Phenolic compounds prevent amyloid ß-protein oligomerization and synaptic dysfunction by site-specific binding.

Cerebral deposition of amyloid ß protein (Aß) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aß aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aß aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aß oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aß. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aß oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aß through different binding.

Anti-amyloidogenic effects of soybean isoflavones in vitro: Fluorescence spectroscopy demonstrating direct binding to Aß monomers, oligomers and fibrils.

Alzheimer's disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid ß-protein (Aß). A growing body of evidence indicates that oligomeric forms of Aß play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aß-induced neurotoxicity in cultured cell systems, the molecular mechanisms remain unclear. Our previous studies demonstrated that red wine-related flavonoids with a flavone backbone are able to inhibit Aß assembly and destabilize preformed Aß aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aß(1-40) and Aß(1-42)in vitro at physiological pH and temperature, by using nucleation-dependent polymerization monitored by thioflavin T fluorescence, atomic force microscopy, electron microscopy, and photo-induced cross-linking of unmodified proteins followed by SDS-PAGE. Our three-dimensional fluorescence spectroscopic analyses demonstrated that glycitein interacted with Aß monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aß species. Glycitein also interacted with different Aß fragments (Aß(1-42), Aß(1-40), Aß(1-16) and Aß(25-35)), exhibiting the highest fluorescence enhancement with Aß(25-35). We speculated that glycitein's anti-amyloidogenic properties are specifically mediated by its binding to Aß monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimer's disease.

Effects of antiparkinsonian agents on ß-amyloid and α-synuclein oligomer formation in vitro.

The aggregation of ß-amyloid protein (Aß) and α-synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimer's disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aß40, Aß42, and αS using a method of photo-induced cross-linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aß and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aß and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression.

Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule.

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered Gq-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of alpha subunit of Gq protein (Galphaq) by inhibiting the GDP release from Galphaq. X-ray crystal structure analysis of the Galphaqbetagamma-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Galphaq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Galpha subunit and an insight into the molecular mechanism of G protein activation.

[Preoperative chemotherapy for advanced gastric cancer].

In the present study, we evaluated the outcome of preoperative treatment with S-1 and CDDP for the treatment of advanced gastric cancer. Fifty-five cases of advanced gastric cancer received pre-operative treatment with S-1 and CDDP. The tumor control rate( PR and CR according to RECIST criteria) was 55%. The clinical response and histological response to the treatment and curative resection were closely related to favorable postoperative survival. We noted that patients who demonstrated CR or PR received S-1 as postoperative treatment, whereas those with SD or PD were more likely to receive paclitaxel as postoperative treatment. Preoperative treatment with S-1 and CDDP was not only an effective initial treatment, but also demonstrated favorable results in a S-1 in vivo sensitivity test.

[Insertion of percutaneous transhepatic biliary endoprosthesis for unresectable lower cholangiocellular carcinoma following cholangitis due to endoscopic biliary plastic stent obstruction].

The patient was an 87-year-old woman who was diagnosed with atrial fibrillation, which was treated with an anticoagulant, and with chronic kidney disease. The patient was diagnosed as having liver dysfunction and lower cholangiocellular carcinoma (cStage I) on ultrasonography and magnetic resonance cholangiopancreatography. Since it was impossible to perform curative resection owing to the patient's decreased cardiac and renal function, we performed palliative endoscopic retrograde biliary drainage (ERBD) with a plastic stent (PS), and the patient was discharged 11 days later. However, the patient was readmitted because of fever (>38.0°C) and vomiting 124 days after ERBD. We assumed that the patient had developed cholangitis due to PS obstruction. Moreover, her blood culture was positive for Klebsiella pneumoniae. We were unable to replace the PS as the tumor had increased in size and hemorrhage from the papilla of Vater continued after the stent had been removed. The signs of inflammation improved after treatment of sepsis with antibiotics and immunoglobulins, and we performed percutaneous transhepatic cholangio drainage( PTCD) and eventually inserted a percutaneous transhepatic biliary endoprosthesis (PTBE) with an expandable metallic stent (EMS). The patient died 2 months later; no stent occlusion was observed. Our experience suggests that endoscopic biliary stents should be selected bearing in mind the patency of the stent and the prognosis.

[A case of successful radical resection of rectal cancer with neo-adjuvant chemoradiotherapy].

In cases of advanced rectal cancer, preoperative chemoradiotherapy( CRT) serves to improve the local control rate, survival rate, radical resection rate, and/or probability of sphincter muscle preservation. According to the Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2010 for the treatment of colorectal cancer, preoperative CRT is the standard treatment for rectal cancer in Europe and the United States. However, there is insufficient evidence in support of its efficacy and safety in Japan, and therefore, CRT needs to be evaluated in properly designed clinical trials. Recently, several studies have reported on the efficacy of preoperative CRT in Japan. Herein, we report a case of rectal cancer in which radical resection was successfully performed with neo-adjuvant CRT.

[Examination of the effect of anesthesia on radiofrequency ablation of hepatocellular carcinoma-a patient survey on anesthesia for radiofrequency ablation].

PURPOSE: Radiofrequency ablation(RFA) is minimally invasive and is easy to perform. In the RFA procedure, puncture and passing of the electrical current are painful. Therefore, some facilities use general anesthesia for RFA. In order to evaluate the use of general anesthesia for RFA of hepatocellular carcinoma, a questionnaire survey was conducted. METHODS: With the cooperation of Tokyo liver-tomo-no-kai(Tokyo Liver Association), a questionnaire survey was conducted for patients who underwent RFA. In the survey, data on the following were obtained "type of anesthesia used", "number of RFA treatment points", "duration of treatment", "length of impact of pain", and "if you need to receive RFA treatment again, how would you feel about this." RESULTS: The ratio of local anesthesia (LA) to general anesthesia (GA) was 113:24. The ratios of the numbers of patients who felt pain to those who felt no pain were 64:49 (LA) and 0:24 (GA). The ratios of the patients who wished to not receive RFA again to the patients who were comfortable with receiving RFA were 65:45 (LA) and 4:20 (GA). CONCLUSION: GA achieves better pain control compared to LA, and the patients who receive GA have greater tolerance of RFA.

[Four cases of airway obstruction successfully treated by airway stenting].

We report 4 cases of malignant airway obstruction treated by airway stenting. Three cases were caused by esophageal cancer and the fourth case by malignant lymphoma. Two patients with esophageal cancer received chemoradiotherapy after airway stenting and survived for 24 months and 54 months, respectively (without cancer recurrence). One patient with esophageal cancer died of airway bleeding 2 months after stent placement. The malignant lymphoma patient was treated by cyclophosphamide+doxorubicin+vincristine+prednisolone(CHOP) with rituximab. Airway obstruction due to malignancy is an urgent oncological situation that should be treated immediately. Airway stenting shows a marked and immediate improvement in dyspnea. Consequently, the induction of chemoradiotherapy or chemotherapy is possible. Two patients were successfully treated by chemoradiotherapy, of which 1 survived for more than 4 years without any evidence of cancer recurrence. Additional chemoradiotherapy for patients who receive esophageal stenting is controversial because of the high frequency of adverse events. However, chemoradiotherapy after airway stenting may demonstrate acceptable anti-cancer effects with fewer adverse events. Airway stenting was an effective treatment for airway obstruction, and additional treatment is essential for longer survival.

[A case of lung metastasis from esophageal cancer resistant to fluorouracil and cisplatin combination therapy but responsive to radiation therapy].

At present, fluorouracil and cisplatin combination therapy is the standard chemotherapy against esophageal cancer, but the choice of second-line chemotherapy is controversial. Furthermore, the effect of radiation therapy against lung metastasis from esophageal cancer is unclear. We report a case of lung metastasis from esophageal cancer resistant to fluorouracil and cisplatin combination therapy but responsive to radiation therapy. The patient was a 55-year-old woman who had undergone an operation for esophageal cancer at another hospital. A single right lung metastasis appeared 1 year after the operation. Combined fluorouracil and cisplatin therapy was administrated for 5 courses, but the lung metastasis increased in size. Afterwards, she was admitted to our hospital. We treated her with 14 courses of S-1 and docetaxel combination therapy administered over 13 months. The lung metastasis was decreased for a period. Furthermore, radiofrequency ablation under computed tomography was performed against the lung metastasis re-growth at another hospital. Although the lung metastasis increased in size, no further metastases were detected during the clinical course. The patient was treated with radiotherapy for the lung metastasis re-growth. The tumor had almost disappeared by 10 months after the completion of radiotherapy. Currently, she is receiving palliative care as an outpatient and the lung metastasis has not been evident for 2 years since the completion of radiotherapy.

[Hepatic arterial infusion with subsequent hepatectomy for a sigmoid colon cancer patient with hepatic metastases-a case report].

It is common to use systemic chemotherapy, instead of hepatic arterial infusion (HAI) of 5-fluorouracil (5-FU) or other cytotoxic agents, for unresectable hepatic metastases in colorectal cancer patients. Nevertheless, systemic administration of anticancer agents such as FOLFOX or FOLFIRI is sometimes difficult to continue for infirm patients. A 71-year-old female who had undergone sigmoidectomy for sigmoid colon cancer received HAI for 12 months because of big bilobar hepatic metastases and poor performance status. Thereafter, a two-stage hepatectomy(first, left lobe: second, S7+8 and S5) was performed successfully. She has been alive for 2.5 years after the first operation but with two small lung metastases in the left lobe. Because of bad performance status and her weak social and familial conditions, treatment with standard systemic chemotherapy could not be continued. In such cases, HAI should be performed if the metastases are limited to the liver.

Familial Parkinson disease mutations influence α-synuclein assembly.

Lewy bodies composed of aggregates of α-synuclein (αS) in the brain are the main histopathological features of Lewy body diseases (LBD) such as Parkinson's disease and dementia with Lewy bodies. Mutations such as E46K, A30P and A53T in the αS gene cause autosomal dominant LBD in a number of kindreds. Although these mutations accelerate fibril formation, their precise effects at early stages of the αS aggregation process remain unknown. To answer this question, we examined the aggregation including monomer conformational dynamics and oligomerization of the E46K, A30P, A53T and A30P/A53T mutations and wild type (WT) using thioflavin S assay, circular dichroism spectroscopy, photo-induced cross-linking of unmodified proteins, electron microscopy, and atomic force microscopy. Relative to WT αS, E46K αS accelerated the kinetics of the secondary structure change and oligomerization, whereas A30P αS decelerated them. These effects were reflected in changes in average oligomer size. The mutant oligomers of E46K αS functioned as fibril seeds significantly more efficiently than those of WT αS, whereas the mutant oligomers of A30P αS were less efficient. Our results that mutations of familial LBD had opposite effects at early stages of αS assembly may provide new insight into the molecular mechanisms of LBD.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

[A case of the long-term survivor of rectal cancer who suffered from successive metastases to ovarium, peritoneum, liver, bone and para-aortic lymphonode].

A 37-year-old female, who had undergone a low anterior resection for lower rectal cancer, had been received chemotherapy (FOLFOX4, FOLFIRI) for 2 years because of right ovarian metastasis occurred and removed 9 months after the first operation. One month after 2 years of continued chemotherapy, progressive metastases happened to occur successively (rt lunge, left ovarium, liver, para-aortic lymphonode, Virchow lymphonode and bone). Right upper lobe pnemonectomy was performed first, then, peritonectomy, total hysterectomy with left oophorectomy and a partial resection of the small bowel were done. IRIS, as postoperative chemotherapy, performed with hepatic arterial infusion (HAI) of CPT-11 and 5- FU resulted in getting a minimal response for about 10 months. Because of the hepatic arterial thrombosis at 10 months after the previous operation, we could not continue HAI with systemic chemotherapy, that was resulted in the progresion of mutiple metastases, and that the patient died 62 months after the first surgery. Immunohistochemical analyses with MIB-1 stainning of four surgical specimens revealed 80% positive cells in the cancerous tissues.

[A case of transverse colon cancer without a recurrence lesion after five years from resection of hepatic metastasis].

The treatment of hepatic metastasis of colon cancer was in progress by new biochemical agents. Generally, a resection was the first alternative treatment against hepatic metastasis of colon cancer, but new antitumor agents were more effective than conventional antitumor agents. Disappearance of metastasis for colon cancer treated with only antitumor agents was commenced to report. We were experienced a case of transverse colon cancer without a recurrence lesion after five years from the resection of hepatic metastasis. A case was a 77-year-old man. He was operated against transverse colon cancer in February 2003. Pathological stage was ss, n0, Stage II. In April 2004, serum CEA was increased. CT examination was not detected a hepatic metastasis but ultrasound examination and MRI detected the metastasis at S7 lesion in the liver. In July 2004, he was admitted to S-1 and PSK until October 2004. In December 2004, the lesion of hepatic metastasis was reduced and serum CEA was decreased. But in September 2005, the metastatic lesion was re-grown. A resection for hepatic metastasis was executed in November 2005. After the resection for hepatic metastasis, he was admitted to UFT/ UZEL from January 2006 to October 2006. Present time( June 2011), the lesion of recurrence was not detected by several examinations (CT, MRI, Ultrasound etc).

[A case of encephalopathy that was suspected to be caused by chemotherapy for colon cancer].

We report a case of encephalopathy that was suspected to be caused by chemotherapy for liver metastasis from sigmoid colon cancer. A 72-year-old male was suspected that he had drug-induced eukoencephalopathy because he was presented with physical disorders during the FOLFOX/bevacizumab therapy. Although a brain MRI revealed Alzheimer disease, leukoencephalopathy was not excluded from the diagnoses due to a fact that his findings could not be compared before and after the chemotherapy. If leukoencephalopathy was suspected, chemotherapy should have been discontinued as soon as possible. Although a partial response was achieved, chemotherapy had to be discontinued in this case. The cases whose physical and neurological disorders were at risk due to a past history need an examination for nervous system in order to make a comparison with the findings before and after chemotherapy.

[A case report of esophageal carcinoma developing esophago-brochial fistula successfully treated by esophageal bypass surgery].

We have experienced a case of esophageal carcinoma developing esophago-bronchial fistula that was successfully treated by esophageal bypass surgery followed by chemo-radiation. A man aged 64 years old with developed esophago- bronchial fistula after initial chemo-radiation was undergone a gastric bypass surgery to separate esophagus and bronchus. Though closure of fistula was just 4 months after definitive chemo-radiation, an oral feeding was possible until the death of the patient. Stent placement for esophageal carcinoma was less invasive treatment though chemo-radiation after a stent placement was accompanied by high incidence of stent associated morbidity. Since esophageal bypass surgery can definitely separate airway from esophagus, chemo-radiation with oral feeding can be easily carried out. Esophageal bypass surgery was a treatment recommendation for the patient with esophageal carcinoma invading trachea or bronchus.

G alpha(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration.

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

Involvement of Gq/11 in both integrin signal-dependent and -independent pathways regulating endothelin-induced neural progenitor proliferation.

We have previously shown that endothelin-B receptor stimulation increases neural progenitor proliferation, partly in G(i) and extracellular matrix molecule-dependent manner. In the present study, we investigated whether G(q/11) is also involved in this response and how G(i) and G(q/11) might regulate the extracellular signal-regulated kinase (ERK) pathway and integrin signaling. Endothelin-induced ERK phosphorylation was independent of integrin ligands, and an inhibitor of G(q/11), YM-254890, as well as pertussis toxin, partially inhibited endothelin-stimulated phosphorylation of Raf-1 and ERK. Endothelin-stimulated protein kinase C (PKC) was partially inhibited by both YM-254890 and pertussis toxin, while only pertussis toxin attenuated endothelin-induced Ras activation. In contrast, endothelin increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in an integrin ligand-dependent manner. Both YM-254890 and pertussis toxin partially inhibited endothelin-stimulated phosphorylation of these proteins. A PKC inhibitor and down-regulation of PKC prevented endothelin-induced phosphorylation of paxillin and ERK. In addition, endothelin-induced proliferation and DNA synthesis were partially inhibited by YM-254890 and pertussis toxin. Taken together, the results indicate that endothelin activates PKC via G(q/11) and G(i), and consequently stimulates the ERK cascade in cooperation with Ras signaling stimulated by G(i). PKC appears to increase tyrosine phosphorylation of paxillin to enhance integrin signaling, which further increases DNA synthesis and proliferation.

Biological properties of a specific Galpha q/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress.

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).