Hierarchical viscosity of aqueous solution of tilapia scale collagen investigated via dielectric spectroscopy between 500 MHz and 2.5 THz.

Aqueous solutions of biomolecules such as proteins are very important model systems for understanding the functions of biomolecules in actual life processes because interactions between biomolecules and the surrounding water molecules are considered to be important determinants of biomolecules' functions. Globule proteins have been extensively studied via dielectric spectroscopy; the results indicate three relaxation processes originating from fluctuations in the protein molecule, the bound water and the bulk water. However, the characteristics of aqueous solutions of collagens have rarely been investigated. In this work, based on broadband dielectric measurements between 500 MHz and 2.5 THz, we demonstrate that the high viscosity of a collagen aqueous solution is due to the network structure being constructed of rod-like collagen molecules surrounding free water molecules and that the water molecules are not responsible for the viscosity. We determine that the macroscopic viscosity is related to the mean lifetime of the collagen-collagen interactions supporting the networks and that the local viscosity of the water surrounded by the networks is governed by the viscosity of free water as in the bulk. This hierarchical structure in the dynamics of the aqueous solution of biomolecules has been revealed for the first time.

Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Microstructures and rheological properties of tilapia fish-scale collagen hydrogels with aligned fibrils fabricated under magnetic fields.

Tilapia fish-scale type I atelocollagen hydrogels with aligned fibril structures were fabricated under a strong magnetic field of 6 or 12 T using two different methods. In the first method, a solution of acid-soluble collagen was neutralized with phosphate buffer saline and maintained in the magnetic field at 28°C for 3h. Under these conditions fibrogenesis occurs, and a hydrogel is formed. The hydrogel was subsequently crosslinked with ethyl-dimethylcarbodiimide (EDC). In the second method, the hydrogels were formed as described above, but in the absence of an applied magnetic field. Only after being crosslinked with EDC were these gels exposed to the magnetic field (28°C for 3h). Both methods led to alignment of the collagen fibrils perpendicular to the magnetic direction, the extent of which depended on the duration of magnetic treatment. Even after EDC treatment, collagen fibrils can align, indicating that crosslinking has taken place within fibrils. Both sorts of aligned hydrogels exhibited similar rheological properties with higher storage and loss moduli than were observed with unoriented gels. The hydrogels treated at 6 T had the best rheological properties. The decrease in tangent angle phase delta indicated that the ratio of elasticity to viscosity was greater in the crosslinked than in the non-crosslinked hydrogels. Atomic force microscopy images showed that magnetic treatment had no effect on the nanostructure of collagen fibrils. Differential scanning calorimetry measurements indicated that collagen hydrogels with and without magnetic treatment had the same denaturation temperature, 48°C, while EDC crosslinking increased the denaturation temperature to 62°C.

Intraperitoneal administration of telomerase-specific oncolytic adenovirus sensitizes ovarian cancer cells to cisplatin and affects survival in a xenograft model with peritoneal dissemination.

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01-100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1-10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1-10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5-1 microM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1-10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg(-1)) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan-Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.

Competitive adsorption of bovine serum albumin and lysozyme on characterized calcium phosphates by polyacrylamide gel electrophoresis method.

Characterizations of hydroxyapatite (HA), biphasic calcium phosphate (BCP) and beta tricalcium phosphate (beta-TCP) ceramic particles were carried out using X-ray diffusion (XRD), Scanning electron micrograph (SEM), Particle Sizer and Zeta potential analyzer. Competitive adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the three calcium phosphates were investigated by polyacrylamide gel electrophoresis (PAGE) method. The results showed that HA, BCP and beta-TCP ceramic particles with irregular shapes and similar size distributions all had negative surface net charges in pH7.4 phosphate buffered saline (PBS) solution and exhibited alike behaviors of BSA and LSZ adsorption. LSZ had higher affinity for calcium phosphate ceramics than BSA and its adsorption on them didn't be almost influenced by the increasing of BSA concentration in the solution. Electrostatic interaction played an important role on the competitive adsorption of BSA and LSZ on the surface of calcium phosphate ceramic particles.

The cross-linkage effect of hydroxyapatite/collagen nanocomposites on a self-organization phenomenon.

Hydroxyapatite(HAp)/collagen nanocomposites were prepared by a coprecipitation method controlling the degree of cross-linkage between collagen molecules using glutaraldehyde. The precipitates filtered were dried in a freeze drier or naturally dried in the air at 25 degrees C. The naturally dried cakes had open channels of 5-15 microm in diameters, which were three-dimensionally and regularly developed over the whole samples, and showed a pretty good mechanical strength. The channels that were formed at spaces among the HAp/collagen particles, cross-linked one another, which had been filled up with water before its evaporation. The ordering state of the open channels depended on the degree of cross-linkage with glutaraldehyde; the optimal self-organized state was found when 30 molecules of glutaraldehyde were added per collagen molecule, though an excess amount of glutaraldehyde suppressed the appearance of the ordered state. From SEM and FT-IR measurements, it was indicated that the self-organization in the HAp/collagen nanocompsites continuously occurred during the drying process together with the removal of water and the increase of the density.

Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice.

We previously reported that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, can induce apoptosis in four kinds of human lung cancer cell lines in vitro. The present study further investigated the in vivo anti-tumor effects of orally administered gallic acid. Gallic acid reduced cell viability of LL-2 mouse lung cancer cells in vitro dose dependently, with a 50% inhibitory concentration (IC50) value of around 200 microM. C57Black mice were transplanted with LL-2 cells, and administered gallic acid (1 mg/ml in drinking water, ad libitum) and/or cisplatin (4 mg/kg i.p. injection, once a week). The average weight of the transplanted tumors, obtained at 29 days after transplantation, in the mice of control, gallic acid-treated cisplatin-treated and cisplatin plus gallic acid-treated groups was 4.02, 3.65, 3.19 and 1.72 g, respectively. The average tumor weight of the mice treated with cisplatin combined with gallic acid was significantly smaller than that of the control group (p<0.05). The amount of apoptotic cells in the tumor tissues of mice treated with gallic acid and/or cisplatin was significantly higher than those of the control mice. Combination of gallic acid and cisplatin increased the tumor cell apoptosis compared with the treatment with cisplatin alone. The present findings suggest that the combination of gallic acid with an anti-cancer drug, including cisplatin, may be an effective protocol for lung cancer therapy.

Experimental and theoretical studies on ferromagnetically coupled metal complexes with imino nitroxides.

Copper(II), zinc(II), and nickel(II) complexes with tridentate imino nitroxyl diradicals, [CuCl(bisimpy)(MeOH)](PF(6)) (1), [ZnCl(2)(bisimpy)] (2), and [NiCl(bisimpy)(H(2)O)(2)]Cl x 2H(2)O (3) (bisimpy = 2,6-bis(1'-oxyl-4',4',5',5'-tetramethyl-4',5'-dihydro-1'H-imidazol-2'-yl)pyridine), were prepared, and their magnetic properties were studied. In 1, the Cu(II) ion has a square pyramidal coordination geometry, of which the equatorial coordination sites are occupied by three nitrogen atoms from the bisimpy and a chloride ion. The coordination geometry of the Zn(II) ion in 2 can be described as a trigonal bipyramid, with two chloride ions and a bisimpy. In 3, the Ni(II) ion has a distorted octahedral coordination geometry, of which four coordination sites are coordinated by the bisimpy and chloride ion, and two water molecules occupy the remaining cis positions. Magnetic susceptibility and EPR measurements revealed that in 1 and 3 the Cu(II) and Ni(II) ions with imino nitroxyl diradicals were ferromagnetically coupled, with the coupling constants J (H = -2J(ij) summation operator S(i)S(j)) of +165(1) and 109(2) cm(-1), respectively, and the intraligand ferromagnetic interactions in 1-3 were very weak. DFT molecular orbital calculations were performed on the diradical ligand, 1, and 2 to study the spin density distribution before and after coordination to the metal ions.

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters.

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.

Effects of quartz addition on the mechanochemical dechlorination of chlorobiphenyl by using CaO.

Grinding a mixture of 3-chlorobiphenyl (BP-Cl) and CaO with or without the addition of quartz was conducted in air by a planetary ball mill to investigate the mechanochemical dechlorination of BP-Cl. The dechlorinating reaction proceeds with an increase in grinding time, and over 99% of BP-Cl is decomposed at 360 min. Washing the ground sample with different solvents results in different products. Addition of quartz to the grinding mixture facilitates dechlorination efficiency, especially in the case of a high weight ratio of BP-Cl to CaO.

[mRNA detection of beta-myosin heavy chain gene in the autopsy cases of hypertrophic cardiomyopathy].

A known trigger of sudden cardiac death, hypertrophic cardiomyopathy (HCM) is associated with a point mutation in or overexpression of MYH7, which encodes the sarcomere protein beta-myosin heavy chain. We used nested RT-PCR to detect MYH7 mRNA in left ventricular myocardial tissue. We extracted total RNA from tissue samples that had been obtained after autopsy of 8 people who had died from HCM-related sudden death and that had been fixed in 10% formalin solution for as long as 4 years. The abundance of total RNA extracted from the 100 mg samples of cardiac muscle ranged from 10 to 24 mg/ml. The products of the nested RT-PCR were electrophoresed on a denaturing 8% polyacrylamide gel, and the MYH7 mRNA product was detected as a 424 bp band. MYH7 mRNA was easier to detect in tissue that had been fixed for shorter rather than longer periods. In addition, the greater the cardiac weight, the higher was the yield of the MYH7 mRNA product. Although mRNA had been detected by using RT-PCR on formalin-fixed paraffin-embedded tissue, no one previously had identified by using RT-PCR or nested RT-PCR on formalin-fixed tissue. By using nested RT-PCR, we were able to detect MYH7 mRNA in myocardial tissue that had been fixed in formalin solution for 4 years. Our results are applicable to retrospective examination into the cause of death in cases of sudden cardiac failure.

[Intratumoral ethanol injection therapy using endoscopic video information system].

Bronchoscopic ethanol injection (BEI) therapy for endobronchial malignant tumor is thought to be an effective modality for airway dilatation and hemostasis without expensive special equipment. But if ethanol leaks into the airway, it causes severe coughing and erosion of bronchial wall. Using endoscopic video information system (EVIS), an assistant operator can observe an ethanol leak and inject an adequate volume. To evaluate the efficacy and adverse effect of BEI therapy, we treated 8 patients with endobronchial malignant tumor using EVIS. Under local anesthesia, a video-bronchoscope (Olympus BF200 or BF 1T200) was inserted orally, a transbronchial aspiration cytology (TBAC) needle was inserted via the biopsy channel, and injections of 0.2 ml of 99% ethanol into the tumor were repeated. After the tumor degenerated to necrotic tissue, it was removed with a biopsy forceps, and this maneuver was repeated for the existing fresh lesion. The total volume of injected ethanol was 4.5 + 1.6 ml (mean + SD). Airway obstruction and atelectasis were relieved in 4 cases, and hemostasis was obtained in 2 cases. In 2 cases, airway dilatation and hemostasis were not obtained. BEI was more effective in the patients with polypoid or nodular tumor protruding into the airway lumen. As for adverse effects, ethanol leaks caused a severe cough in 2 cases, although they were tolerable and treatment could be continued. BEI therapy using EVIS was thought to be useful to control airway obstruction by endobronchial polypoid tumor with good cost-effectiveness and fewer adverse effects.

Inactivation of proprotein convertase, PACE4, by alpha1-antitrypsin Portland (alpha1-PDX), a blocker of proteolytic activation of bone morphogenetic protein during embryogenesis: evidence that PACE4 is able to form an SDS-stable acyl intermediate with alpha1-PDX.

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.

A case of Crush syndrome with giant negative T waves and reversible left ventricular dysfunction.

A 54-year-old male experienced the Hanshin earthquake at 5:46 am on Jan. 17, 1995. He was rescued after being buried under his house for 20 h. After being treated at two hospitals, he was admitted to our hospital with acute renal failure caused by crush syndrome. The maximal serum creatinine kinase level was 35,000 IU/L (CK-MM: 100%), and the maximal myoglobin level was 12,600 ng/ml. An electrocardiogram showed inverted T waves in V5,6 which later became giant negative T waves, and a QS pattern in a VL and V1-6. Two-dimensional echocardiography showed a hypokinetic left ventricle. He was treated with hemodialysis 13 times. Cardiac contraction gradually improved to the normal level. Coronary angiography revealed normal coronary arteries and acetylcholine did not induce coronary spasm. Biopsy specimens from the right ventricular septum showed slight degeneration. 201Tl imaging showed hypoperfusion except in the anterior wall and 123I-MIBG imaging showed a perfusion defect except in the anterior wall. After about 1 month, only 201Tl imaging showed improvement. We believe that myocardial ischemia, cardiac sympathetic nerve damage and/or cardiac contusion caused giant negative T waves and left ventricular dysfunction. This is a rare case which showed significant myocardial damage with crush syndrome.

[Bucillamin induced lung injury in rheumatoid arthritis].

Thirteen cases with rheumatoid arthritis who experienced lung injury during the treatment with bucillamine (Bc), about whom the questionnaires were answered by the physicians and whose X-ray films could be rechecked, were studied. Nine cases out of the 13 showed patchy mottled infiltrates in the bilateral center sparing the periphery, and the other 4 showed diffuse infiltrates. In these 9, serum gamma-globulin level decreased when lung injury appeared. The gamma-globulin level before the start of Bc administration, the level when lung injury appeared, the gamma-globulin decrease, and its ratio to the level before Bc were 1790 +/- 661 mg/dl, 1297 +/- 666 mg/dl, 459 +/- 320 mg/dl and 29.1 +/- 18.0%, respectively. In 5 out of the 9, gamma-globulin level reincreased when they recovered from the injury: 2 out of the 5 showed the reincrease even after steroid therapy. The data were obtained only from 2 out of the 4 with diffuse pattern in X-ray, and the decrease and the decrease ratio were 200 mg/dl (5.8%) in 1, and 49 mg/dl (3.6%) in the other. The characteristics of Bc-induced lung injury might be mottled infiltrates in the center appearing concurrently with serum immunoglobulin decrease.

Follow-up observation of a patient with left ventricular thrombus by echocardiography.

We report a case of left ventricular thrombus which caused systemic embolism during warfarin therapy. A 66-year-old man admitted to our hospital with intermittent loss of consciousness and incomplete palsy. The patient had a past history of cerebral infarction, hypertension and diabetes mellitus. Electrocardiography showed abnormal Q waves at II, III, and aVF. Echocardiography showed a mobile protruding thrombus at the left ventricular apex. Left ventricular cineangiography showed a filling defect at the left ventricle. Coronary angiography confirmed total occlusion of the right coronary artery and significant stenosis of the left coronary artery. After administration of warfarin, the patient suddenly fell down to the comatose state. His left arm became pale without pulsation. The thrombus in the left ventricle disappeared by echocardiography and systemic embolism was suspected. Thromboembolectomy from his left arm was performed. Two weeks after the day brain computed tomography showed low density area at the posterior lobe of the cerebrum. Although the eyesight of the patient was lost, he could stand and walk with some help two months later. This is a rare case of systemic embolism during warfarin therapy due to left ventricular thrombus whose process was constantly observed by echocardiography.