Yurika Saitoh

Role of the CXCL12/CXCR4 axis in Peritoneal Carcinomatosis of Gastric Cancer

Peritoneal carcinomatosis is a frequent cause of death in patients with advanced gastric carcinoma. Because chemokines are now considered to play an important role in the metastasis of various malignancies, we hypothesized that they may be involved in the development of peritoneal carcinomatosis by gastric carcinoma. Human gastric carcinoma cell lines, which were all highly efficient in generating malignant ascites in nude mice upon i.p. inoculation, selectively expressed CXCR4 mRNA and protein. In particular, NUGC4 cells expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its ligand CXCL12. CXCL12 enhanced proliferation and rapid increases in phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase of NUGC4 cells. We also showed that AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor growth and ascitic fluid formation in nude mice inoculated with NUGC4 cells. Additionally, we examined human clinical samples. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high concentrations of CXCL12 (4.67 ng/mL). Moreover, immunohistochemical analysis showed that 22 of 33 primary gastric tumors with peritoneal metastasis were positive for CXCR4 expression (67%), whereas only 4 of 16 with other distant metastasis were positive (25%). Notably, 22 of 26 CXCR4-expressing primary tumors developed peritoneal metastases (85%). CXCR4 positivity of primary gastric carcinomas significantly correlated with the development of peritoneal carcinomatosis (P < 0.001). Collectively, our results strongly suggest that the CXCR4/CXC12 axis plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. Thus, CXCR4 may be a potential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

Role of CX3CL1/Fractalkine in Osteoclast Differentiation and Bone Resorption

The recruitment of osteoclast precursors toward osteoblasts and subsequent cell-cell interactions are critical for osteoclast differentiation. Chemokines are known to regulate cell migration and adhesion. CX3CL1 (also called fractalkine) is a unique membrane-bound chemokine that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in osteoblast-induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of bone marrow cells containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteoblasts. Anti-CX3CL1 also significantly suppressed bone resorption in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis, osteoporosis, and cancer bone metastasis.

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

ABSTRACT Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures.

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.

Immunoreactivity of Glutamate in Mouse Retina Inner Segment of Photoreceptors With In Vivo Cryotechnique

The purpose of this study was to clarify a previously controversial issue concerning glutamate (Glu) immunoreactivity (IR) in the inner segment (IS) of photoreceptors by using in vivo cryotechnique (IVCT) followed by freeze substitution (FS), which enabled us to analyze the cells and tissues reflecting living states. Eyeballs from anesthetized mice were directly frozen using IVCT. The frozen tissues were processed for FS fixation in acetone containing chemical fixatives, and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. The strongest Glu-IR was obtained in the specimens prepared by FS with paraformaldehyde or a low concentration of glutaraldehyde, whereas no Glu-IR was obtained without the chemical fixatives. The Glu was immunolocalized in the IS, outer and inner plexiform and ganglion cell layers. Thus, the immunolocalization of Glu in the IS was clearly demonstrated using IVCT.

Involvement of the Tyro3 receptor and its intracellular partner Fyn signaling in Schwann cell myelination

Schwann cells—peripheral myelin-forming glial cells—wrap around individual neurons. Tyro3 tyrosine kinase is a Schwann cell receptor that regulates myelination. Furthermore, Tyro3 interacts with the Fyn nonreceptor tyrosine kinase to promote myelination, representing a new receptor-linked signaling mechanism that controls myelination.

Immunohistochemical Distribution of Serum Proteins in Living Mouse Heart with In Vivo Cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods.

Involvement of Src in the membrane skeletal complex, MPP6–4.1G, in Schmidt–Lanterman incisures of mouse myelinated nerve fibers in PNS

Schmidt–Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). In this study, we report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite to those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the membrane protein palmitoylated 6 (MPP6) in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate that the Src–MPP6–4.1G protein complex in SLIs has a role in signal transduction in the PNS.

Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique

Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an “in vivo cryotechnique” (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofixed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffinized sections were immunostained for P-PK-S, Na+/HCO3− cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identified the same 40-kDa bands. The IVCT is useful for time-dependent analysis of the immunodistribution of P-PK-S and P-Akt-S.