A method for the staining of intraosseous nerve fibers using Sihler's staining technique.

Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler's staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler's staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques.

Bifid mandibular canal: confirmation of limited cone beam CT findings by gross anatomical and histological investigations.

OBJECTIVES: The aims of this study were (1) to assess the validity of limited cone beam CT (CBCT) in detecting the distribution of bifid mandibular canals in the retromolar region by comparing its findings with those of panoramic radiography and spiral CT imaging, and (2) to confirm the contents of such canals depicted on limited CBCT images by using gross anatomical and histological methods. METHODS: Bilateral bifid mandibular canals of a Japanese cadaver were investigated. The canals depicted on panoramic radiography, spiral CT and limited CBCT images were compared. Cross-sectional limited CBCT images of these canals were compared with gross anatomical sections of the mandible and their contents were confirmed histologically. RESULTS: The spiral CT and limited CBCT images showed the bilateral bifid mandibular canals in the retromolar region whereas the panoramic radiographs indicated the presence of only the left bifid mandibular canal. The canal distribution was more distinct in the limited CBCT images than in the spiral CT images and the cross-sectional limited CBCT images were consistent with the gross anatomical sections. Histologically, the canals contained several nerve bundles and arteries among which the largest nerve and artery were of a similar size. CONCLUSION: Limited CBCT is valuable for assessing the distribution of bifid mandibular canals. It is clinically significant to accurately localize a bifid mandibular canal of the retromolar region because it contains a nerve bundle and artery.

Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin beta4.

We previously found an inverse relationship between sialidase Neu1 expression and metastatic potential of murine cancer cells. To elucidate the mechanism underlying the cellular events, the human sialidase gene NEU1 was overexpressed or silenced in colon cancer HT-29 cells. When NEU1-overexpressing cells were injected transsplenically into mice, in vivo liver metastasis was significantly reduced. NEU1 suppressed cell migration, invasion and adhesion in vitro, whereas the silencing resulted in the opposite. One of the major molecular changes by NEU1 was decreased sialylation of integrin beta4, assessed by PNA- and MAL-II-lectin blotting of immunoprecipitates with anti-integrin beta4 antibody. The desialylation was accompanied by decreased phosphorylation of the integrin followed by attenuation of focal adhesion kinase and Erk1/2 pathway. Moreover, NEU1 caused downregulation of matrix metalloproteinase-7, overexpression of which is associated with cancer metastasis. Treatment of the cells with GalNAc-alpha-O-benzyl, an inhibitor of O-glycosylation, showed increased PNA-positive integrin beta4 with its decreased phosphorylation, indicating that sialic acid removal from the integrin O-glycans results in the decreased phosphorylation. Biotinylation and immunofluorescence staining exhibited some NEU1 molecules to be at the cell surface accessible to the integrin. These results suggest that NEU1 is important in regulation of integrin beta4-mediated signaling, leading to suppression of metastasis.

A crucial role of plasma membrane-associated sialidase in the survival of human cancer cells.

Human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is markedly upregulated in human cancers, leading to apoptosis suppression. To define molecular mechanisms and the possible target for NEU3, its encoding gene was silenced by small interference RNA (siRNA) or overexpressed in human cells. NEU3 siRNA-induced apoptosis with no special stimuli in HeLa cells, accompanied with decreased Bcl-xL and increased mda7 and GM3 synthase mRNA levels, whereas overexpression resulted in the opposite. Carcinoma HT-29 and MCF-7 cells appeared to be similarly affected, but normal cell lines demonstrated no significant changes. NEU3 siRNA was found to inhibit and NEU3 overexpression to stimulate Ras activation with consequent influence on extracellular signal-regulated kinases and Akt. Ras activation by NEU3 was abrogated by PP2 (src inhibitor) or AG1478 (epidermal growth factor receptor (EGFR) inhibitor), and NEU3 actually enhanced EGF-stimulated tyrosine-phosphorylation of EGFR, suggesting that the upstream targets might be tyrosine kinases including src and EGFR, and the subsequent stimulation of Ras cascade leads to the inhibition of cell apoptosis. Glycolipid changes observed seemed to be one of the causes of the cell effects. NEU3 may thus be an essential gene for cancer cell survival and siRNAs targeting this protein could have utility for gene-based therapy of human cancers.

Bioassay monitoring of waste PCB samples during chemical destruction treatments.

Capacitor oil samples (PCBs > 90%wt) were treated in a bench scale experiment to investigate the destruction of PCBs during chemical destruction processes (a catalytic hydrodechlorination treatment with palladium carbon and additional treatment with potassium tert-butyloxide). Using those results, this study confirmed the decrease of PCBs and other undesirable dioxin-like compounds such as PCDD/Fs in treated samples during the treatment. Dioxin-responsive chemical-activated luciferase expression (DR CALUX) AhR reporter gene bioassay was used to evaluate dioxin-like activity in the samples. During the treatment, the efficiency for PCB capacitor oil was around 99.99% or more in WHO-TEQ and CALUX-TEQ, whereas the sum of PCBs was reduced at a resulting efficiency of >99.9999%. In this study, a new cleanup procedure for separating PCBs from the mineral oil matrix was also developed for DR CALUX. The procedure consists of dimethylsulphoxide partitioning followed by silica gel-44% sulphuric acid reflux treatment and activated carbon chromatography. With the cleanup, CALUX-TEQ values were in good agreement with WHO-TEQ values and were as much as 3.3 times higher than WHO-TEQs for untreated/treated PCB-containing insulating oil samples. The DR CALUX results of mineral oil samples containing various PCB concentrations of 0.5-50 mg/kg (corresponding WHO-TEQs: 0.012-1.2 microg-TEQ/g) also correlated well with WHO-TEQs (CALUX-TEQ/WHO-TEQ ratio = 1.0-3.0), which was consistent with the theoretical quantification limit of the CALUX. These results supported the validity of the proposed clean-up method.

Alterations of muscarinic acetylcholine receptors in atypical Pick's disease without Pick bodies.

BACKGROUND: Atypical Pick's disease without Pick bodies is a type of frontotemporal dementia characterised by semantic dementia and temporal dominant lobar atrophy with ubiquitinopathy. No neurochemical analyses have ever been reported in this condition. OBJECTIVE: To investigate muscarinic acetylcholine receptors (mAchR) and their subtypes (M1-M4) in atypical Pick's disease. SUBJECTS: Five cases of atypical Pick's disease were studied. They were compared with nine control cases, 11 cases of Alzheimer's disease, and seven cases of dementia with Lewy bodies. METHODS: A [(3)H]quinuclidinyl benzilate (QNB) binding assay and an immunoprecipitation assay using subtype specific antisera were used. RESULTS: The total amount of mAchR in the temporal cortex was lower in atypical Pick's disease than in controls or Alzheimer's disease cases, but there were no significant differences between the three groups in the frontal cortex. In the temporal cortex, there was a smaller proportion of M1 receptors in atypical Pick's disease than in the controls or in the patients with Alzheimer's disease and dementia with Lewy bodies. In contrast, the proportion of M2 receptor was higher in atypical Pick's disease than in the other three groups. CONCLUSIONS: Depletion of postsynaptic cholinoreceptive neurones in the temporal cortex is more severe in atypical Pick's disease than in other neurodegenerative dementing disorders.

Fungal histidine kinases.

Eukaryotic cells predominantly use serine, threonine, and tyrosine phosphorylation in various intracellular signal transduction pathways. In contrast, prokaryotic organisms employ numerous "two-component" systems, in which signaling is achieved by transferring a phosphoryl group from phosphohistidine in the "sensor kinase" component to aspartate in the "response regulator" component. In the last several years, genetic screens and genome projects have identified sensor kinases and response regulators in lower eukaryotes and plants, revealing that eukaryotic organisms also make use of His-Asp phosphotransfer in a limited number of signaling pathways. Extensive studies in yeasts have demonstrated that a variation of the two-component system, a multistep "phosphorelay," is the prevailing mechanism among distantly related yeast species. In the budding yeast Saccharomyces cerevisiae, a His-Asp-His-Asp phosphorelay transmits osmotic stress signals to a mitogen-activated protein kinase (MAPK) cascade to induce adaptive responses. A phosphorelay in the fission yeast Schizosaccharomyces pombe, analogous to the S. cerevisiae phosphorelay, is responsible for MAPK activation in response to peroxide stress. Mammalian cells do not have any two-component or phosphorelay systems, although protein histidine kinases unrelated to the sensor kinase may be involved in cellular signaling. Because some phosphorelay proteins are essential for virulence of microbial pathogens, including the yeast fungus Candida albicans, novel antibiotics targeted to phosphorelays may be effective against eukaryotic pathogens without causing host cell damage.

Distribution of m1 muscarinic acetylcholine receptors in the hippocampus of patients with Alzheimer's disease and dementia with Lewy bodies-an immunohistochemical study.

Of the five subtypes (m1-m5) of muscarinic acetylcholine receptors (mAChR), the m1 subtype is the most abundant in the human cerebral cortex and hippocampus. Impairment of the muscarinic cholinergic system in the brain may cause cognitive dysfunction in patients with Alzheimer's disease (AD), and choline esterase inhibitors (ChE-I) are used to improve cognitive dysfunction. Severe impairment of the cholinergic system has also been reported in the brains of subjects with dementia with Lewy bodies (DLB). There have been a few reports about the distribution of mAChR subtypes in the human brain. In the present study, we investigated the distribution of m1 mAChR in the human hippocampus using an antibody against the m1 subtype. In the control brains, m1 immunoreactivity was observed in the apical dendrites and cell bodies of granular neurons of the dentate gyrus and pyramidal neurons of CA1-3 and the subiculum. The dendrites and the cell bodies of the pyramidal neurons in layers III and V of the parahippocampal cortex and other temporal cortices were also positive for m1 immunoreactivity. This m1 immunoreactivity was markedly reduced in AD and DLB brains.

Human stomach-specific gene, CA11, is down-regulated in gastric cancer.

To reveal the implication in gastric cancer pathogenesis of the novel human gene referred to as CA11, which was recently isolated by a differential display technique using normal gastric mucosa and gastric cancer tissue, we examined CA11 expression in 50 primary gastric cancers and also introduced the CA11 gene into gastric cancer cells. RNA dot blot analysis against various human organs and developmental stages demonstrated that CA11 was intensively expressed especially in normal stomach tissue. Northern blot analysis showed that expression of the CA11 gene in cancer tissue was down-regulated compared with normal tissue. Semi-quantitative RT-PCR also demonstrated that CA11 gene expression was decreased in 41 out of 50 (82%) of the gastric cancer tissues, when compared with normal stomach tissues, while no relationship was found between CA11 expression and various clinicopathological characteristics including histological type, depth of invasion, lymph node metastasis, and clinical stage. Immunohistochemical analysis with anti CA11 antibody showed that CA11-positive staining was observed in the surface regions of normal gastric epithelium, but was found faintly or not at all in cancer tissues. CA11 transfected MKN28 cells also displayed a marked decrease in the number of colony formations when compared to double normal controls. These findings suggest that the loss of CA11 expression in gastric tissues may play an important role in gastric carcinogenesis.

Splenic and pulmonary metastases from renal cell carcinoma: report of a case.

We report herein the case of a patient in whom pulmonary and splenic metastases from renal cell carcinoma (RCC) were successfully treated by surgical excision. A 69-year-old man who underwent left nephrectomy for RCC 17 months before was suspected to have a pulmonary metastasis based on computed tomography (CT) findings. Partial resection of the left lower lobe was performed with thoracoscopic assistance. However, 4 months later, a splenic tumor, 6 cm in diameter, was detected by CT and ultrasonography, and a splenectomy was performed. Histologically, both resected specimens were diagnosed as metastasis from RCC. A second pulmonary metastasis of the left upper lobe was resected 4 years 8 months later. The patient was in good health when last seen 11 months after his last operation. Malignant neoplasms rarely metastasize to the spleen and most cases are found at autopsy, or feature multiple distant metastases. Only four other cases of splenic metastases from RCC have been reported. The prognosis associated with splenic metastasis is favorable when only a solitary lesion exists.

Estrogenic and dioxin-like potency in each step of a controlled landfill leachate treatment plant in Japan.

The estrogenic activity (by E-screen bioassay), the concentrations of PCBs, PCDDs/PCDFs (and their resulting toxicity equivalents, TEQ) and several endocrine disrupting chemicals (EDCs: e.g., bisphenol A, nonylphenol, Butyl benzylpthalate (BBP), di-n-butylphthalate (DBP), 17alpha-ethynyl-estradiol or 4-octylphenol) have been analyzed from leachates of each step (before treatment, after biodegradation/sedimentation and after charcoal treatment) of a controlled landfill leachate treatment plant. The comparison of the effluent of the examined landfill leachate treatment plant with water from a nearby river in this study indicated no additional dioxin-like (e.g., TEQ: 0.027 compared to 1.01 pg TEQ/l; PCBs: 1.2 compared to 3.9 ng/l) or estrogenic impact (2.8 compared to 3.5 ng estradiol equivalents EE/l; analyzed by E-screen bioassay) from the leachate treatment plant into the surrounding water environment. The impact of dioxin-like compounds from uncleaned leachates into the final cleaned effluents could be sufficiently reduced by the leachate treatment plant for PCDDs (75%), PCDFs (62%), dioxin-like PCBs (97%), and the sum of TEQ (78%). The leachate treatment plant also achieved a reduction of the estrogenic activity as determined by E-screen (from 4.8 to 2.8 ng EE/l = 42%), by GC/MS for bisphenol A (>96% and nonylphenol (>98%) or by ELISA for estradiol (>80%). Additionally, for the validation of the E-screen, five known endocrine disrupting chemicals (EDCs: bisphenol A, BBP, DBP, 17 alpha-ethynyl-estradiol, 4-octylphenol) were analyzed. The EC50 values and estradiol equivalents factors (EEFs) for the five EDCs determined in this study were comparable to previously published data. The combined biological and chemical trace analysis data have provided valuable information on the relative contribution of natural, synthetic, and non-steroidal anthropogenic chemicals to the estrogenic and dioxin-like activity in leachates from a wastewater treatment plant, and water from a nearby river.

Prednisolone suppresses ischemia-reperfusion injury of the rat liver by reducing cytokine production and calpain mu activation.

BACKGROUND: We investigated the effects of prednisolone on cytokine production and calpain mu activation during hepatic ischemia-reperfusion (IR) injury. METHODS: The hilar area of the left lateral and median lobes of rat liver was clamped for 60 min. Prednisolone was administered at 1.0, 3.0, or 10 mg/kg at 30 min before ischemia. In addition to biochemical and microscopic analyses, IL-beta and TNF-alpha production was evaluated by RT-PCR. Calpain mu activation and talin degradation were determined by Western blotting, using specific antibodies. RESULTS: In the control and prednisolone (1.0 mg/kg) groups, serum AST and ALT levels were elevated, and cell membrane bleb formation was observed after 2 h of reperfusion. Moreover, calpain mu activation, talin degradation, and overexpression of IL-beta and TNF-alpha mRNAs were detected. Infusion of prednisolone at 3.0 or 10 mg/kg significantly suppressed biochemical and microscopic changes. At 10 mg/kg, prednisolone markedly suppressed IL-beta and TNF-alpha transcription and calpain mu activation and talin degradation, consistent with the improved 7-day survival after total hepatic ischemia (75% vs. 25% in control group, P = 0.039). CONCLUSIONS: Cytoprotective effect of prednisolone in hepatic IR injury was closely associated with suppression of IL-beta/TNF-alpha production and calpain mu activation.

Combustion of brominated flame retardants and behavior of its byproducts.

The substance flow rate of PBDDs/DFs into flue gas and incineration residues from incineration of three types of waste samples containing brominated flame retardants were examined. The samples used consisted of PBDEs (a typical retardant), used TV casing materials (actual waste materials), and waste printed circuit boards. PBDDs/DFs concentrations in the experimental samples of PBDEs/PE, waste TV casing materials and printed circuit boards ranged between 3000 and 130,000 ng/g. These values are very high when compared to other investigations. The increase of chlorine concentration in input sample reduced the ratio of PBDDs/DFs in flue gas and raised the ratio of PCDDs/DFs. With adequate combustion control and flue gas treatment, the amount of PBDDs/DFs released from the incineration of resin containing brominated flame retardants was lower than the input amount. The presence of PBDDs/DFs in incineration residues dominated the total amount of dioxins released. When PBDDs/DFs, PCDDs/DFs and PXDDs/DFs were considered as a total, the total amount released was lower than the total amount input.

[Neurally mediated syncope in association with small cell lung carcinoma].

A 66-year-old woman was admitted to our hospital because of frequent syncopal episodes and for treatment of small cell lung carcinoma. Neurally mediated syncope was diagnosed by the head-up tilt test, which evoked early severe hypotension (after 12 min at the 80-degree tilt position). Treatment of carcinoma by chemotherapy and radiotherapy promptly eliminated the syncopal episodes. This was an unusual case of neurally mediated syncope associated with small cell lung carcinoma.

Effect of Ca(2+)-independent mechanisms on the hypoxic relaxation of guinea-pig tracheal rings.

Hypoxia induces bronchodilation in vivo and in vitro, but the mechanisms are still unclear. To evaluate whether an extra- or intracellular free Ca(2+) ion is involved in the mechanisms of hypoxic relaxation, we simultaneously measured cytosolic Ca(2+)levels and tensions in both intact and denuded guinea-pig tracheal strips precontracted with histamine (100 microM), and assessed the effect of hypoxia on guinea-pig tracheal rings precontracted with okadaic acid (10 microM) and calyculin-A (0.1 approximately 10 microM) under an extracellular Ca(2+)-free state. The exposure of tracheal rings to hypoxia induced an immediate decrease of tracheal tension without decrease in intracellular free Ca(2+)levels. In the presence of okadaic acid but not calyculin-A, hypoxic air exposure caused significant transient reductions in tracheal tone. Further, thapsigargin (5 microM or 10 microM) did not affect hypoxic bronchodilation, suggesting that the release of intracellular Ca(2+) does not take a role in hypoxic bronchodilation. Hypoxic dilation decreased ATP content in epithelium-intact rings but not epithelium-denuded rings, indicating a relationship between hypoxic dilation and change of adenine nucleotide in epithelium-intact rings. Our findings indicate that the epithelium dependent mechanisms of hypoxic relaxation of guinea pig tracheal rings preconstricted with histamine may not be related to the mobilization of extra and intra-cellular Ca(2+).

Outcome of pancreatic cancer patients based on genetic lymph node staging.

We prospectively assessed the clinical value of genetic staging of lymph node metastasis in patients with pancreatic adenocarcinoma who underwent curative surgery. K-ras gene mutations were detected in the primary tumors in 18 of 25 patients with pancreatic adenocarcinoma. Among these 18 patients, mutated K-ras gene was also found in at least one lymph node in 13 patients. Of these 13 patients, seven had no evidence of histological nodal involvement and six had histological lymph node metastasis. Although there was no significant difference in overall survival rates between the pathological node-negative and -positive patients, overall survival of the five patients with nodes-negative for the mutated K-ras gene were significantly better than that of the 13 patients with genetically metastasis-positive nodes (p<0.001). Furthermore, overall survival of the six patients with genetically metastasis-positive nodes limited to peripancreatic area was significantly better than that of seven patients with genetical metastasis in lymph nodes beyond the peripancreatic areas (p=0.018). These findings suggest that detection of K-ras gene mutations in lymph nodes may be clinically useful to assess the accurate tumor staging and to stratify the patient with pancreatic adenocarcinoma who are at high or low risk for recurrence after curative surgery.

Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase.

In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe. The fission yeast mpr1(+) gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative stress is severely impaired in the Deltampr1 mutant as well as in the mpr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine. In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1. Unexpectedly, when exposed to hydrogen peroxide, Deltampr1 cells can induce the catalase gene ctt1(+), one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation. We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1(+) expression upon oxidative stress independently of the Spc1 stress-activated protein kinase. These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression.

Assessment of Stanniocalcin-1 mRNA as a molecular marker for micrometastases of various human cancers.

We assessed Stanniocalcin-1 (STC-1) mRNA expression in human normal tissues, various types of human cancer cell lines, and cancer tissues obtained during surgery. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect STC-1 mRNA, the transcripts were detected in 20 out of 21 cancer cell lines and in all tumor tissues from various types of cancer. Semi-quantitative analyses with multiplex RT-PCR showed that STC-1 mRNA tended to be enhanced in cancer tissues of hepatocellular carcinoma and colorectal cancer compared to background cancer-free tissues. Analysis of blood samples obtained from 11 patients with hepatocellular carcinoma before, after and during hepatectomy showed STC-1 mRNA expression in 8 out of 11 patients at least one time. Normal donor blood samples (n=31) were all-negative for STC-1 mRNA expression. Our results indicate that STC-1 mRNA might be a useful molecular marker for detection of tumor cells in blood from patients with various types of malignancies.

Heat-shock-induced activation of stress MAP kinase is regulated by threonine- and tyrosine-specific phosphatases.

In eukaryotic species from yeast to human, stress-activated protein kinases (SAPKs), members of a MAP kinase (MAPK) subfamily, regulate the transcriptional response to various environmental stress. It is poorly understood how diverse forms of stress are sensed and transmitted to SAPKs. Here, we report the heat shock regulation of the fission yeast Spc1 SAPK, a homolog of human p38 and budding yeast Hog1p. Although osmostress and oxidative stress induce strong activation of the Wis1 MAPK kinase (MEK), which activates Spc1 through Thr-171/Tyr-173 phosphorylation, activation of Wis1 upon heat shock is relatively weak and transient. However, in heat-shocked cells, Pyp1, the major tyrosine phosphatase that dephosphorylates and inactivates Spc1, is inhibited for its interaction with Spc1, which leads to strong activation of Spc1. Subsequently, Spc1 activity is rapidly attenuated by Thr-171 dephosphorylation, whereas Tyr-173 remains phosphorylated. Thr-171 dephosphorylation is compromised in a strain lacking functional type 2C serine/threonine phosphatases (PP2C), Ptc1 and Ptc3. Moreover, Ptc1 and Ptc3 can dephosphorylate Thr-171 of Spc1 both in vivo and in vitro. These observations strongly suggest that PP2C enzymes play an important role in the attenuation of Spc1 activity in heat-shocked cells. Thus, transient activation of Spc1 upon heat shock is ensured by differential regulation of threonine and tyrosine phosphorylation.