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In this paper, we design and implement A4 sized, web-based POP(Print On Demand) system using multi-layer technique. We Propose the framework of POP system, which uses multi-layer technique, uses highly accessible web-service, produces template, enables a user to easily edit, and allows the user to instantly print edited files. This system will be widely used in web-based electronic publication and e-book.

Recently, Development of high-speed internet networks can be a real-time video conference system. SD-quality still images using the software codec system exists, and two-way system with mobile systems, but in most video only send and receive video system. In this study, HD web-cam using the two-way live broadcasts simultaneously in the education system and the mobile system has developed an audible system. In the study using the H.264 standard video compression techniques were applied to high-bandwidth compression technology, smart phones, to develop a streaming server for broadcasting were applied. Video sharing, document, web-sharing was made possible in real time. In addition, the private IP for WiFi zone, available anytime, anywhere that can be converted to a public IP technology, IP tunneling technology applied.

During the design process of a terminal, the handling capacity of a container yard needs to be evaluated in advance. This study suggests formulas for estimating the expectations and the variances of cycle times for various types of operations of a yard crane. Statistical analysis is used to estimate the expectations and variances. The main focus of this study is to show the impact of interdependencies among handling time elements on the expectation and variance of the cycle times; these interdependencies have not been considered in previous studies. Numerical experiments are done for evaluating the difference in the variance of cycle times and the waiting of trucks between the cases with and without the consideration of interdependencies.

In this study, we suggest the system to analyze network users’ action patterns by using Data-Mining Technique. We installed Network Tap to implement the analysis system of network action and copied the network packet. The copied packet is stored at the database through MainMemoryDB(MMDB) of the high-speed. The stored data analyze the users’ action patterns by using Data-Mining Technique and then report the results to the network manager on real-time. Also, we applied the standard XML document exchange method to share the data between different systems. We propose this action pattern analysis system operated embedded type of SetToBox to install easily and support low price.Keywords:Action Patterns, Network Tap, Data-Mining, Embedded System, Standard XML 1. 서 론 1) 인터넷의 발달은 개인은 물론 기업의 업무에도 많은 영향을 주고 있다. 사내에서 근무하는 직원은 기본적으로 인터넷을 통해 업무에 도움이 되는 정보를 검색하여 활용하기도 하고, 홈쇼핑 및 각종 블로그를 통해 개인 활동을 하기도 한다. 이런 개인이 속한 대부분의 단체에는 업무의 종류로 부서를 구분한다. 부서 각각은 그 업무의 형태에 따라 일정한 패턴을 가지고 활동하게 된다. 물론 그 활동의 패턴은 인터넷 사용에도 반영 된다. 영업 부서에서 개발 부서가 사용하는 패턴의 움직임이 생긴다면 분명 이상 행위로 분류할 수 있다. 최악의 경우 사내의 기밀이 유출될 가능성도 예측

To assess the diagnostic accuracy of prospective ECG-triggering 64-slice multidetector computed tomography (MDCT) coronary angiography for evaluation of coronary artery disease (CAD). Forty-two patients (31 males, 11 females, mean age 64 years) underwent cardiac CT and invasive coronary angiography (ICA). Patients with a heart rate of

Yard cranes are important pieces of handling equipment in container yards. This study analyzes cycle times of various handling operations of yard cranes, including receiving, loading, discharging, and delivery operations. An operation cycle is divided into elementary movements, and formulas for expected time and variance of each elementary movement are derived analytically. Cycle times of various types of yard crane are compared with each other analytically and numerically. The accuracy of the formulas is evaluated through a simulation study.

A block is the basic unit of storage space in container terminals. This study proposes two methods for optimizing the block size, by considering the throughput requirements of yard cranes (YCs) and the block storage requirements. To estimate the YC performance, cycle-time models of various handling operations of YCs are analytically derived. Two types of container yards are examined: those with blocks that are laid out parallel to the quay and those laid out vertical to the quay.

The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell growth inhibition were examined using cell cycle analysis, immunoblotting and immunoprecipitation. The BCH treatment induced cell cycle arrest at G1 phase in KB cells. The expression of cyclin D3 was remarkably decreased by BCH treatment. The BCH inhibited the expression of cyclin-dependent protein kinase 6 (CDK6) in a time-dependent manner. In addition, the expression of CDK inhibitor p27 was increased by BCH treatment in KB cells, but not CDK inhibitors p21 and p15. These results suggest that, in KB cells, the inhibition of LAT1 by BCH causes cell cycle arrest at G1 phase by inhibiting cyclin D3-CDK6 complex whereas increasing expression of a CDK inhibitor p27.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins,more » can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.« less

We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.

This study uses a structural equation model to examine the effects of noise on self-rated job satisfaction and health in open-plan offices. A total of 334 employees from six open-plan offices in China and Korea completed a questionnaire survey. The questionnaire included questions assessing noise disturbances and speech privacy, as well as job satisfaction and health. The results indicated that noise disturbance affected self-rated health. Contrary to popular expectation, the relationship between noise disturbance and job satisfaction was not significant. Rather, job satisfaction and satisfaction with the environment were negatively correlated with lack of speech privacy. Speech privacy was found to be affected by noise sensitivity, and longer noise exposure led to decreased job satisfaction. There was also evidence that speech privacy was a stronger predictor of satisfaction with environment and job satisfaction for participants with high noise sensitivity. In addition, fit models for employees from China and Korea showed slight differences.This study is motivated by strong evidence that noise is the key source of complaints in open-plan offices. Survey results indicate that self-rated job satisfaction of workers in open-plan offices was negatively affected by lack of speech privacy and duration of disturbing noise.

Almost of industrial measuring instruments usually are equipped only with serial communication devices. In order to connect these instruments to internet, we implement an embedded translator. This device has the hardware components composed of one WAN port, two LAN ports, and two UARTs, and functions as a communication translator between serial and internet communication. it also provides web-based monitoring function that is absent from existing serial-to-ethernet converter. The hardware is implemented using the KS8695 network processor which s an ARM922T as processor core. We have installed the boa web server and utilized the CGI function for internet-based remote control, added the IP sharing function which allows the network with private IP addresses to access the internet, and developed a serial-to-ethernet translation program. Finally, we show an application example of the developed translator that remotely monitors the solar energy production system.

Adsorption data for CH4, C2H6, C2H4, H2, and N2 on the zeolite 5A adsorbent (W. R. Grace & Davision Co.) were obtained at 293 K, 303 K, and 313 K. The pressure range studied was 0 to 2 MPa, which covers the operating pressure range in pressure swing adsorption (PSA) units. Experimental data were correlated by the Langmuir and Langmuir−Freundlich (L−F) equations for several isotherm models. This paper compares the Langmuir and L−F equations. Despite the relative simplicity of both isotherms, the experimental data were fit very well. In addition, the Clausius−Clapeyron equation was employed to calculate the isosteric enthalpy of adsorption.

A fluidized bed reactor made of quartz with 0.055 m i.d. and 0.65 m in height was employed for the thermocatalytic decomposition of methane to produce CO2 free hydrogen. The fluidized bed reactor was proposed in order to overcome the reactor plugging problem due to carbon deposition, which was resulted in the shut-down of the fixed bed reactor system. Several kinds of activated carbons were employed as the catalyst to examine the reaction activity. The experimental results in this study were compared with that of fixed bed reactor. The reaction rate and deactivation of carbon catalyst were similar to that of lab scale fixed bed reactor system. The deactivation due to carbon deposition was verified with the SEM images of fresh and used carbon catalysts. The effects of gas velocity, reaction temperature and particle size on the reaction rates were also investigated. From the experimental results of this study, the optimum operating conditions for fluidized bed were also determined.

This paper explains the construction of the filesystems which could be utilized in embedded systems as an implementation of ubiquitous computing. It includes the formal architecture of filesystem hierarchy for the DOC (Disk-On-Chip) filesystem and the flash filesystem based on the MTD (Memory Technology Devices). For DOC, the root filesystem and the user filesystem are constructed by the TrueFFS supported by the M-Systems. For MTD filesystem, the root filesystem is implemented in the fast RAM disk, and the user filesystem is implemented in the JFFS2 that supports large capacity. In order to support the GUI filesystem, the porting process of Qt/E is also included in this paper.

In order to optimize the performance of an adsorption column, the adsorption and regeneration dynamic characteristics were studied for 20% methane and 80% hydrogen binary system on nonisothermal and nonadiabatic conditions. The adsorption dynamic characteristics were studied at various flow rates, 7.2 LPM to15.8 LPM, and at various adsorption pressures, 6 to 12 atm. Also, regeneration dynamic characteristics were studied at various purge rates, 1.5 to 3.5 LPM, and constant pressure, 1.2 atm. Nonisothermal and nonadiabatic models, considering linear driving force model and Langmuir-Freundlich adsorption isotherm model, were considered to compare between prediction and experimental data.

Several domestic activated carbons were tested as the catalyst for decomposition of methane. All the activated carbons showed similar deactivation pattern. Mass transport effect in the catalyst particles was observed and the pore mouth blocking appeared to occur noticeably especially in large particles. Between different kinds of the activated carbons, no discernible trend was observed between the initial activity and the surface area, and the initial activities lay in a relatively narrow range. The reaction orders were 0.5 and the same for different samples of the activated carbon. The activation energies were also about the same, ca. 200 kJ/mol , regardless of the kind and suppliers. These results indicate that the reaction mechanism is the same for all the activated carbons. Ashes in the activated carbons do little affect the catalytic activity.

Catalytic characteristics of carbon blacks for methane decomposition were investigated. The reaction was carried out under atmospheric pressure at 1023–1323 K. Carbon blacks exhibited stable catalytic activity despite of carbon deposition. No trend could be induced between the activity and the surface area of fresh samples. The reaction order was near unity and the activation energies over carbon blacks were significantly lower than those over activated carbons. It was observed that the deposited carbon formed many protrusions on the particles and made the particles bigger and stuck together. In spite of marked changes of the carbon morphology during the reaction, it is noteworthy that the activity of carbon blacks was maintained nearly constant. The nature of active sites was discussed.

The amount of deposited carbon on activated carbons was adjusted by varying the space time and the time on stream. Carbon nuclei formation appeared to occur initially but was terminated soon and then the carbon crystallite growth became dominant. The methane decomposition rate over activated carbons had a nearly linear relationship with the amount of carbon deposited. This suggests that the carbon deposition occurs uniformly and the activity decreases due to pore blocking, resulting in loss of accessibility to the active sites. The rate was also nearly proportional to the surface area for the same kind of activated carbon, which is contrasted with the previous finding that no discernible trend was observed between the initial activity and the surface area among different kinds of the activated carbons.

Adsorption equilibria of single components for pure methane, ethane, ethylene, hydrogen, and nitrogen onto activated carbon adsorbent (Calgon Co.) were measured. The results were obtained with a static volumetric method at 293.15 K, 303.15 K, and 313.15 K and at pressures up to 2 MPa. Experimental data were correlated by the Langmuir−Freundlich equation. Isosteric enthalpies of adsorption were calculated and shown according to surface loading.

Floor vibrations induced by humans walking barefoot were investigated in heavyweight buildings. Six floating floors with different floor structures and thick resilient isolators were built in laboratories with the same dimensions and boundary conditions. Subjective tests were performed to assess the vibration serviceability of the floor structures. In the first test, subjects were asked to walk across a floor and then rate the intensity of the vibrations, and the acceptability and serviceability of the floors. In the second test, subjects were seated on a chair in the middle of the floor and asked to rate the floor vibrations when a walker passed by the subjects. Floor vibrations induced by human walking were analysed using peak acceleration, root-mean-square (r.m.s.) acceleration, and the vibration dose value (VDV), with four frequency weighting functions (Wb , Wk , Wg, and Wm ). Significant differences in the measured floor vibrations were found across the floor structures with greater floor vibration leading to greater perceived vibration intensity, lower acceptability, and lower serviceability. The VDV was correlated with perceptions of floor vibration when used with all four frequency weighting functions. The impact noise induced by walking did not influence subjective evaluations of floor vibration. A heavy/soft impact source (a standard impact source) provided a useful prediction of differences between the perception of the vibration on different floors.

In fuzzy inferencing, most of conventional fuzzy controllers have problems of speed down in floating point operations of fuzzy membership functions in (0,1) as compared with integer operations. Therefore, in this paper, we propose a high-speed fuzzy controller with only integer operations. In this, for fast fuzzy computations, we use a scan line conversion algorithm to convert lines of each fuzzy linguistic term to the set of the closest integer pixels. We also implement a GUI (Graphic User Interface) application program for the convenient environments to modify and input fuzzy membership functions.

In this paper, we propose an integrated GUI environment of parallel fuzzy inference system fur pattern classification of remote sensing data. In this, as 4 fuzzy variables in condition part and 104 fuzzy rules are used, a real time and parallel approach is required. For frost fuzzy computation, we use the scan line conversion algorithm to convert lines of each fuzzy linguistic term to the closest integer pixels. We design 4 fuzzy processor unit to be operated in parallel by using FPGA. As a GUI environment, PCI transmission, image data pre-processing, integer pixel mapping and fuzzy membership tuning are considered. This system can be used in a pattern classification system requiring a rapid inference time in a real-time.

The neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) was originally discovered as a transcriptional repressor of neuronal genes in non-neuronal cells. However, it was recently reported to be abundantly expressed in several types of aggressive cancer cells, as well as in mature neurons. In the present study, the role of NRSF/REST in the human oral squamous cell carcinoma (SCC) KB cell line was evaluated. NRSF/REST was expressed at a higher level in KB cells when compared with that in normal human oral keratinocytes (NHOKs). Knockdown of NRSF/REST by siRNA reduced cell viability only in KB cells in a time-dependent manner, and this effect was due to the activation of apoptosis components and DNA fragmentation. In addition, knockdown of NRSF/REST disrupted the mTOR signaling pathway which is a key survival factor in many types of cancer cells. For example, the phosphorylation of elF4G, elF4E and 4E-BP1 was significantly reduced in the KΒ cells upon NRSF/REST knockdown. These results imply that NRSF/REST plays an important role in the survival of oral cancer cells by regulating the mTOR signaling pathway.

Mitral stenosis (MS) and mitral regurgitation (MR) are the most frequent conditions that cause a dilation and dysfunction of the left atrial appendage (LAA). Despite similarly dilated LAA in patients with MS and MR, the incidence of LAA thrombi and the risk of thromboembolism is different between these patients. The purpose of this study was to characterize the filling pattern of LAA by using intravenous administration of perfluorocarbon-exposed dextrose albumin (PESDA) during transesophageal echocardiographic examination in patients with MS and MR. Twenty-four patients with moderate to severe MS, 12 patients with severe MR, and a control group including 30 patients with conditions other than mitral valve disease underwent transesophageal echocardiographic examination with an intravenous bolus injection of PESDA. LAA emptying and filling velocities and maximal and minimal areas of LAA and LAA ejection fraction were measured. Digital gray-scale intensity (GSI) of the left atrial (LA) and LAA cavity after PESDA injection was measured by off-line analysis. Compared with control patients, patients with MS or MR had larger maximal and minimal areas of LAA and reduced LAA ejection fraction. LAA peak emptying flow velocity was significantly lower in patients with MS compared with those of MR or control patients. LAA peak filling velocity was significantly lower in patients with MS compared with that of control patients. However, there was no significant difference of LAA peak filling velocity between the patients with MS and MR. There was no significant difference of GSI ratio of LAA and LA between patients with MR and control patients; however, GSI ratio of LAA and LA was significantly lower in patients with MS compared with that of MR. The incidence of LAA spontaneous echo contrast and LAA thrombi in patients with MS was significantly higher than that of the patients with MR and control subjects (P.005). Despite similarly dilated LAA area and depressed contractile function of LAA in patients with MS and MR compared with control patients, profoundly impaired LAA filling with resultant flow stasis was demonstrated by contrast echocardiography in patients with MS. These findings may explain the higher incidence of LAA spontaneous echo contrast and thrombus in patients with MS.

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.

The differential expression of specialized voltage-gated potassium (Kv) channel subtypes probably reflects the wide range of functions in the nervous system. In the present study, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry. Immunoreactivities for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 were observed in the pyramidal cells of the CA1-CA3 areas. In addition, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1-CA3 were strongly immunoreactive for Kv1.4. Although there was some overlap, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies of the pyramidal cells and granule cells. As a whole, the present study has clearly shown Kv1 channel distributions in the gerbil hippocampus, which were variant between species and therefore more or less functionally significant. This study may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy.

A role for p53-mediated modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. P53 gene upregulation precedes apoptosis in many cell types, and a potential role for this molecule in apoptosis of neurons has already been demonstrated in Alzheimer's disease. Recent studies suggest that p53-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In the present study, we examined changes in p53-immunoreactive (IR) neurons in the brains of aged rats for the first time employing immunocytochemical and in situ hybridization methods. P53-IR neurons were found in the CA1 region of hippocampus, septal region and cerebellum in the aged rats, but there was no p53-IR cell in the brains of adult rats. In the hippocampus of the aged rat, p53-IR cells predominated in the stratum oriens and pyramidal layers, while the molecular layer contained relatively few p53-IR cells. The most prominent population of immunoreactive labeling in cerebellar cortex was localised within the cell bodies of Purkinje cells and dendrites in molecular layers. Upregulation of p53 in the Purkinje cells observed in this study suggests that significant loss of Purkinje cells with aging may be regulated with several apoptosis-controlling factors including p53 and oxidative stress mechanism. Further investigations are required to establish whether direct functional relations exist between p53 and the apoptotic neuronal death in normal aging or Alzheimer brains.

Analogs of the alpha-factor tridecapeptide mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr13 was replaced with Phe, p-F-Phe, m-F-Phe, p-NO2-Phe, p-NH2-Phe or Ser were synthesized and purified to >99% homogeneity. These analogs were bioassayed using a growth arrest assay and a gene induction assay and evaluated for their ability to compete with binding of tritiated alpha-factor to its receptor Ste2p. The results showed that the phenolic OH of Tyr13 is not required for either biological activity or receptor recognition. Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80-120% of the biological activity of the parent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of alpha-factor. In contrast, substitution of Ser or Ala at position 13 resulted in a >100-fold decrease in receptor affinity suggesting that the aromatic ring is involved in binding to the receptor. The lack of a strict requirement for Tyr13 allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of the peptide. These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay. One potential receptor ligand [Tyr(125I)1,Nle12, Phe13] alpha-factor exhibited saturable binding with a KD of 81 nM and was competed by alpha-factor for binding in a whole-cell assay. Thus a new family of radioactive ligands for the alpha-factor receptor has been revealed. These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies.

Supplemental Digital Content is available in the text., Background: Treatments for severe blepharoptosis are well documented and include the most common operations for restoring upper eyelid ptosis, which are levator surgery and frontal muscle transfers; however, the choice of treatment is still controversial. There are different approaches to the restoration of upper eyelid ptosis, and the choice will be based on ptosis severity and the surgeon’s skill and experience. Methods: Two hundred and fourteen patients presenting with a levator function of between 2 and 4 mm received ptosis correction between 1991 and 2010 at our clinic. Of these, 71 patients underwent Müller aponeurosis composite flap advancement for correction of 89 eyelids, and frontalis muscle transfer was performed on 143 patients (217 eyelids). Postoperative results were evaluated with an average follow-up period of 23 months. Results: The preoperative average for marginal reflex distance (MRD1) in the Müller aponeurosis composite flap advancement group was 1.25 mm, and in the frontal muscle transfer group, it was 0.59 mm. The area of corneal exposure (ACE) was 57.2% in the Müller aponeurosis composite flap advancement group and 53.6% in the frontal muscle transfer group. The postoperative average distance was not significantly different for the 2 techniques. In the Müller aponeurosis composite flap advancement group, MRD1 was 2.7 mm and ACE was improved to 73.5%. In the frontal muscle transfer group, MRD1 was 2.3 mm and ACE was 71.2%. Undercorrection and eyelid asymmetry were the most frequently observed postoperative complications for both techniques. Conclusions: In our study, we confirmed that Müller aponeurosis composite flap advancement and the frontalis transfer technique are both effective in the correction of severe blepharoptosis; our results showed no significant differences between the 2 techniques.

This paper studies a new automated container terminal (ACT) system which utilizes multistory frame bridges and rail-mounted trolleys to transport containers between the quay and the yard. Beside typical ACT systems use trucks or automated guided vehicles for transporting containers between quay cranes and yard cranes, the new design uses three types of handling machines, namely, ground trolleys (GTs), transfer platforms (TPs), and frame trolleys (FTs). These three types of handling machines collaborate with one another to transport containers. This study decomposes the system into several subsystems. Each subsystem has one TP and several FTs and GTs dedicated to this TP. Then, a Markov chain model is developed to analyze the throughput of TPs. At last, the performance of the new ACT system is estimated. Sensitivity analyzes the numbers, and the processing rates of trolleys are conducted through the numeric experiments.

In recent years, natural polymers such as cellulose, alginate and chitosan have been used worldwide as biomedical materials and devices, as they offer more advantages over synthetic polymers. The aim of this study was to clarify the usefulness of microbial cellulose (MC) for use as a dressing and scaffold material. For evaluating the biodegradability and toxicity of MC, we divided the rats (n = 12) into two groups (the implanted group and the non-implanted group). In the implanted group, we implanted the film type of MC in the backs of six rats. In the non-implanted group, however, we did not implant the film type of MC in the backs of the six rats. Four weeks later, we compared two groups by the gross, histological and biochemical characteristics by using blood and tissue samples. To evaluate the wound healing effects of MC, three full-thickness skin defects were made on the backs of each rat (n = 20). Three wounds on the backs of the same rats were treated with other dressing materials, namely, Vaseline gauze (group Con), Algisite M(®) (group Alg) and MC (group MC). We analysed the gross, histological and biochemical characteristics by western blotting. MC was found to be biodegradable and non-toxic. On day 3, the MC film was visible under the subcutaneous tissue; however, after 4 weeks, no remnants of the film were visible under the subcutaneous tissue. Furthermore, there was no evidence of MC-induced toxicity. Moreover, group MC showed more rapid wound healing compared with group Con. On day 14 after skin excision, group MC showed greater decrease in wound size compared with group Con (33% versus 7·2%). The wound healing effects were also substantiated by the histological findings (greater reduction in inflammation and rapid collagen deposition as well as neovascularisation) and western blotting (decreased expression of vascular endothelial growth factor and transforming growth factor-β1 in group MC on day 14 after skin excision, unlike group Con). This study showed that, in addition to having wound healing effects, MC is biodegradable and non-toxic and can, therefore, be used as a dressing and scaffold material.

PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor. Introduction: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand–receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. Materials and Methods: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. Results: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (ICmax and ECmax) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. Conclusions: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N. Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464-46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator.

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.

We have revealed age-related changes in the expression patterns of Kv1.1 and Kv1.2 in the rat cerebellum for the first time. In the aged rat, immunoreactivity for Kv1.1 was increased in the cell bodies of Purkinje cells, while the staining intensity was significantly decreased in the granule cells. The cell bodies of cerebellar output neurons showed strong Kv1.1 immunoreactivity in the nucleus medialis, interpositus and lateralis of the aged rat. Kv1.2 immunoreactivity was found in some interneurons with their processes in this region of the aged rat. Image analysis demonstrated that immunoreactivities for Kv1.1 and Kv1.2 were increased specifically in the cell bodies of cerebellar output neurons of the aged rat. This study may provide useful data for future investigations on the channels that cause brain diseases and age-related disorders.

Voltage-gated K(+) (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum.

The α-factor receptor (Ste2p) from Saccharomyces cerevisiae belongs to the family of G-protein coupled receptors (GPCRs) that, upon the binding of a ligand, transduce a signal via an associated guanine nucleotide binding protein (G protein). The binding of the tridecapeptide pheromone a-factor to Ste2p initiates a cascade of events that leads to the mating of haploid yeast cells. To understand the signaling process, one must first understand how ligands are recognized by and associated with their receptor. Fluorescence techniques have been widely used to investigate ligand-receptor interactions [1]. This work reports the synthesis, bioactivity, binding affinity of a-factor tagged with a fluorescent probe at various positions in the peptide backbone, and a fluorescence analysis of the binding environment of these ligands bound to Ste2p.

G protein-coupled receptors (GPCRs) are multifunctional proteins involved in cell-cell communication and represent the largest gene family in eukaryotes. Signal molecules recognized by these receptors include odorants, biogenic amines, peptides and proteins, and are involved in regulation of a plethora of biological processes including pain sensation, growth, blood pressure regulation and intermediary metabolism. Despite the importance of GPCRs, few details are available on the molecular aspects of ligand recognition or signal transduction. Ongoing studies in our laboratory use mating in Saccharomyces cerevisiae as a paradigm to understand various aspects of GPCR function. One specific goal of our work is to understand the nature of the interaction of the pheromone α-factor (WHWLQLKPGQPMY) with its GPCR (Ste2p). In this report, we discuss a multi-pronged approach utilizing fluorescence spectroscopy, site-directed mutagenesis, and photoaffinity labeling to define the interaction of this peptide pheromone with Ste2p.

Chronic infection and inflammation have recently been implicated as important etiologic agents for atherosclerosis in general and, in particular, ischemic heart disease. Several agents have been suggested as possible candidates for the chronic inflammation including cytomegalovirus, Helicobacter pylori and Chlamydia pneumoniae. We hypothesized that a vascular infection with C. pneumoniae may induce a chronic inflammatory reaction in the host vascular tissue and activated inflammatory cells may express inflammatory mediators such as cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs). At first, we evaluated the relationship between C. pneumoniae infection and atherosclerosis indirectly by serologic study, and then, to confirm our hypothesis, we performed an immunohistochemical study of atherosclerotic plaques. The seropositive rate of anti-Chlamydia pneumoniae IgG was higher in the disease group (Group I, 59.8%, n = 254) than in the negative control group (Group III, 47.4%, n = 97) (p = 0.041), but the anti-Chlamydia pneumoniae IgA was not different in seropositivity between the two groups (Group I, 64.6%; Group III, 57.7%). The simultaneous seropositive rates of both IgG and IgA were 56.7% in Group I and 43.3% in Group III (p = 0.033). In subgroups without the conventional risk factors of atherosclerosis, these findings were more prominent. Furthermore, we performed immunohistochemical staining on the atherosclerotic aortic tissues obtained from patients that were seropositive to C. pneumoniae (n = 5), by using antibodies to C. pneumoniae, COX-2, and MMP-9. The immunoreactivity for COX-2 and MMP-9 increased in the atherosclerotic plaques itself, predominantly in the surrounding area of immunoreactive C. pneumoniae. These findings support our hypothesis and C. pneumoniae may participate in a pathogenetic mechanism for atherogenesis or progression of atherosclerosis. The present study may open a promising perspective concerning future therapeutic trials of chronic inflammation related atherogenesis under pathophysiological conditions.

Although it has been shown that the percentage of bone marrow blasts in myelodysplastic syndrome (MDS) constitute the only independent determinant of survival and progression to acute leukemia, the great variability in survival among patients with MDS of similar percentage of blasts has prompted us to investigate new objective, independent prognostic parameters for the selection of high-risk patients. It was suggested that CD34 antigen expression adversely affected the prognosis of acute myelogenous leukemia. However, no study has been published so far on clinical and prognostic significance of CD34 antigen expression in MDS. Bone marrow biopsies from 58 patients diagnosed as primary MDS were studied using QBEND/10, a monoclonal antibody which recognized the human progenitor CD34 antigen on routine aldehyde-fixed, paraffin-embedded samples. The high percentage of CD34-positive cells (above 3% of total bone marrow nucleated cells) was predominantly observed in cases with RAEB-T, CMML, and to a lesser degree in RAEB. But neither age, hemograms, bone marrow findings including percentage of blasts, ALIP, nor leukemic transformation correlated with the percentage of CD34-positive cells. The median actuarial survival time in the high positive group was significantly shorter (12.0 months) than that of the low group (30.0 months; p = 0.028). The high CD34 aggregate was selectively found in cases with RAEB, RAEB-T, and CMML. The percentage of bone marrow blasts (p = 0.007) and ALIP (p = 0.030) significantly correlated with number of CD34 aggregates. The median survival time in the high CD34 positive group (25.0 months; p = 0.003). On multivariate analysis, the positivity of CD34 cells (p