Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis.

BACKGROUND: Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen. OBJECTIVE: To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations. METHODS: Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma. RESULTS AND CONCLUSIONS: A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.

BMP10 expression in the adult rat central nervous system.

Bone morphogenetic protein 10 (BMP10), is a member of the transforming growth factor ß (TGFß) superfamily. Although BMP10 plays pivotal roles during development, including vascular development and cardiogenesis, little information is available for BMP10 expression in the central nervous system (CNS). We, thus, investigated BMP10 expression in the adult rat CNS using immunohistochemistry. BMP10 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express BMP10 protein. These data indicate that BMP10 is widely expressed throughout the adult CNS, and this abundant expression strongly supports the idea that BMP10 also plays important roles in the adult CNS.

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes.

The reactivation of X-linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X-linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist-inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X-linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X-linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.

BMP9 expression in the adult rat brain.

Bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2 (GDF2), is a member of the transforming growth factor ß (TGF ß) superfamily. Although BMP9 plays pivotal roles during development, including angiogenesis, hematopoiesis, hepatogenesis, osteogenesis, and glucose metabolism, little information is available for BMP9 expression in the central nervous system (CNS). We, thus, investigated BMP9 expression in the adult rat CNS using immunohistochemistry. BMP9 was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes and ependymal cells also express BMP9 protein. These data indicate that BMP9 is widely expressed throughout the adult CNS, and this abundant expression strongly supports the idea that BMP9 also plays important roles in the adult brain.

Follistatin expression in the central nervous system of the adult rat.

Follistatin was initially cloned as a monomeric polypeptide that inhibits the release of follicle-stimulating hormone. Although follistatin also plays pivotal roles in skeletal muscle hypertrophy and immunoregulation in the epididymis, little information is available regarding follistatin function in the adult central nervous system (CNS). Hence, we investigated follistatin expression in the adult rat CNS using immunohistochemistry. Follistatin was intensely expressed in most neurons and their axons. Furthermore, oligodendrocytes, ependymal cells, and some astrocytes also expressed follistatin protein. These data indicate that follistatin is widely expressed throughout the adult CNS. The abundant expression of follistatin in the adult brain suggests that this protein plays important roles in the CNS.

BMP6 expression in the adult rat central nervous system.

BMP6, a member of the TGF-ß superfamily, is known to be involved in many diseases, such as Alzheimer's disease, suggesting that BMP6 plays pivotal roles in the central nervous system (CNS), however, there's no information about the distribution of BMP6 in the adult CNS. Therefore, we investigated BMP6 expression in the CNS using immunohistochemistry. BMP6 was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes, astrocytes and ependymal cells also express BMP6 protein. These data indicate that BMP6 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that BMP6 plays important roles in the adult brain.

Myostatin expression in the adult rat central nervous system.

Myostatin (also called as growth and differentiation factor 8 or GDF8), a member of the transforming growth factor ß (TGF-ß) superfamily of secreted differentiation and growth factors, is a potent inhibitor of skeletal muscle mass in mammals. Although myostatin also plays pivotal roles in cardiac growth and metabolism, postnatal glucose metabolism and adipogenesis, little information is available for myostatin function in the adult central nervous system (CNS). We, thus, investigated myostatin expression in the adult rat CNS using immunohistochemistry. Myostatin was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes, astrocytes and ependymal cells also express myostatin protein. These data indicate that myostatin is widely expressed throughout the adult CNS, and its abundant expression in the adult brain suggests the idea that myostatin plays important roles in the CNS.

GDF11 expression in the adult rat central nervous system.

Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a member of the transforming growth factor ß (TGF-ß) superfamily. Although GDF11 plays pivotal roles during development, including anterior/posterior patterning, formation of the kidney, stomach, spleen and endocrine pancreas, little information is available for GDF11 expression in the adult central nervous system (CNS). We, thus, investigated GDF11 expression in the adult rat CNS using immunohistochemistry. GDF11 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express GDF11 protein. These data indicate that GDF11 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that GDF11 plays important roles in the adult brain.

Chordin and noggin expression in the adult rat trigeminal nuclei.

Bone morphogenetic proteins (BMP) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators, such as chordin and noggin. Although the deep involvement of BMP signaling in the development and functions of the trigeminal nuclei has been postulated, little information is available for its expression in the trigeminal nuclei. We, thus, investigated chordin and noggin expression in the adult rat trigeminal nuclei using immunohistochemistry. Chordin and noggin were intensely expressed throughout the trigeminal nuclei. In addition, interesting differences are observed between chordin expression and noggin expression. For example, chordin prefers dendritic expression than noggin, suggesting that chordin is involved in the regulation of dendritic morphology and synaptic homeostasis. Furthermore, chordin and noggin were differentially expressed in the neuropil of the trigeminal nuclei. Since BMP signaling is known to play a pivotal role to make precise neural network, theses differences might be important to keep precise interneuronal connections by regulating local BMP signaling intensity in each region. Interestingly, we also detected chordin and noggin expression in axons of the trigeminal nerves. These data indicate that chordin and noggin play pivotal roles also in the adult trigeminal system.

Three-dimensional computed tomographic analysis of variations of the carotid artery.

BACKGROUND: Although the terms tortuous, coiling, and kinking have been used to describe the curvature of the carotid artery, the prevalence rates of these patterns have differed among studies. We morphologically evaluated the characteristics of the carotid artery by means of three-dimensional computed tomography (3DCT) to clarify the prevalence of tortuosity, coiling, and kinking. We present our results and discuss the clinical impact of our findings. METHODS: A total 148 patients underwent contrast-enhanced CT (including 55 patients who underwent dynamic CT), and anatomical variations were analyzed on the basis of 3DCT images. RESULTS: Among the 296 arteries, tortuosity was present in 254 (85.8%), coiling in 9 (3.0%), kinking in 3 (1.0%), and occlusion in 2 (0.7%). CONCLUSION: 3DCT image reconstruction is an effective means for classifying morphological variations of the ICA and detecting abnormalities of the carotid artery. It can thereby potentially reduce the risk of serious complications during neck surgery.

E3 Ubiquitin Ligases as Molecular Targets in Human Oral Cancers.

The ubiquitin-proteasome pathway is involved in various biological processes. Several oncogenic E3 ligases target tumor suppressor proteins for ubiquitin-mediated degradation. Alternatively, some other E3 ligases play as a tumor suppressor specifically targeting oncogene products. Deregulation of these E3 ligases induces unbalance between oncogenic signal and tumor suppressor pathway and leads to cellular transformation, tumor growth and metastasis in various human malignancies including oral, and head and neck cancers. Facilitated degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) has been observed in oral, and head and neck cancers, and is correlated with their poor prognosis. SCF(Skp2), KPC complex, Pirh2 and CRL4(DDB2-Artemis) have been reported as E3 ligases targeting p27(Kip1) for degradation. In oral cancers, it is reported that overexpression of Skp2 and Pirh2 is associated with poor prognosis. Thus, chemical inhibitors against these E3 ligases are applicable for oral cancer therapy. Some potential compounds that inhibit E3 ligase activity of SCF(Skp2) have been reported. Moreover, the HECT-type E3 ligase WWP family and Smurf1 are also involved in the development and growth of human oral cancers. Therefore, small molecule inhibitors against HECT-type E3 ligases are discussed as anti-oral cancer drugs.

Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of THR176 in GATA-binding protein 2.

A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

Improved technique for evaluating oral free flaps by pinprick testing assisted by indocyanine green near-infrared fluorescence angiography.

In head and neck surgery, free-flap reconstruction using a microvascular anastomosis is an indispensable option after tumor ablation. Because the success of free-flap reconstruction is enhanced by rapid identification and salvage of failing flaps, postoperative monitoring of free flaps is essential. We describe a new technique using indocyanine green (ICG) near-infrared angiography and pinprick testing to monitor intraoral free flaps. A solution of ICG (Diagnogreen, 5 ml) was intravenously injected, and scanning was performed with a near-infrared video camera system. Thirty seconds after ICG injection, a pinprick test was performed by placing a 24-gage needle through the dermis to the subcutaneous fat of the flap. Pinprick testing during ICG fluorescence imaging was performed in 30 patients. Flap perfusion was confirmed in all patients, and all flaps survived postoperatively. ICG fluorescence imaging demonstrated that flap perfusion was maintained.

Matrix-assisted laser desorption/ionization imaging mass spectrometry revealed traces of dental problem associated with dental structure.

Periodontal disease is a serious dental problem because it does not heal naturally and leads to tooth loss. In periodontal disease, inflammation at periodontal tissue is thought as predominant, and its effect against tooth itself remains unclear. In this study, we applied matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS) to teeth for the first time. By comparing anatomical structure of tooth affected with periodontal disease with normal ones, we analyzed traces of the disease on tooth. We found signals characteristic of enamel, dentin, and dental pulp, respectively, in mass spectra obtained from normal teeth. Ion images reconstructed using these signals showed anatomical structures of the tooth clearly. Next, we performed IMS upon teeth of periodontal disease. Overall characteristic of the mass spectrum appeared similar to normal ones. However, ion images reconstructed using signals from the tooth of periodontal disease revealed loss of periodontal ligament visualized together with dental pulp in normal teeth. Moreover, ion image clearly depicted an accumulation of signal at m/z 496.3 at root surface. Such an accumulation that cannot be examined only from mass spectrum was revealed by utilization of IMS. Recent studies about inflammation revealed that the signal at m/z 496.3 reflects lyso-phosphatidylcholine (LPC). Infiltration of the signal is statistically significant, and its intensity profile exhibited the influence has reached deeply into the tooth. This suggests that influence of periodontal disease is not only inflammation of periodontal tissue but also infiltration of LPC to root surface, and therefore, anti-inflammatory treatment is required besides conventional treatments.

Imaging mass spectrometry distinguished the cancer and stromal regions of oral squamous cell carcinoma by visualizing phosphatidylcholine (16:0/16:1) and phosphatidylcholine (18:1/20:4).

Most oral cancers are oral squamous cell carcinoma (OSCC). The anatomical features of OSCC have been histochemically evaluated with hematoxylin and eosin. However, the border between the cancer and stromal regions is unclear and large portions of the cancer and stromal regions are resected in surgery. To reduce the resected area and maintain oral function, a new method of diagnosis is needed. In this study, we tried to clearly distinguish the border on the basis of biomolecule distributions visualized by imaging mass spectrometry (IMS). In the IMS dataset, eleven signals were significantly different in intensity (p < 0.01) between the cancer and stromal regions. Two signals at m/z 770.5 and m/z 846.6 were distributed in each region, and a clear border was revealed. Tandem mass spectrometric (MS/MS) analysis identified these signals as phosphatidylcholine (PC) (16:0/16:1) at m/z 770.5 in the cancer region and PC (18:1/20:4) at m/z 846.6 in the stromal region. Moreover, the distribution of PC species containing arachidonic acid in the stromal region suggests that lymphocytes accumulated in response to the inflammation caused by cancer invasion. In conclusion, the cancer and stromal regions of OSCCs were clearly distinguished by use of these PC species and IMS analysis, and this molecular identification can provide important information to elucidate the mechanism of cancer invasion.

Photodynamic therapy with intradermal administration of 5-aminolevulinic acid for port-wine stains.

OBJECTIVE: To investigate whether intradermal administration of 5-aminolevulinic acid (5-ALA) could achieve a sufficient amount of protoporphyrin IX (PpIX) to induce photochemical reaction in chicken comb, the animal model of port wine stains (PWSs). METHODS: PpIX accumulation after 5-ALA administration through intradermal or intravenous injection was monitored for 24 hours. Localization of PpIX was observed under a confocal microscope. The comb was exposed to red light after intradermal 5-ALA injection, and the subsequent changes were observed grossly and microscopically. RESULTS: In the comb, PpIX accumulation achieved the peak level at 5 and 4 hours after intravenous or intradermal injection of 5-ALA, respectively, and was almost completely eliminated within 24 hours. A similar amount of PpIX was observed in both groups. While in the body skin, a lower level of PpIX was observed after intradermal injection. A confocal microscope showed that PpIX distributed evenly in comb dermis without significant difference between the two groups. The vascular structure in comb was disrupted after laser irradiation based on intradermal administration of 5-ALA. CONCLUSIONS: Intradermal injection of 5-ALA is a safer administration route that could achieve the equivalent of PpIX accumulation and destroy vasculature after PDT. It might be applicable to the clinical treatment of PWSs.

Sufficient PpIX production for PDT even with short contact time of topically applied 5-ALA in rabbit tongues.

Although effectiveness of photodynamic therapy (PDT) after application of 5-aminolevulinic acid ointment to oral mucosal lesions has been reported, a consensus regarding recontact time of ALA applied to a lesion has been unreached. Hence, we determined the contact time of ALA required for protoporphyrin IX (PpIX) production efficient for full-blown PDT reaction. ALA ointment was topically applied to healthy rabbits' tongues for different periods and then washed out. On the surface of the tongue, 10-min contact of ALA maximized the PpIX-derived fluorescence. PpIX yield in a tissue specimen with 10-min contact of ALA reached 73% of that in a tissue specimen with 240-min contact. Histological observation showed that PpIX accumulation predominated in the basal layer, and the PDT effects were confined in the mucosal epithelium regardless of contact time. These results suggest that 5-aminolevulinic-acid-ointment-mediated PDT with short contact of ALA is potentially applicable for treating tongue epithelial lesions.

Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma.

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5-fluorouracil (5-FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5-FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5-FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC.