excitation from the right and left bundle branches to the ventricular myocardium. Recently, anatomists succeeded in photographing the Purkinje fibers of a sheep, which clearly showed the mesh structure of the Purkinje fibers. In this study, we present a technique for modeling the mesh structure of Purkinje fibers semiautomatically using an extended L-system. The L-system is a formal grammar that defines the growth of a fractal structure by generating rules (or rewriting rules) and an initial structure. It was originally formulated to describe the growth of plant cells, and has subsequently been applied for various purposes in computer graphics such as modeling plants, buildings, streets, and ornaments. For our purpose, we extended the growth process of the L-system as follows: 1) each growing branch keeps away from existing branches as much as possible to create a uniform distribution, and 2) when branches collide, we connect the colliding branches to construct a closed mesh structure. We designed a generating rule based on observations of the photograph of Purkinje fibers and manually specified three terminal positions on a three-dimensional (3D) heart model: those of the right bundle branch, the anterior fascicle, and the left posterior fascicle of the left branch. Then, we grew fibers starting from each of the three positions based on the specified generating rule. We achieved to generate 3D models of Purkinje fibers of which physical appearances closely resembled the real photograph. The generation takes a few seconds. Variations of the Purkinje fibers could be constructed easily by modifying the generating rules and parameters.

Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer

Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer.

Morphological characteristics of the dermal papillae in the development of pressure sores.

Mechanisms of skin break down in the development of human pressure sores are still unclear. This study was undertaken to clarify the morphological characteristics of the dermal papillae in the skin associated with pressure sores. Skin tissues were excised from the sacrum of a Japanese subject post mortem, where a superficial pressure sore had developed. Light microscopic and transmission and scanning electron microscopic examinations were performed. It was found that the atrophic, irregular contour and alignment of the dermal papillae were characteristic of the boundary area between healthy and damaged areas. In addition, a relatively dense network of collagen fibres in the papillary layer of the boundary area was observed when compared with the healthy area. These findings suggest that the morphological changes of the papillae observed in the boundary area affect microcirculation, impairing tissue viability by inhibiting nutritive blood supply and by accumulating metabolic byproducts which predispose to tissue damage.

Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ( [125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [125I]EGF in lysosomes and the disappearance of [125I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not beta-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 micrograms/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.

Preclinical Efficacy and Safety of 1-Deoxygalactonojirimycin in Mice for Fabry Disease

Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of α-galactosidase A (α-Gal A) activity. It has been shown that protein misfolding is primarily responsible for the enzyme deficiency in a large proportion of mutations identified in Fabry patients with residual enzyme activity, and 1-deoxygalactonojirimycin (DGJ) can effectively increase the residual enzyme activity in cultured patients cells. Herein, we demonstrate the preclinical efficacy and safety of DGJ in transgenic mice that express human mutant α-Gal A activity. α-Gal A activity in heart, kidney, spleen, and liver was increased dose- and time-dependently. The mutant α-Gal A was increased in cardiomyocytes and distal convoluted tubules of the transgenic mice in a null background after 2 weeks of DGJ treatment. Globotriaosylceramide storage was remarkably reduced in kidney of mice after a 4-week treatment at a dosage of approximately 3 mg/kg body weight/day. The half-life of DGJ was less than 1 day in all major issues and that of the enzyme synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at ∼30 mg/kg body weight/day for 9 weeks. Furthermore, no change was observed in appearance, growth, fertility, and life span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and is well tolerated in mice.