Pathogenesis of selective damage of granule cell layer in cerebellum of rats exposed to methylmercury.

Granule cell-selective toxicity of methylmercury in the cerebellum is one of the main unresolved issues in the pathogenesis of Minamata disease. Rats were orally administered methylmercury chloride (10 mg/kg/day) for 5 consecutive days, and their brains were harvested on days 1, 7, 14, 21, or 28 after the last administration for histological examination of the cerebellum. It was found that methylmercury caused a marked degenerative change to the granule cell layers but not to the Purkinje cell layers. The generative change of the granule cell layer was due to cell death, including apoptosis, which occurred at day 21 and beyond after the methylmercury administration. Meanwhile, cytotoxic T-lymphocytes and macrophages had infiltrated the granule cell layer. Additionally, granule cells are shown to be a cell type susceptible to TNF-α. Taken together, these results suggest that methylmercury causes small-scale damage to granule cells, triggering the infiltration of cytotoxic T-lymphocytes and macrophages into the granule cell layer, which secrete tumor necrosis factor-α (TNF-α) to induce apoptosis in granule cells. This chain is established based on the susceptibility of granule cells to methylmercury, the ability of cytotoxic T lymphocytes and macrophages to synthesize and secrete TNF-α, and the sensitivity of granule cells to TNF-α and methylmercury. We propose to call the pathology of methylmercury-induced cerebellar damage the "inflammation hypothesis."

Methylmercury induces hyaluronan synthesis in cultured human brain microvascular endothelial cells and pericytes via different mechanisms.

In a cerebrum damaged by methylmercury, where neuropathological lesions tend to localize along deep sulci and fissures, edematous changes in white matter have been proposed as the cause of such localization. Since hyaluronan has a high water-retention capability and can contribute to the progression of edematous changes, we hypothesize that methylmercury increases hyaluronan in brain microvascular cells. Our experimental results indicate that methylmercury induces the expression of hyaluronan in cultured human microvascular endothelial cells and pericytes through the induction of expressed UDP-glucose dehydrogenase and hyaluronan synthase 2, respectively. After exposure to methylmercury, hyaluronan largely accumulates in perivascular space, where it contributes to the progression of edematous changes.

[A Case of Advanced Rectal Cancer Resected Successfully after Induction Chemotherapy with Modified FOLFOX6 plus Panitumumab].

We report a case of advanced colon cancer that was effectively treated with mFOLFOX6 plus panitumumab combination chemotherapy. The patient was a 54-year-old man who had type 2 colon cancer of the rectum. An abdominal CT scan demonstrated rectal cancer with bulky lymph node metastasis and 1 hepatic node (rectal cancer SI [bladder retroperitoneum], N2M0H1P0, cStage IV). He was treated with mFOLFOX6 plus panitumumab as neoadjuvant chemotherapy. After 4 courses of chemotherapy, CT revealed that the primary lesion and regional metastatic lymph nodes had reduced in size (rectal cancer A, N1H1P0M0, cStage IV). Anterior rectal resection with D3 nodal dissection and left lateral segmentectomy of the liver was performed. The histological diagnosis was tubular adenocarcinoma (tub2-1), int, INF a, pMP, ly0, v0, pDM0, pPM0, R0. He was treated with 4 courses of mFOLFOX6 after surgery. The patient has been in good health without a recurrence for 2 years and 5 months after surgery. This case suggests that induction chemotherapy with mFOLFOX6 plus panitumumab is a potentially effective regimen for advanced colon cancer.

Expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes after exposure to methylmercury.

The localization of neuropathological lesions along deep sulci and fissures is one of the characteristics of a cerebrum damaged by methylmercury. Edematous changes in white matter have been proposed as the cause of the localization of lesions; however, the molecular mechanisms underlying methylmercury-induced edema remain unclear. Since the vascular endothelial growth factor (VEGF) system regulates vascular permeability and can be involved in the progression of edematous changes, we examined the effect of methylmercury on the expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes. After methylmercury exposure, mRNA and protein levels of VEGF-A in pericytes and placenta growth factor (PlGF) and VEGF-receptor-1/-2 in endothelial cells were elevated. The induction of pericyte VEGF-A expression was independent of hypoxia-inducible factor-α and hypoxia-response element signaling. Taken together, these results suggest that methylmercury activates the VEGF system in brain microvessels in a paracrine fashion. When the activation occurs in narrow areas such as along the deep sulci in the cerebrum, hyperpermeability and subsequent edematous changes would cause a circulatory disturbance and result in neural cell damage. We propose this as a reason for the localization of the neuropathological lesions along the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, in patients with Minamata disease.

1,635 Endoscopic submucosal dissection cases in the esophagus, stomach, and colorectum: complication rates and long-term outcomes.

BACKGROUND: Endoscopic submucosal dissection (ESD) enables en bloc resection of early gastrointestinal neoplasms; however, most ESD articles report small series, with short-term outcomes performed by multiple operators on single organ. We assessed short- and long-term treatment outcomes following ESD for early neoplasms throughout the gastrointestinal tract. METHODS: We performed a longitudinal cohort study in single tertiary care referral center. A total of 1,635 early gastrointestinal neoplasms (stomach 1,136; esophagus 138; colorectum 361) were treated by ESD by single operator. Outcomes were complication rates, en bloc R0 resection rates, and long-term overall and disease-specific survival rates at 3 and 5 years for both guideline and expanded criteria for ESD. RESULTS: En bloc R0 resection rates were: stomach: 97.1 %; esophagus: 95.7 %; colorectum: 98.3 %. Postoperative bleeding and perforation rates respectively were: stomach: 3.6 and 1.8 %; esophagus: 0 and 0 %; colorectum: 1.7 and 1.9 %. Intra criteria resection rates were: stomach: 84.9 %; esophagus: 81.2 %; colorectum: 88.6 %. Three-year survival rates for lesions meeting Japanese ESD guideline/expanded criteria were for all organ-combined: 93.4/92.7 %. Five-year rates were: stomach: 88.1/84.6 %; esophagus: 81.6/57.3 %; colorectum: 94.3/100 %. Median follow-up period was 53.4 (range, 0.07-98.6) months. Follow-up rate was 94 % (1,020/1,085). There was no recurrence or disease-related death. CONCLUSIONS: In this large series by single operator, ESD was associated with high curative resection rates and low complication rates across the gastrointestinal tract. Disease-specific and overall long-term prognosis for patients with lesions within intra criteria after curative resection appeared to be excellent.

[A case of emergency resection of a giant gastrointestinal stromal tumor of the stomach associated with hemorrhagic shock].

We report a case of emergency resection of a giant gastrointestinal stromal tumor of the stomach associated with hemorrhagic shock. A 79-year-old woman was admitted to our hospital because of massive hematemesis. Laboratory analysis revealed a hemoglobin level of 6.5 g/dL. Abdominal computed tomography (CT) and upper gastrointestinal endoscopy revealed a submucosal tumor, 12 cm in diameter, in the fornix of the stomach. As a Dieulafoy-like lesion was present, we attempted coagulation hemostasis in the exposed blood vessels. Endoscopic hemostasis was not successful. The patient went into hemorrhagic shock. Emergency surgery was performed: total gastrectomy with distal pancreatosplenectomy. The resected specimen measured 10×12×7 cm and was hard. Immunohistologically, the tumor was positive for c-kit and CD34 and negative for alpha smooth muscle actin (αSMA), desmin, and S-100. Histological examination revealed that the patient had a high-risk gastrointestinal stromal tumor of the stomach with no nodal metastasis. The postoperative course was uneventful and the patient has remained alive without recurrence for 4 years.

The cytotoxicity of methylmercury in human microvascular endothelial cells and pericytes in culture.

Methylmercury is an environmental neurotoxin that induces severe neurological damage in the brain of humans and animals. The main pathological characteristic of methylmercury neurotoxicity is the location of the damage; lesions are localized around the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, and the granule cell layer of the cerebellum. Since the localization of the damage is suggested to be a result of secondary damage occurring due to edematous change in the white cortex, the toxicity of methylmercury to cells that compose the microvessels--endothelial cells and pericytes--may be important for understanding the neurotoxicity of methylmercury. We investigated the toxicity of methylmercury to human brain microvascular endothelial cells and pericytes using a cell culture system. It was revealed that the toxicity of methylmercury to microvascular cells depends on the cell type and density. It is suggested that vascular tissue is one of the targets of methylmercury toxicity and that this may contribute to the progression of edematous change in the brain. Methylmercury may also be involved in the progression of cardiovascular diseases.

Resistance of human brain microvascular endothelial cells in culture to methylmercury: cell-density-dependent defense mechanisms.

Vascular toxicity is important for understanding the neurotoxicity of methylmercury, because microvessels strongly influence the construction of microenvironment around neurons. Previously, we found that low density-human brain microvascular pericytes are markedly susceptible to methylmercury cytotoxicity due to high expression levels of the L-type amino acid transporter 1 (LAT-1) that transports methylmercury into the cells. Although LAT-1 can be, in general, highly expressed in sparse cells that require amino acids for growth, we found that human brain microvascular endothelial cells, regardless of cell density, were resistant to methylmercury cytotoxicity. To investigate the mechanisms underlying this resistance, we exposed the endothelial cells at low and high cell densities to methylmercury and determined the extent of nonspecific cell damage, intracellular accumulation of methylmercury, expression of LAT-1 and LAT-2 mRNAs, and intracellular expression of reduced glutathione and metallothionein. These experiments indicate that sparse endothelial cells intracellularly accumulate more methylmercury via the highly expressed LAT-1, but are resistant to methylmercury cytotoxicity by higher expression of the protective sulfhydryl peptides, namely, reduced glutathione and metallothionein. It is suggested that both nonspecific and functional damage is caused in pericytes, whereas functional abnormalities rather than nonspecific damage may occur to a greater extent in the endothelial cells in the brain microvessels exposed to methylmercury. The previous and present data also suggest that methylmercury exhibits toxicity in endothelial cells in a manner different from that in pericytes in the brain microvessels.

Cell-density-dependent methylmercury susceptibility of cultured human brain microvascular pericytes.

The knowledge of vascular toxicity is important for understanding the neurotoxicity of methylmercury. In the present study, we investigated the cell-density-dependent susceptibility of human brain microvascular pericytes to methylmercury-induced toxicity by using a cell-culture system. The susceptibility of sparse pericyte cultures to methylmercury was greater than that of the dense cultures. In addition, the sparse cultures were more susceptible to methylmercury than to inorganic mercury and cadmium. The intracellular accumulation of methylmercury in the sparse cells was significantly higher than that in the dense cells. Methylmercury is transported through the L-type large neutral amino acid transporter (LAT 1) in the form of a complex with cysteine. The mRNA- and protein-level expressions of LAT 1 in the sparse cells were markedly higher than those in the dense cells; in addition, the LAT 1 expression was increased by methylmercury. However, there was no reduction in the levels of glutathione and metallothionein, which are involved in the defense mechanisms against methylmercury, in the sparse cells. The present data revealed that pericytes are markedly susceptible to methylmercury-induced cytotoxicity at low cell densities. The susceptibility of the sparse pericytes is postulated to be due to the not only constitutively higher but also methylmercury-induced expression of LAT 1, which increased the intracellular accumulation of methylmercury.

Suppression of fibroblast growth factor-2 expression: possible mechanism underlying methylmercury-induced inhibition of the repair of wounded monolayers of cultured human brain microvascular endothelial cells.

Vascular toxicity is an important feature of the neuropathy induced by methylmercury. Methylmercury does not cause nonspecific cell damage, but rather retards the repair of wounded monolayers of cultured human brain microvascular endothelial cells by inhibiting their proliferation. Since vascular endothelial cell proliferation during the repair process strongly depends on the fibroblast growth factor-2 (FGF-2) system, we investigated the effects of methylmercury on the expression of FGF-2 and related proteins (i.e., FGF receptor 1 and perlecan) in cultured human brain microvascular endothelial cells. Of the mRNAs examined, FGF-2 mRNA expression was significantly lowered by methylmercury in not only wounded monolayers but also dense and sparse cultures of endothelial cells; a lower expression of FGF-2 protein in the cells was confirmed. In addition, exogenous FGF-2 partially abrogated the proliferation-inhibitory effect of methylmercury. The results of this study suggest that suppression of FGF-2 expression is one of the mechanisms underlying the inhibitory effect of methylmercury in damaged endothelial cell monolayers. The FGF-2 system may be one of the important biological systems behind the vascular toxicity of methylmercury.

Arsenite but not arsenate inhibits general proteoglycan synthesis in cultured arterial smooth muscle cells.

Epidemiological and experimental studies have suggested that exposure to metalloid arsenic constitutes a risk factor for vascular disease associated with atherosclerosis. Since in atherosclerosis, types of proteoglycans (PGs) present change depending on the stage, we investigated the effect of 2 chemical forms of inorganic arsenic-a trivalent sodium arsenite and a pentavalent sodium arsenate-on the synthesis of PGs in cultured arterial smooth muscle cells. The results indicate that arsenite but not arsenate, at a noncytotoxic level, inhibits general PG synthesis independent of cell density. Arsenite may be one of the chemical forms of inorganic arsenic that influences the PG composition in blood vessel walls during the progression of vascular disorders such as atherosclerosis.

A novel method using cyanobacteria for ecotoxicity test of veterinary antimicrobial agents.

The effect of antimicrobial agents for veterinary use on the growth of cyanobacteria was investigated by measuring minimum inhibitory concentration, medium effective concentration (EC50), and no-observed-effect concentration of seven antimicrobial agents for eight cyanobacteria. The results demonstrated that the seven antimicrobial agents, even at low concentrations, inhibited the growth of cyanobacteria. Microcystis aeruginosa and Synechococcus sp. had the highest sensitivity to the antimicrobial agents used in the present study. It is considered that the utilization of cyanobacteria would enable easy and highly sensitive assessment of the toxicity of such chemicals as antimicrobial agents. We suggest that cyanobacteria be used for ecotoxicity test in addition to the hitherto established method that uses green algae.

Flubendiamide, a novel Ca2+ channel modulator, reveals evidence for functional cooperation between Ca2+ pumps and Ca2+ release.

Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca2+ pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca2+ pump activity (EC50=11 nM) of an insect that resequesters Ca2+ to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca2+ release with Ca2+ pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca2+ release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca2+ is an important mediator for the functional coordination of RyRs and Ca2+ pumps. However, kinetic factors for Ca2+ pumps, including ATP and cytoplasmic Ca2+, failed to affect the Ca2+ pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca2+ pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca2+ binding site on the Ca2+ pump. This mechanism should play an essential role in precise control of intracellular Ca2+ homeostasis.

Biodegradation of bisphenol A and disappearance of its estrogenic activity by the green alga Chlorella fusca var. vacuolata.

Bisphenol A (BPA) is known as an endocrine disruptor and often is found in landfill leachates. Removal of BPA by green alga, Chlorella fusca, was characterized, because we previously found that various phenols were well removed by this strain, including BPA. Chlorella fusca was able to remove almost all BPA in the concentration range from 10 to 80 microM for 168 h under continuous illumination at 18 W/m2. At the low light intensity of 2 W/m2, 82% of 40 microM BPA was removed, and only 27% was removed in the dark. Moreover, C. fusca could remove 90% of 40 microM BPA under the 8:16-h light:dark condition, which was almost as high as that under the continuous-light condition. The amount of BPA contained in the cells was less than the amount of BPA removed from the medium. Monohydroxybisphenol A was detected as an intermediate of BPA degradation. Moreover, estrogenic activity that originated from BPA in the culture medium also completely disappeared. Based on these results, BPA was finally degraded to compounds having nonestrogenic activity. Therefore, C. fusca can be considered a useful organism to remove BPA from landfill leachates.

Bone mineral density in adult patients with Turner's syndrome: analyses of the effectiveness of GH and ovarian steroid hormone replacement therapies.

To analyze the effects of treatments with GH and cyclic estrogen/progesterone (E/P) replacement on bone mineralization in patients with Turner's syndrome (TS), bone mineral density (BMD) was measured longitudinally. BMDs of the whole body and the lumbar spine in 16 adult female patients with TS (17-38 year old; 0-20 years by length of E/P treatment) were assessed using dual energy X-ray absorptiometry one to 5 times over a treatment period of up to 7 years maximum. GH treatment was performed in 9 cases (GH group), but not in the remaining 7 (non-GH group). E/P replacement therapy was initiated in all patients after they finished GH administration. The BMDs of both the whole body and the lumbar spine in the patients with TS were significantly less than those in age-matched normal subjects, and did not improve with E/P treatment. Although there were no differences in final body height and age at the beginning of E/P administration between the GH and non-GH groups, whole body BMD in the GH group was significantly lower than that in the non-GH group. These results indicate that GH administration in childhood and adolescence and E/P treatment in adulthood did not increase bone mineralization in the TS patients. Therefore, we can conclude that the optimal protocol of hormonal replacement therapy with GH and E/P during childhood and adolescence should be established as soon as possible.

Photosynthesis-dependent removal of 2,4-dichlorophenol by Chlorella fusca var. vacuolata.

Of 7 green algae, Chlorella fusca var. vacuolata removed about 23% of 2,4-dichlorophenol (DCP) at 10-80 microM after 4 d when grown photoautotrophically. Removal of DCP was growth-dependent and was suppressed dose-dependently by the photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea.

Removal of hazardous phenols by microalgae under photoautotrophic conditions.

Various algae were screened for their ability to decrease the concentration of 2,4-dinitrophenol (DNP), as a model compound of hazardous phenols, under photoautotrophic conditions. Chlorella fusca var. vacuolata and Anabaena variabilis grew well and showed high DNP removal ability over the concentration range of 5 to 40 microM. Their abilities to remove various phenols were investigated. More than 90% of 40 microM o- and m-nitrophenol and DNP was removed during the cultivation period of 5 d. o-, p-Chlorophenol and 2,4-dichlorophenol could be removed, but not to a significant extent. C. fusca also removed 85% of bisphenol A, suspected to be an endocrine disrupter. It was found that microalgae would be applicable to the removal of hazardous phenols without the addition of any organic carbon sources.