MAP2 is required for dendrite elongation,PKA anchoring in dendrites,and proper PKA signal transduction

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.     

Halothane attenuated haloperidol and enhanced clozapine-induced dopamine release in the rat striatum

The effect of halothane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA)) induced by neuroleptics was studied using in vivo microdialysis techniques. Halothane attenuated haloperidol-induced dopamine release and enhanced clozapine-induced dopamine release in the rat striatum.A microdialysis probe was implanted into the right striatum of male SD rats. Rats were given saline or the same volume of 200 microg kg(-1) haloperidol (D(2) receptor antagonist), 10 mg kg(-1) sulpiride (D(2) and D(3) antagonist), or 10 mg kg(-1) clozapine (D(4) and 5-HT(2) antagonist) intraperitoneally with or without 1-h halothane anesthesia (0.5 or 1.5%). Halothane anesthesia did not change the extracellular concentration of DA, but increased the metabolite concentrations in a dose-dependent manner. The increased DA concentration induced by haloperidol was significantly attenuated by halothane anesthesia, whereas the metabolite concentrations were unaffected. Halothane had no effect on the changes in the concentrations of DA or its metabolites induced by sulpiride. The clozapine-induced increases in DA and its metabolites were enhanced by halothane anesthesia.Our results suggest that halothane anesthesia modifies the DA release modulated by antipsychotic drugs in different ways, depending on the effects of dopaminergic or serotonergic pathways.     

Optimizing transport protocol parameters for large scale PC cluster and its evaluation with parallel data mining

Recently, PC clusters have come to be studied intensively for large scale parallel computers of the next generation. ATM technology is a strong candidate as a de facto standard of high speed communication networks. Therefore, an ATM-connected PC cluster is a promising platform from the cost/performance point of view, as a future high performance computing environment. Data intensive applications, such as data mining and ad hoc query processing in databases, are considered very important for massively parallel processors, as well as for conventional scientific calculations. Thus, investigating the feasibility of applications on an ATM-connected PC cluster is meaningful. In this paper, an ATM-connected PC cluster consisting of 100 PCs is reported, and characteristics of a transport layer protocol for the PC cluster are evaluated. Point-to-point communication performance is measured and discussed, when a TCP window size parameter is changed. Parallel data mining is implemented and evaluated on the cluster. Retransmission caused by cell loss at the ATM switch is analyzed, and parameters of retransmission mechanism suitable for parallel processing on the large scale PC cluster are clarified. Default TCP protocol cannot provide good performance, since a lot of collisions happen during all-to-all multicasting executed on the large scale PC cluster. Using TCP parameters with the proposed optimization, performance improvement is achieved for parallel data mining on 100 PCs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age-related changes in blood coagulation and fibrinolysis in mice fed on a high-cholesterol diet.

To investigate the pathogenesis of hyperlipidemia-induced atherosclerosis, we examined age-dependent changes in platelet activity, blood coagulation and fibrinolysis in susceptibility to a high cholesterol diet (HCD) feeding in male ICR mice. Pretreatment of platelet-rich-plasma from HCD feeding mice for 3 days with epinephrine (300 microM) resulted in a marked enhancement of adenosine 5'-diphosphate (ADP: 0.1 microM) or collagen (0.7 microgram/ml)-stimulated aggregation compared with the same in control mice. Yohimbine as alpha 2-adrenergic blocker antagonized these aggregations in a dose-dependent manner. A significant increase in plasma total cholesterol and VLDL (very low-density lipoprotein)-LDL (low-density lipoprotein)-cholesterol and the liver/body weight ratio was observed in mice fed on HCD for 3 months (3-month HCD mice). In the early phase of this experiment, a significant increase in fibrinogen was observed. In the middle phase, increases in the activity of antithrombin III (ATIII) and alpha 2-plasmin inhibitor (alpha 2-Pl) followed. Plasminogen content gradually decreased in both normal diet and HCD mice throughout the experiment. The activity of plasminogen activator inhibitor (PAI) decreased in 3-month HCD mice. Morphological observation of the aortic arch from 3-month HCD mice revealed apparent atheromatous plaques not seen in control mice. These results suggest that 3-month HCD mice can be a convenient hyperlipidemia-induced atherosclerotic model and the changes in platelet activity, coagulation and fibrinolysis in the early phase may be a cause of pathologic changes in this model.     

Effects of surfactant distribution and ventilation strategies on efficacy of exogenous surfactant

The effectsof both surfactant distribution patterns and ventilation strategiesutilized after surfactant administration were assessed in lung-injuredadult rabbits. Animals received 50 mg/kg surfactant via intratrachealinstillation in volumes of either 4 or 2 ml/kg. A subset ofanimals from each treatment group was euthanized for evaluation of theexogenous surfactant distribution. The remaining animals wererandomized into one of three ventilatory groups: group1 [tidal volume(VT) of 10 ml/kg with 5 cmH₂O positive end-expiratorypressure (PEEP)]; group 2 (VT of 5 ml/kg with 5 cmH₂O PEEP); orgroup 3 (VT of 5 ml/kg with 9 cmH₂O PEEP). Animals wereventilated and monitored for 3 h. Distribution of the surfactant wasmore uniform when it was delivered in the 4 ml/kg volume. When thedistribution of surfactant was less uniform, arterial PO₂ values were greater ingroups 2 and3 compared with group1. Oxygenation differences among the differentventilation strategies were less marked in animals with the moreuniform distribution pattern of surfactant (4 ml/kg). In bothsurfactant treatment groups, a high mortality was observed with theventilation strategy used for group 3.We conclude that the distribution of exogenous surfactant affects theresponse to different ventilatory strategies in this model of acutelung injury.

     

Sorafenib Neoadjuvant Therapy in the Treatment of High Risk Renal Cell Carcinoma

ObjectiveTo evaluate the clinical efficacy of sorafenib as preoperative neoadjuvant therapy in patients with high risk renal cell carcinoma (RCC).ResultsAmong the 18 patients there were 13 male and 5 female, with a median age of 54.6 years. The objective response rate (ORR) of the operation on the selected patients is very high (94.4%), including 4 cases (22.2%) of partial response (PR) and 13 cases (72.2%) of stable disease (SD). After preoperative sorafenib treatment, the average tumor size of the 18 patients decreased from 7.8 cm (ranging from 3.6 to 19.2 cm) to 6.2 cm (ranging from 2.4 to 16.8 cm), and the median value of average tumor CT value decreased from 61HU to 52 HU. Among the 5 patients who had IVC tumor thrombi, the grades of tumor thrombi in 2 patients who were grade II before sorafenib treatment became grade I and grade 0 respectively, 2 patients of grade III both became grade II.ConclusionPreoperative neoadjuvant therapy with sorafenib for high risk RCC patients can significantly decrease primary tumor volume as well as tumor thrombus, which could help the nephron-sparing surgery (NSS) or radical nephrectomy to be done successfully.     

Investigation of Susceptibility Genes Triggering Lachrymal/Salivary Gland Lesion Complications in Japanese Patients with Type 1 Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) is a unique form of chronic pancreatitis characterized by high serum IgG4 concentration and a variety of complicating extra-pancreatic lesions. In particular, lachrymal/salivary gland lesions tend to manifest in a highly active AIP disease state, and several genes are speculated to be associated with the onset of this complication. We therefore searched for candidate susceptibility genes related to lachrymal/salivary gland lesions in a genome-wide association study (GWAS) with the GeneChip Human Mapping 500k Array Set (Affymetrix, CA) that was followed by fine mapping of additional single nucleotide polymorphisms (SNPs) in strongly significant genes with TaqMan assays. Venous blood samples were obtained from 50 type 1 AIP patients with lachrymal/salivary gland lesions (A group) and 53 type 1 AIP patients without (B group). The mean values of IgG and IG4 were both significantly different (P<0.05) between the groups. SNPs that showed a significant association with the A group at the genome-wide level (P<0.0001) were identified and subsequently used in fine SNP mapping of candidate genes. In total, five SNPs had a positive association with complicated AIP (most notably rs2284932 [P=0.0000021]) and five SNPs possessed a negative association (particularly rs9371942 [P=0.00000039]). Among them, KLF7, FRMD4B, LOC101928923, and MPPED2 were further examined for complication susceptibility using additional SNPs that were not included in the GWAS. Individual genotyping of KLF7 rs2284932 revealed that the frequency of the minor C allele was significantly increased (P=0.00062, Pc=0.0018, OR=2.98, 95%CI=1.58-5.65) in group A. The minor T allele of rs4473559 in FRMD4 demonstrated a significant association in the A group (P=0.00015, OR=3.38, 95%CI=1.77-7.65). In the LOC101928923 gene, the frequency of the minor C allele of rs4379306 was significantly decreased in group A in both TaqMan and GWAS analyses. Lastly, the minor C allele of MPPED2 rs514644 carried a significantly increased risk of complications. These four genes may be linked with the onset of lachrymal/salivary gland lesions in type 1 AIP patients and require further study.     

A rapid and affordable screening platform for membrane protein trafficking

Background

Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed.

Results

This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane.

Conclusion

The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.

     

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.     

Decomposition Analysis of Waste Generation From Stocks in a Dynamic System

We conducted a decomposition analysis of material flows in a dynamic system, focusing on factors in the generation of waste consumer durables. A methodology for the analysis of consumer durables was developed and applied to three common consumer durables: cathode ray tube TVs, refrigerators, and passenger cars. The methodology decomposed changes in the numbers of waste products into three factors: changes in lifespan distribution, past trends in replacement sales, and past trends in sales for additional purchases. The decomposed equation clearly showed that the number of waste products would not necessarily be reduced by lifespan extension alone. This is because the number of waste products generated is affected not only by current lifespan distribution but also by past trends in sales for replacement and in additional purchases. The results show that changes in past replacement sales influence the current generation of waste, even if current replacement sales are declining. To reduce the generation of waste products on a short‐term basis, lifespan must be extended until the waste‐reducing effect of lifespan extension exceeds the waste‐increasing effect of the other two factors. From a long‐term perspective, controlling current replacement and additional purchases can be used to prevent future waste product generation.