Nrf2 activation attenuates both orthodontic tooth movement and relapse.

During orthodontic tooth movement, osteoclasts resorb the alveolar bone at the compress side of periodontium. Reactive oxygen species (ROS) works as intracellular signaling molecules of RANKL during osteoclastogenesis, although ROS has cytotoxicity against cells such as lipid oxidation. To deal with oxidative stress, cells have a defense system that is scavenging ROS by augmented antioxidative stress enzymes via transcriptional regulation with nuclear factor E2-related factor 2 (Nrf2). Previously, we reported that augmented antioxidative stress enzymes by Nrf2-gene transfer inhibited bone destruction. In the present study, we examined the effects of Nrf2 activation on osteoclastogenesis and, thereby, orthodontic tooth movement and orthodontic relapse. Mouse macrophage cell line RAW264.7 cells were used as osteoclast progenitor cells and stimulated with recombinant RANKL (100 ng/mL) with or without Nrf2 activator sulforaphane (SFN) and epigallocatechin gallate (EGCG) or ROS scavenger catechin. Osteoclastogenesis, resorption activity, and osteoclast marker gene expression were examined. Intracellular ROS was analyzed by flow cytometry. Maxillary first molars of C57BL6 male mice were moved palatally with 0.012-inch NiTi wire (100-mN force); SFN or EGCG was injected into the palatal gingiva once a week; and phosphate buffered saline was injected on the contralateral side. Tooth movement was monitored using a stone model with precise impression, and the amount of the tooth movement was compared among groups. SFN and EGCG significantly, but catechin weakly, inhibited RANKL-mediated osteoclastogenesis in vitro. Western blot analysis revealed that SFN and EGCG augmented the nuclear translocation of Nrf2 and the expression of anti-oxidative stress enzymes such as HO-1, although catechin did not. SFN and EGCG significantly, but catechin weakly, attenuated the intracellular ROS. Finally, animal experiment revealed that both SFN and EGCG successfully inhibited the orthodontic tooth movement. Additionally, SFN inhibited the relapse. These results suggest that Nrf2 activation could be therapeutic target for the anchorage enforcement in orthodontic treatment and pharmacologic retention against relapse.

Multifocal granulomatous jejunitis associated with an argyrophilic gram-positive segmented filamentous bacterium in a Holstein cow.

Multifocal, raised, ulcerated firm nodules accompanied by an intussuscepted area were detected in the jejunum of an 8-year-old Holstein cow. The cut surfaces of the nodules were yellow-white. Microscopically, the lamina propria was expanded by an intense infiltration of epithelioid cells, multinucleate giant cells, macrophages, lymphocytes and neutrophils. Numerous bacteria were found within the granulomatous lesions. These were argyrophilic, gram-positive, periodic acid-Schiff-positive, segmented, rarely branched, elongate filamentous bacteria (2-28 µm in length, 0.2-0.35 µm in diameter). Ultrastructurally, a cell wall with an electron-transparent zone was detected. The present pathogen was clearly different from the argyrophilic, gram-negative, non-segmented, filamentous bacterium previously reported in a Holstein cow with jejunal granuloma. Comparative 16S rDNA gene sequencing analysis revealed that the organism was an unpublished species (GenBank accession number AB539875). This is the first report of bovine jejunal granuloma associated with an argyrophilic gram-positive segmented filamentous bacterium.

Lawsonia intracellularis and virulent Rhodococcus equi infection in a thoroughbred colt.

A 26-month-old thoroughbred colt with a 4-month history of continuous diarrhoea and weight loss was subject to necropsy examination. The small intestinal mucosa was thickened and this change particularly affected the terminal ileum. Microscopical examination revealed multifocal epithelial hyperplasia, with multifocal granulomas and marked lymphocytic infiltration of the lamina propria. Numerous gram-negative argyrophilic curved bacilli were observed within the cytoplasm of affected enterocytes. Macrophages and epithelioid cells forming the granulomas had abundant, lightly eosinophilic, foamy cytoplasm, with occasional large, clear vacuoles containing gram-positive coccobacilli. Immunohistochemical studies suggested that the argyrophilic bacilli were Lawsonia intracellularis and the gram-positive coccobacilli were Rhodococcus equi. L. intracellularis-specific DNA fragments were amplified from the affected ileocaecal mucosa by polymerase chain reaction. Virulent R. equi (VapA positive) was isolated in pure culture from the liver and mesenteric lymph nodes. These results suggested that the two intracytoplasmic organisms had induced multifocal proliferative and granulomatous enteritis accompanied by severe and extensive lymphocytic infiltration.

Is frozen section effective for diagnosis of unsuspected gallbladder cancer during laparoscopic cholecystectomy?

BACKGROUND: Although frozen section is recommended to prevent tumor dissemination following laparoscopic cholecystectomy (LC) for unsuspected gallbladder cancer, there are no reports concretely demonstrating its effectiveness and outcome. METHODS: Frozen section during LC was performed in 990 patients with gallstones. The sensitivity, specificity of frozen section, and false-negative cases were evaluated in comparison with postoperative entire cross sections. RESULTS: In frozen section, 983 cases were diagnosed as benign and 7 cases as malignant. Of the benign cases, cancer was discovered in 4 patients postoperatively in which frozen section was diagnosed as regenerative epithelial severe atypia. Sensitivity was 64% and specificity was 100%. Concerning the results of frozen section by p-TNM classification, cancer was diagnosed in 40% of Tis lesions, whereas it was found in 83% of T2 or T3 lesions. CONCLUSION: Frozen section is effective in cases with T2 or greater lesions for which conversion to radical surgery should be required.

Enzymatic degradation of atactic poly(R,S-3-hydroxybutyrate) induced by amorphous polymers and the enzymatic degradation temperature window of an amorphous polymer system.

The phase structure and biodegradability were investigated for amorphous blends of chemosynthetic fully amorphous atactic poly(R,S-3-hydroxybutyrate) (a-PHB) with atactic poly(methyl methacrylate) (PMMA) and atactic poly(R,S-lactide) (a-PLA). The differential scanning calorimetry thermal analysis indicated that a-PHB/PMMA blends were partially miscible while a-PHB/a-PLA blends were miscible in the studied composition range. The biodegradations of the blends were carried out in phosphate buffer solution in the presence of bacterial poly(R-3-hydroxybutyrate) extracellular depolymerases purified from Alcaligenes faecalis T1 and P. stutzeri. Although a-PHB in the pure state was not degraded by these depolymerase, it was degraded by blending with PMMA and a-PLA. The results demonstrated that the enzymatic degradation of a-PHB can be induced by amorphous polymers such as PMMA and a-PLA. Also, the biodegradation rate of a-PHB in the blends decreased drastically when the degradation temperature is too much away from the polymer glass transition temperatures. On the basis of these results, a temperature window of the enzymatic degradation was first proposed for the blend and the essence of induced degradation was discussed.

Dye-staining stereomicroscopic examinations for fine mucosal structures of the gallbladder.

BACKGROUND: In order to diagnose an unsuspected gallbladder carcinoma and to examine whether a differential diagnosis could be made between cancer and noncancerous lesions during surgery, we evaluated the findings of fine structures of various types of gallbladder mucosa. METHODS: We used stereomicroscopy with a dye-contrast technique under water and measured the maximum blood vessel diameters of the gallbladder mucosa: normal gallbladder, chronic cholecystitis, and carcinoma. RESULTS: All normal gallbladders showed fine-reticular-type findings. In chronic cholecystitis, 5.8% of the specimens (n = 69) had fine reticular type, 87.0% had rough reticular type, and 7.2% had atrophic type. All the cases of adenomyomatosis (n = 16) showed rough reticular type. In eight specimens of pancreaticobiliary maljunction, 75% of them showed high reticular type, and the other 25% showed papillary type. The two adenoma specimens showed fine granular type. In five gallbladder carcinomas, the lattice-like pattern completely disappeared and showed rough granular type. The average of maximum vessel diameters in the gallbladder mucosa were 41.0 microm in normal gallbladders, 99.1 microm in patients with chronic cholecystitis, and 614.8 microm in patients with a carcinoma. There were significant differences among them (p < 0.05). CONCLUSION: This study showed that differential diagnosis between cancer and noncancerous lesion is possible by dye-staining mucosal pattern and measurement of maximum vessel diameters by stereoscopic examination.

Is preventive resection of the extrahepatic bile duct necessary in cases of pancreaticobiliary maljunction without dilatation of the bile duct?

BACKGROUND: No consensus has been reached on whether preventive resection of the extrahepatic bile duct is necessary in cases of pancreaticobiliary maljunction (PBM) without dilatation of the extrahepatic bile duct (undilated type). METHODS: Sixty-eight patients with PBM underwent corrective surgery and several clinical characteristics and pathological findings including K-ras point mutation were evaluated. RESULTS: Unlike dilated bile duct, none of the patients with undilated type duct had clinical symptoms in early childhood. In patients with either cystic or spindle type duct, amylase levels in the bile duct were >10(4) U/l, whereas those in patients with undilated type duct were <10(4) U/l. Postoperative scintigraphy of the biliary system of undilated type revealed no evidence of cholestasis. After surgery, eight patients with undilated type duct, in whom the bile duct had been preserved, had no further clinical symptoms and no evidence of malignancy. Bile duct tissue specimens revealed no hyperplasia, dysplasia or cancerous lesions and they had no K-ras mutation in undilated type. CONCLUSION: The results showed that there was little bile stasis, injury to the mucosa was mild and less genetic changes could be seen in patients with undilated type duct. Therefore, in patients without dilatation of bile duct and advanced cancer, cholecystectomy alone is sufficient.

Relation between K-ras codon 12 mutation and p53 protein overexpression in gallbladder cancer and biliary ductal epithelia in patients with pancreaticobiliary maljunction.

It is well known that the incidence of biliary cancer is higher in patients with pancreaticobiliary maljunction (PBM) than in individuals without PBM. However, the relationship between PBM and the carcinogenesis remains unclear. The purpose of the present study was to examine histopathologic changes in the mucosa of the gallbladder and bile duct in patients with PBM, and to investigate K-ras oncogene mutation and overexpression of p53 protein in the mucosa. We examined 47 surgical specimens of gallbladder and 36 surgical specimens of bile duct obtained from 48 patients with PBM. The 48 patients were divided into three age groups: group A (0-3 years), group B (4-39 years), and group C (40 years or more). Investigation of K-ras mutation and overexpression of p53 protein was performed using an enriched polymerase chain reaction (PCR) and enzyme-linked mini-sequence assay (ELMA), and by the streptavidin-biotin (SAB) method, using DO-7 antibodies, respectively. Hyperplastic changes in the gallbladder mucosa were observed in patients in the three groups. However, metaplastic or dysplastic changes were observed in the mucosa of only groups B and C. K-ras gene mutation in the gallbladder mucosa was found in 18.8% of the hyperplastic mucosae in group B and in 20% in group C. The mutation was found in 33.3% of lesions with metaplastic change associated with hyperplastic changes and in 25% of lesions with dysplastic changes in group C. No mutation was observed in the non-cancerous mucosae of gallbladders and bile ducts without congenital dilatation of the bile duct. Overexpression of p53 protein was observed only in carcinoma of the gallbladder; in seven of nine advanced carcinomas and in two of three carcinomas in situ. We concluded that the mucosal epithelia of the biliary system in patients with PBM showed a high frequency of gene mutations and the carcinogenesis appeared in involve a multistage process of mutation in the K-ras gene and the p53 suppressor gene.

Nuclear cyclin D1 overexpression is a critical event associated with cell proliferation and invasive growth in gallbladder carcinogenesis.

Cyclin D1 overexpression is remarkably frequent in several human carcinomas and is believed to be a critical event in oncogenesis. We examined cyclin D1 expression, p53 expression, and the Ki-67 labeling index by immunostaining in human gallbladder mucosa in conditions varying from normal to malignant tissue. We also examined K-ras codon 12 mutations in these tissues with a two-step polymerase chain reaction. Nuclear cyclin D1 overexpression was observed in 48% of carcinomas occurring independently of adenoma, but not in adenomas, carcinomas arising in adenomas, or nonneoplastic lesions. Cytoplasmic cyclin D1 overexpression was observed in about 15% of abnormal specimens, irrespective of the type of epithelial abnormality. Carcinomas showing nuclear cyclin D1 overexpression had significantly higher Ki-67 labeling indexes than those with no overexpression. Moderately to poorly differentiated adenocarcinomas showed a higher incidence of nuclear cyclin D1 overexpression than papillary to well differentiated carcinomas. Specimens with cyclin D1 overexpression showed a high incidence of lymph permeation, venous permeation, and lymph node metastasis. We conclude that nuclear cyclin D1 overexpression is a critical event importantly associated with cell proliferation and invasive growth in gallbladder carcinogenesis, and that cyclin D1 immunostaining may become a useful marker for evaluating gallbladder carcinomas.

Identification of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase.

A poly[(R)-3-hydroxybutyrate] (P(3HB))-degrading marine bacterium (strain NK-1, JCM10458) was isolated from the Pacific Ocean deep-sea floor (1165 m in depth) in Japan. The organism was a motile and Gram negative, aerobic, and rod-shaped bacterium, and its DNA had a guanine-plus-cytosine content of 57.7 mol%. On the basis of several phenotypic characters and a phylogenetic analysis of the gene coding for 16S rRNA, this strain was identified as Marinobacter sp. The strain required sodium salt for growth in the medium and secreted a P(3HB) depolymerase into the supernatant when it was cultivated on (S)-3-hydroxybutyric acid or P(3HB) as the sole carbon source. The P(3HB) depolymerase (PhaZMsp) was purified to homogeneity from the culture supernatant of Marinobacter sp. by hydrophobic and ion exchange column chromatography and showed a molecular mass of 70 kDa. PhaZMsp was stable at temperatures below 37 degrees C and at pH values of 7.5-10.0. The N-terminal amino acid sequences of both the purified enzyme and the truncated one shared high homologies to the N-terminal and internal sequences of Pseudomonas stutzeri depolymerase, respectively. High-performance liquid chromatography analysis revealed that the enzymatic products of P(3HB) yielded monomer, dimer, and trimer of 3-hydroxybutyric acid. PhaZMsp was capable of hydrolyzing P(3HB), poly(3-hydroxypropionate), and poly(4-hydroxybutyrate).

Function of the catalytic domain of poly(3-hydroxybutyrate) depolymerase from Pseudomonas stutzeri.

The mechanism of enzymatic hydrolysis for (R)-3-hydroxybutyrate (3HB) oligomers with poly[(R)-3-hydroxybutyrate] [P(3HB)] depolymerase (PhaZpst) from Pseudomonas stutzeri was investigated by two deletion mutants lacking the substrate-binding domain and linker region, PhaZpst delta sbd and PhaZpstcore. The two deletion mutants had no ability for hydrolysis of water-insoluble P(3HB), while the hydrolysis activities of two deletion mutants for water-soluble 3HB oligomer and its derivatives (dimer, trimer, and tetramer) were identical with those of the wild type, indicating that the function of catalytic domain is independent of its substrate-binding domain and linker region. The hydrolyzed products analysis of 3HB oligomers by HPLC showed that the active site of catalytic domain recognizes at least two 3HB units for hydrolysis. The initial rates of hydrolysis of dimer derivative were lower by 2 orders of magnitude than those of trimer and tetramer derivatives, suggesting that 3HB oligomer derivatives larger than trimer are favorite substrates for PhaZpst.

[Percutaneous ethanol injection therapy with hepatic arterial infusion chemotherapy for liver metastases from gastric cancer].

Ten patients with liver metastases from advanced gastric cancer received percutaneous ethanol injection therapy (PEI) and chemotherapy by hepatic arterial infusion (HAI) via implantable reservoir. A 90% ethanol solution including 10%. Lipiodol was injected in the liver as PEI.5-FU, EPIR and MMC were used as the regimen for HAI chemotherapy. We have performed this therapy (PEI + HAI) for ten patients with liver metastases since February, 1997. These patients have received this therapy for 4-36 months and three patients died within 16 months. However, three patients did not develop any liver failure after this therapy. The median survival rate was 25.2 months. There are statistically significant differences between upto ss and over se of invasion, and between INF alpha and gamma (p = 0.005).

Assessment of cerebrospinal fluid levels of serum amyloid P component in patients with Alzheimer's disease.

Serum amyloid P component (SAP) is a normal plasma constituent that is observed both in senile plaque and in neurofibrillary tangle in brains of patients with Alzheimer's disease (AD). In this study, we evaluated the SAP levels in cerebrospinal fluid (CSF) of 72 patients with AD, 11 frontotemporal dementia and nine normal control subjects. There was no significant difference in the SAP levels between the AD group and other groups. However, among AD patients, cognitive function was rated using the Mini-Mental State Examination and was correlated with the SAP level (R = 0.38, P < 0.05). Our results suggest that measurement of the SAP levels in CSF can be useful for assessing the degree of cognitive impairment in AD patients.

Malignant paraganglioma metachronously recurring at short duration in different paraganglions: report of a case.

We report the rare case of a 57-year-old man with retroperitoneal malignant paraganglioma. He was referred to our hospital complaining of left lower abdominal dull pain. Computer tomography (CT), magnetic resonance imaging, and aortography showed a mass measuring 5 x 4 cm in size on the left side of the abdominal aorta below the renal artery. The resected tumor was confirmed histologically to be malignant paraganglioma. Irradiation of the resected area was performed. Four months after the operation, an abdominal CT scan showed a 5.5 x 3.0 cm mass on the right side of the aorta below the diaphragm. This tumor was thereafter also resected. Three months after the second operation, a soft subcutaneous nodule measuring 4.5 x 2.0 cm in size was palpable above the left clavicle and was visible on a CT scan. A resection was again performed. All tumors showed the same histological findings. This is the first case reported in the Japanese literature with such a short-term demonstration of multiple metachronous recurrences in different paraganglions.

Poly(aspartic acid) degradation by a Sphingomonas sp. isolated from freshwater.

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.

Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases.

The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ Afa), Pseudomonas stutzeri (PhaZ Pst), and Comamonas acidovorans (PhaZ Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ Afa, PhaZ Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12+/-4 nm2/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains.

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst) of Pseudomonas stutzeri was cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

An enzyme-linked immunosorbent assay and reference ranges for bisphosphoglycerate mutase in human erythrocytes.

We established an enzyme-linked immunosorbent assay (ELISA) system for the determination of human bisphosphoglycerate mutase (BPGM) protein content in human erythrocytes using a polyclonal anti-BPGM antibody, we determined reference ranges for BPGM protein content, synthase activity, and specific activity in human erythrocytes. We produced a recombinant human BPGM (rBPGM) by gene manipulation using E. coli and then obtained the polyclonal antibody by immunizing rabbits with purified rBPGM. The reproducibility of the ELISA was in an acceptable range with a coefficient of variation under 1.5%. The ELISA was reliable in the range of 0.1 to 10 ng/mL. The polyclonal anti-rBPGM antibody did not show any cross-reaction with recombinant human B type phosphoglycerate mutase, which is highly homologous to rBPGM. The ELISA was found to be practical for the determination of BPGM protein content in human erythrocytes. The mean BPGM protein content was 56.3+/-9.7 microg/mL in whole blood (mean+/-SD, n = 50). The ELISA can be used to examine various hematologic disorders with abnormal red cell size and cell counts, and to detect BPGM enzymopathy in human erythrocytes.