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By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Exploring biological systems from highly complex datasets is an important task for systems biology. The present study examined co-expression dynamics of mouse heart transcriptome by spectral graph clustering (SGC) to identify a heart transcriptomic network. SGC of microarray data produced 17 classified biological conditions (called condition spectrum, CS) and co-expression patterns by generating bi-clusters. The results showed dynamic co-expression patterns with a modular structure enriched in heart-related CS (CS-1 and -13) containing abundant heart-related microarray data. Consequently, a mouse heart transcriptomic network was constructed by clique analysis from the gene clusters exclusively present in the heart-related CS; 31 cliques were used for constructing the network. The participating genes in the network were closely associated with important cardiac functions (e. g., development, lipid and glycogen metabolisms). Online Mendelian Inheritance in Man (OMIM) database indicates that mutations of the genes in the network induced serious heart diseases. Many of the tested genes in the network showed significantly altered gene expression in an animal model of hypertrophy. The results suggest that the present approach is critical for constructing a heart-related transcriptomic network and for deducing important genes involved in the pathogenesis of various heart diseases.

Building on our discovery that mutations in the transmembrane serine protease, TMPRSS3, cause nonsyndromic deafness, we have investigated the contribution of other TMPRSS family members to the auditory function. To identify which of the 16 known TMPRSS genes had a strong likelihood of involvement in hearing function, three types of biological evidence were examined: 1) expression in inner ear tissues; 2) location in a genomic interval that contains a yet unidentified gene for deafness; and 3) evaluation of hearing status of any available Tmprss knockout mouse strains. This analysis demonstrated that, besides TMPRSS3, another TMPRSS gene was essential for hearing and, indeed, mice deficient for Hepsin (Hpn) also known as Tmprss1 exhibited profound hearing loss. In addition, TMPRSS2, TMPRSS5, and CORIN, also named TMPRSS10, showed strong likelihood of involvement based on their inner ear expression and mapping position within deafness loci PKSR7, DFNB24, and DFNB25, respectively. These four TMPRSS genes were then screened for mutations in affected members of the DFNB24 and DFNB25 deafness families, and in a cohort of 362 sporadic deaf cases. This large mutation screen revealed numerous novel sequence variations including three potential pathogenic mutations in the TMPRSS5 gene. The mutant forms of TMPRSS5 showed reduced or absent proteolytic activity. Subsequently, TMPRSS genes with evidence of involvement in deafness were further characterized, and their sites of expression were determined. Tmprss1, 3, and 5 proteins were detected in spiral ganglion neurons. Tmprss3 was also present in the organ of Corti. TMPRSS1 and 3 proteins appeared stably anchored to the endoplasmic reticulum membranes, whereas TMPRSS5 was also detected at the plasma membrane. Collectively, these results provide evidence that TMPRSS1 and TMPRSS3 play and TMPRSS5 may play important and specific roles in hearing.

In wireless personal area network, a channel is shared by a number of nodes. Therefore packet collision may take place and it degrades the throughput performance. To improve the throughput of slotted CSMA/CA in IEEE 802.15.4, we propose knowledge-based exponential backoff (KEB) scheme for enhancing channel utilization solely based on the locally available channel state information. In addition, we propose an analytical model of slotted channel access mechanism with finite retry and derive the theoretical throughput limit. In simulation experiments, we show that the existing MAC scheme, binary exponential backoff (BEB) scheme, operates very far from the theoretical limits due to increased time for negotiating channel access. Also performance results indicate that KEB shows significant improvement in throughput performance over BEB with the knowledge of the network status.

The function of the frizzled (fz) locus is required to coordinate the cytoskeletons of pupal epidermal cells so that a parallel array of cuticular hairs and bristles is produced. We report here the molecular cloning and characterization of the fz locus. The locus is very large. Mutations that inactivate the gene are spread over 100 kb of genomic DNA. The major mRNA product of the gene is a 4-kb RNA that is encoded by 5 exons spread over more than 90 kb of genomic DNA. Conceptual translation of this mRNA indicates that it encodes an integral membrane protein that is likely to contain both extracellular and cytoplasmic domains.

1. The aim of the present study was to examine the acute and chronic effects of adenoviral gene transfer on cardiac function in terms of left ventricular (LV) mechanoenergetic function. Recombinant adenoviral vector carrying beta-galactosidase and green fluorescent protein genes (Ad.betagal-GFP) was used. Cardiac function was examined in cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relationships (ESPVR/EDPVR), systolic pressure-volume area (PVA), LV relaxation rate, equivalent maximal elastance at mid-range LV volume (eE(max) at mLVV), coronary blood flow, coronary vascular resistance and myocardial oxygen consumption (VO(2)) were also measured. 2. To examine the ex vivo acute effects of the adenoviral vector, data were obtained before and 30-90 min after intracoronary infusion of Ad.betagal-GFP in the excised, cross-circulated hearts that underwent serotonin pretreatment. To examine the in vivo chronic effects of adenoviral gene transfer, normal rat hearts received Ad.betagal-GFP or saline by a catheter-based technique and data were obtained 3 days after the injection of Ad.betagal-GFP or saline. 3. The ESPVR, EDPVR, LV relaxation rate, eE(max) at mLVV, coronary blood flow and coronary vascular resistance remained unchanged in Ad.betagal-GFP-transfected hearts in both ex vivo acute and in vivo chronic experiments. Moreover, the ex vivo and in vivo transfection caused no change in the slope and VO(2) intercept of the VO(2)-PVA relationship, VO(2) for basal metabolism and for Ca(2+) handling in excitation-contraction coupling and O(2) costs of LV contractility. 4. These results indicate that adenoviral gene transfer has neither acute nor chronic toxic effects on LV mechanical and energetic function. A special combination of in vivo adenoviral gene transfer and a cross-circulation experimental system may provide a useful novel strategy to explore the functional and mechanoenergetic role of specifically targeted genes in the diseased heart.

A wireless sensor network consists of many micro-sensor nodes distributed throughout an area of interest. Each node has a limited energy supply and generates information that needs to be communicated to a sink node. The basic operation in such a network is the systematic gathering and transmission of sensed data to a base station for further processing. During data gathering, sensors have the ability to perform in-network aggregation (fusion) of data packet routes to the base station. The lifetime of such a sensor system can be defined as the time during which the sensor information is gathered from all of the sensors and combined at the base station. Given the location of the sensors, the base station and the available energy at each sensor, the main interest is to find an efficient manner in which data can be collected from the sensors and transmitted to the base station at a given rate, so as to maximize the system lifetime. A zone based data aggregation scheduling scheme is presented to accomplish this. The experimental results demonstrate that the proposed protocol significantly outperforms other methods in terms of the energy saving and system lifetime.

This paper describes the design and implementation of an IPv6 multicast forwarder as the preliminary step for developing a high-speed IPv6 multicast-enabled router for use in an environment where multicast packets occupy some portion of the total network traffic using the programmable IXP2400 network processor. We validate our implementation and evaluate its performance with hardware experiments. Our experimental results show that the measured forwarding rate is 86% of full line rate in the minimum IPv6 packet size and the latency of IPv6 multicast packet forwarding compared with IPv6 unicast increases 25% in average. These are due to the processing overhead of IPv6 multicast route lookup in IPv6 multicast forwarder microblock, the packet copy in packet replication microblock, and the packet buffer management in packet TX microblock

The aim of this study was to examine whether short- and long-term gene transfer of Ca 2+ handling proteins restore left ventricular (LV) mechanoenergetics in aortic banding-induced failing hearts. Aortic-banded rats received recombinant adenoviruses carrying sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) (Banding + SERCA), parvalbumin (Banding + Parv) or β-galactosidase (Banding + βgal), or an adeno-associated virus carrying SERCA2a (Banding + AAV.SERCA) by a catheter-based technique. LV mechanoenergetic function was measured in cross-circulated hearts. “Banding”, “Banding + βgal” and “Banding + saline” groups showed lower end-systolic pressure at 0.1 ml intraballoon water (ESP 0.1 ), higher end-diastolic pressure at 0.1 ml intraballoon water (EDP 0.1 ) and slower LV relaxation rate, compared with “Normal” and “Sham”. However, “Banding + SERCA” and “Banding + Parv” showed high ESP 0.1 , low EDP 0.1 and fast LV relaxation rate. In “Banding”, “Banding + βgal” and “Banding + saline”, slope of relation between cardiac oxygen consumption and systolic pressure–volume area, O 2 cost of total mechanical energy, was twice higher than normal value, whereas slope in “Baning + SERCA” and “Banding + Parv” was similar to normal value. Furthermore, O 2 cost of LV contractility in the 3 control banding groups was ∼ 3 times higher than normal value, whereas O 2 cost of contractility in “Banding + SERCA”, “Banding + AAV.SERCA” and “Banding + Parv” was as low as normal value. Thus, high O 2 costs of total mechanical energy and of LV contractility in failing hearts indicate energy wasting both in chemomechanical energy transduction and in calcium handling. Improved calcium handling by both short- and long-term overexpression of SERCA2a and parvalbumin transforms the inefficient energy utilization into a more efficient state. Therefore enhancement of calcium handling either by resequestration into the SR or by intracellular buffering improves not only mechanical but energetic function in failing hearts.

Recently issued standard IEEE 802.15.4 is a novel standard to achieve some critical features of wireless sensor networks. In this paper, we propose the algorithm to allocate GTSs according to the packet arrival rate from end devices and the number of devices in beacon enabled mode of IEEE 802.15.4 star topology networks. From simulation results, we show that our proposed algorithm enhances throughput comparing with the original IEEE 802.15.4 that uses only CAP duration for transmission.

It is a challenge to prototype network applications such as NAT that needs compute-intensive packet header processing while keeping the line speed on programmable network processors. In this paper, we design, implement, and evaluate a NAT subsystem capable of run-time adaptation on an experimental board containing a pair of Intel IXP2400 network processors, which operates in switch-over mode (NAT or NAPT) based on the fullness of the available global addresses or user configuration. We evaluate and validate our system through simulations and hardware experiments. It is found that the bottleneck of the system is due to the DRAM access latency. Also, we demonstrate that our NAT subsystem can support more than five hundreds of thousands of concurrent TCP/UDP sessions and sustain the full line rate on two Gigabit Ethernet links. Our experimental results and architecture can contribute to the other designs and implementations of network services over programmable network processors since they have similar architectures, functionalities and components.

Network Mobility (NEMO) is concerned with managing the mobility of an entire network and included one or more Mobile Routers (MRs) which are connected as gateways to the Internet. This paper proposes the optimal handoff decision mechanisms, not only avoiding the ‘ping-pong effect’ and frequent handoffs, but also reducing handoff latency under multi-hop network mobility. The simulation results demonstrate that the proposed method is well adapted for supporting network mobility over traditional handoff decision algorithms.

Network Mobility (NEMO) is concerned with managing the mobility of an entire network and included one or more Mobile Routers (MRs) which are connected as gateways to the Internet. This paper proposes a mechanism for route optimization in nested NEMO by using local source routing with uni-direction tunneling. Our scheme focuses on minimizing the number of tunnels required outside the NEMO when there are multiple levels of nesting. The simulation results demonstrate that the proposed scheme is well adapted for supporting route optimization over existing NEMO’s bi-directional tunneling scheme.

In recent years, Wireless Local Area Networks (WLANs) have become extremely popular. IEEE 802.11 protocol is the dominating standards for WLANs employing the Distributed Coordination Function (DCF) as its Medium Access Control (MAC) mechanism. In the literature, several papers have stuided performance of IEEE 802.11 protocol and assume that a station always have a packet available for transmission. This paper presents a Markov chain model to compute IEEE 802.11 DCF performance taking into account the packet arrival rates. Throughput and packet delay analysis are carried out in order to study the performance of IEEE 802.11 DCF under various traffic conditions. We present the analytical results of throughput and packet delay through network size and arrival rates. Results indicate that throughput and packet delay depends on the number of stations and packet arrival rates.

Engineering of secreted protease variants exhibiting altered substrate specificity is a challenging task because effective screening methods for the desired property are not available yet. In this study, we sought to obtain variants of Kex2, a yeast Golgi protease, which exhibit altered P2 specificity. We first randomly mutated three Asp residues (D176, D210, and D211) that constitute the S2 pocket of Kex2 and then isolated from the resulting library Kex2 variants that preferred substrates with Met (poorly preferred by wild type Kex2) at the P2 position using a yeast-based screening method. The Kex2 variants isolated from this initial screening were further tested against various substrate sequences. Four out of the 16 isolated Kex2 variants showed greater preference for Met than for Lys (preferred by the wild-type Kex2) at the P2 position. We therefore suggest that our method might serve as an efficient tool for engineering and directing the evolution of secreted proteases.

To obtain greater insight into atrial remodeling at the molecular level we analyzed the changes in gene expression in human atrial tissue between patients with chronic atrial fibrillation (AF) and those with normal sinus rhythm (NSR). cDNA microarray analysis was used to identify genes differentially expressed during sustained AF of more than 6 months (n = 9, mean age, 45 +/- 12, 6 males and 3 females) as compared to those with NSR (n = 9, mean age, 47 +/- 13, 6 males and 3 females). Western blot analysis was performed to confirm the altered gene expression and to establish the changes in protein expression. DNA gel electrophoresis to establish DNA ladder formation, which was associated with apoptosis in response to chronic AF, was performed. Microscopic findings were observed via electron microscopy. In the microarray analysis, out of 8,167 candidate genes, 66 genes showed a significant change in the expression level in the patients with chronic AF, which was in contrast to those with NSR. Among those, 31 genes were consistently down-regulated and 35 up-regulated more than 2-fold. The relative amounts of the Bcl-2 and p27 in the atrial tissue were decreased and angiotensin II type 2 (AT2) receptor and p21 were increased in the patients with chronic AF as compared to those with NSR. The atrial cardiomyocytes in chronic AF showed a prominent DNA ladder, which is a biochemical hallmark of apoptosis. The expression of Bcl-2, AT2 receptor, p21, and p27 were consistent with a significant role in the apoptosis of cardiac myocytes in the patients with chronic AF.

We have identified TMPRSS6, a novel type 2 transmembrane serine protease. TMPRSS6 possesses all the signature motifs of the family of transmembrane serine proteases (TMPRSSs), including a transmembrane domain, an LDL receptor class A (LDLRA) domain, a scavenger receptor cysteine-rich (SRCR) domain, and a serine protease domain. The substrate specificity of TMPRSS6 is slightly different from those of other TMPRSS family members. Combined with the finding that TMPRSS6 is expressed strongly in the thyroid and weakly in the trachea, this may indicate that TMPRSS6 has a specialized role.

Recent demands for real time applications have given rise to a need for QoS in the Internet. DiffServ is one of such efforts currently being developed by IETF. In a DiffServ network, previous researchers have proposed several marking mechanisms that are adapted to operate in intra-domain rather than in multi-domain. In this paper, we propose an enhanced marking mechanism based on a color-aware mode using the conventional tswTCM for flows crossing one or more DiffServ domains in order to guarantee effectively enhanced end-to-end QoS. The key concept of the mechanism is that the marking is carried out adaptively based on the initial priority information stored at the ingress router of the first DiffServ domain and the average queue length for promotion of packets. By statistics based on simulations, we show that the proposed marking mechanism improves an end-to-end QoS guarantee for most cases.

In this paper, the contact force between two colliding bodies is modeled by using Hertz’s force-displacement law and nonlinear damping function. In order to verify the appropriateness of the proposed contact force model, the free drop type impact test is carried out for different impact velocities and different materials of the impacting body, such as rubber, plastic and steel. The drop type impact experiment are installed to measure the velocity before impact more accurately, which verify the characteristics of contact force model. The parameters of the contact force model are estimated using the optimization technique. Finally the estimated parameters are used to predict the impact force between two colliding bodies in opening action of the spring actuated linkage system, a kind of switch mechanism for switching electric circuits.Copyright © 2005 by ASME

A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca(2+) release process during muscle contraction. The histidine-rich Ca(2+)-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5-20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes.

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.

Identifying cellular substrates repertoire of individual proteases will facilitate our understanding of their physiological and pathological roles. In this article, we employed a yeast-based screening method to isolate CED-3 substrates. This method uses a transcription factor anchored to the plasma membrane by fusion to a library of cellular protein sequences. When a fusion protein is cleaved by CED-3, the transcription factor is released from the plasma membrane and enters the nucleus where it turns on the expression of reporter genes. We identified seven candidate clones by screening a genomic library using this method. Of these seven clones, two were cleaved by purified CED-3 in vitro. Therefore, the method described here may be generally used for genomewide screening to isolate potential substrates of specific proteases.

The interactions between C99, presenilin, and nicastrin were investigated by a split-ubiquitin assay. We found that C99 homodimerizes and binds weakly to presenilin and strongly to nicastrin. Domain mapping assays revealed the transmembrane and cytoplasmic carboxy-terminal region of C99 is sufficient for the dimerization of C99 and the interaction between C99 and nicastrin. The extracellular domain of C99 is responsible for binding to presenilin. Nicastrin bound to C99 via its transmembrane domain and carboxy-terminal region. These observations suggest that dimerized (or oligomerized) C99 directly interacts with presenilin, and that this interaction is facilitated by nicastrin.

The recent advancement of wireless communication technology provides us with new opportunities, but it initiates new challenges and demands as well. Especially, there is an increasing demand for supporting user mobility and service mobility transparently. In this paper, we propose a new mechanism for service migration based on sensor networks of wireless sensor nodes. This mechanism is composed of a component-based server-side model for service mobility and Mobile IP technology for supporting user mobility among sensor networks.

We report here a genetic assay suitable for detecting site-specific proteolysis in secretory pathways. The yeast enzyme invertase is linked to the truncated lumenal region of the yeast Golgi membrane protein STE13 via a protease substrate domain in a Saccharomyces cerevisiae strain lacking invertase. When the substrate is cleaved by a specific protease, the invertase moiety is released into the periplasmic space where it degrades sucrose to glucose and fructose. Therefore, site-specific proteolysis can be detected by monitoring the growth of yeast cells on selective media containing sucrose as the sole carbon source. We confirmed the validity of this assay with yeast Kex2 and human TMPRSS2 proteases. Our data suggest that this in vivo assay is an efficient method for the determination of substrate specificity and mutational analysis of secreted or membrane proteases.

Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of beta-lapachone (beta-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD(+) level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of beta-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of beta-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, beta-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. beta-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, beta-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that beta-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. beta-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy.

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.

The RNA genome of hepatitis G virus (HGV) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. The HGV NS3 protease is responsible for the cleavage of the HGV polyprotein at four different locations. No conserved sequence motif has been identified for the cleavage sites of the NS3 protease. To determine the substrate specificity of the NS3 protease, amino acid sequences cleaved by the NS3 protease were obtained from randomized sequence libraries by using a screening method referred to as GASP (Genetic Assay for Site-specific Proteolysis). Based on statistical analyses of the obtained cleavable sequences, a consensus substrate sequence was deduced: Gln-Glu-Thr-Leu-Val downward arrow Ser, with the scissile bond located between Val and Ser. The relevance of this peptide as a cleavable substrate was further supported by molecular modeling of the NS3 protease. Our result would provide an insight on the molecular activity of the NS3 protease and may be useful for the design of substrate-based inhibitors.

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.

Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy. We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease. A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence. In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved. This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media. Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/Pro)-Cys / (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined. This is largely consistent with the previous results obtained by biochemical methods. An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease. These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.

HRC (histidine-rich Ca2+ binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ with low affinity and high capacity that is reminiscent of calsequestrin. The physiological role of HRC is largely unknown. In this study, we show that HRC exists as a multimeric complex (probably larger than a pentamer) under physiological conditions. At higher Ca2+ concentrations, the complex appeared to dissociate into dimers or trimers that form a more relaxed structure. This is in striking contrast to the characteristics of calsequestrin. An earlier immuno-electron microscopic study showed that HRC resides in the lumen of the sarcoplasmic reticulum (SR), but this conclusion has been challenged by other data. By tryptic digestion and biotinylation of SR vesicles, we provide compelling evidence showing that HRC is indeed present in the lumen of the SR.

The frizzled (fz) gene is required for development of planar tissue polarity in the epidermis of Drosophila melanogaster; it likely encodes an integral membrane protein that functions as a receptor for a tissue polarity-signaling molecule. On the basis of hydropathy analyses, it has been postulated that Fz protein contains seven transmembrane domains. Consistent with that model, the amino terminus of Fz was found to be extracellular, whereas the carboxyl terminus was intracellular. In the present study, the membrane topology of Fz was further examined in vivo by constructing various fz-suc2 fusion genes and analyzing their invertase activity in transformed yeast. We observed that plating efficiency and growth rate on sucrose media, as well as invertase secretion, were consistent with the proposed seven-pass model of Fz. This study provides the first experimental assessment of overall Fz topology.

The related Drosophila Suppressor 2 of zeste [Su(z)2] and Posterior sex combs (Psc) proteins are both locus-specific chromosome binding proteins. They are found at many of the same polytene chromosome loci as other Polycomb-group proteins. The 1,365 amino acid Su(z)2 protein and the 1,603 amino acid Psc protein share a conserved 200 amino acid domain, the homology region (HR). To identify the protein domain responsible for locus-specific chromosome binding, we made a series of Hsp70:cDNA deletion constructs of the Sz(z)2 gene and transformed these into flies. We found that the HR is necessary and sufficient for Su(z)2 locus-specific polytene chromosome binding. The murine Bmi-1 protein also shares the conserved HR domain. When expressed in flies, the Bmi-1 protein showed a locus-specific chromosome binding pattern similar to that of the Su(z)2 and Psc proteins. These results argue that a locus-specific chromosome binding function resides in the HR domain. Other results show that a second, low affinity, non-specific chromosome binding function is localized outside the HR in the Su(z)2 protein, and that the Su(z)2 protein contains at least two nuclear localization signals.

Autocatalytic processing mediated by the carboxy-terminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing in biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Mutations have been reported for several craniosynostotic disorders in exon IIIa (exon U or 7) or IIIc (exon B or 9) of the fibroblast growth factor receptor 2 gene (FGFR2). Among the conditions with FGFR2 mutations are two autosomal dominant syndromes, Crouzon and Jackson-Weiss. In this study, 24 Crouzon and one Jackson-Weiss syndrome patients were screened for mutations in the two exons by direct sequencing, and mutations were detected in 28% (7/25) of all cases. Five different mutations were found including two novel (W290G, C342W) and two previously reported, recurrent mutations for Crouzon syndrome (A344A, S354C), and one new mutation for Jackson-Weiss syndrome (C342R). The W290G mutation was found in exon IIIa which is common to both alternatively spliced forms of FGFR2, BEK (expressed predominantly in primordial bones) and KGFR (expressed preferentially in epithelia). Atypical Crouzon syndrome features of epithelial-derived anal and/or external ear anomalies were present in the two affected family members with the mutation. This phenotype possibly reflects the expression of both mutant BEK and KGFR. In addition, the Jackson-Weiss syndrome mutation, C342R, in exon IIIc was observed previously in other craniosynostotic syndromes, Crouzon and Pfeiffer. These results underscore the allelic heterogeneity of these conditions and the complexity of the phenotypic consequences of FGFR2 mutations.

HRC is a high capacity Ca2+ binding protein located in the lumen of sarcoplasmic reticulum (SR). Previously, we had shown that HRC knock-down not only led to enhanced Ca2+ release and Ca2+ uptake in SR, but exacerbated cardiac function after transverse aortic constriction (TAC), suggesting its important role in Ca2+ cycling and cardiac function. HRC-KO mice were generated and used for the present study. Pressure overload induced by TAC in the KO mice heart resulted in severe cardiac hypertrophy with 27% increase of heart weight (HW) per body weight (BW) ratio and 25% increase of tibia length per BW ratio as compared to WT TAC mice. HRC KO mice also showed decreased fractional shortening (FS) by 37%, increased TGF-β expression by 2 folds, severe cardiac fibrosis and highly increased number of TUNEL positive signals in heart tissue compared to WT TAC mice. The electrocardiogram (ECG) study showed shorter RR interval, faster heart rate and decreased R amplitude in HRC KO TAC mice. The incidence of arrhythmia was significantly increased in HRC KO mice after intraperitoneal injection of caffeine (120 mg/kg BW) and epinephrine (2 mg/kg BW),indicating that HRC KO mice are more susceptible to epinephrine and caffeine injection, consistent with the previous report (Jaehnig et al. Mol. Cell. Biol., 2006). The survival rate of HRC KO mice was significantly decreased after TAC. Taken together, our results suggest that ablation of HRC could lead to altered SR Ca2+ cycling and deterioration of cardiac function under pathological condition. (Supported by Korea MEST NRF Grant (20110002144), the 2011 GIST System Biology Infrastructure Establishment Grant and KISTI-KREONET).

The adult cuticular wing of Drosophila is covered with an array of distally pointing hairs. Mutations in the inturned (in) gene result in both abnormal hair polarity (i.e., hairs no longer point distally), and, in most cells forming more than one hair. We have isolated and characterized a collection of in alleles. Among this collection of alleles are a number of rearrangements that enable us to assign in to 77B3-5. Almost all of the in alleles, including putative null alleles, result in a stronger phenotype on the wing at 18 degrees than 29 degrees. The data argue that the in-dependent process is cold-sensitive. Temperature shift experiments with a hypomorphic allele show that this cold sensitivity can be relieved by several hours of incubation at the permissive temperature at a variety of times in the early pupae, but that this ability ends prior to the start of hair morphogenesis. One new allele showed a dramatic heat sensitivity. Temperature shift experiments with this allele revealed a very short temperature-sensitive period that is a few hours prior to the start of hair morphogenesis. That the temperature during hair morphogenesis is irrelevant for the phenotype of in is consistent with the hypothesis that the only role that in has in wing hair development is to regulate the initiation of hair morphogenesis.

The protein encoded by the Drosophila tissue polarity gene, frizzled (fz), is required for both the intercellular transmission and the intracellular transduction of a tissue polarity signal. In order to study the biochemical characteristics of this rare protein, we constructed a hs-fz fusion gene and transferred this to Drosophila tissue culture cells and embryos. Cell fractionation experiments and immunostaining experiments showed that the Fz protein is an integral membrane protein containing an odd number of transmembrane domains, consistent with previous suggestions that it contains seven transmembrane domains. Immunostaining of pupal wings showed that the Fz protein is evenly distributed throughout the wing arguing that the Fz protein does not act as a graded morphogen.

Almost every cell in the Drosophila pupal wing forms a single, distally pointing cuticular hair. The function of the frizzled (fz) gene is essential for the elaboration of the normal wing hair pattern. In the absence of fz function hairs develop, but they display an abnormal polarity. We have examined the developmental expression of the fz gene at the RNA level via in situ hybridization and at the protein level via Western blotting. We have found that fz is expressed in all regions of the epidermis before, during, and after the fz cold sensitive period. We have also found that fz function is not required for normal fz expression. We have further found that mutations in several other tissue polarity genes do not noticeably alter the expression or the modification state of the Fz protein.

HRC is a SR luminal protein that binds to both triadin and SERCA, and affects Ca2+ cycling in the SR (Arvanitis et al. Am. J. Physiol. Heart. Circ. Physiol. 293: H1581, 2007). In the present study, we attempted to characterize the functions of HRC by AAV-mediated KD using TAC-HF model. We expected that HRC KD could recover cardiac function in failing heart (FH), because HRC KD by itself could enhance Ca2+ uptake and Ca2+ release by increased activities of SERCA2 and RyR2 in HL-1 cells. Unexpectedly, AAV9-mediated HRC KD in TAC-FH showed decreased fractional shortening and increased cardiac fibrosis compared with control TAC-FH. We found that phospho-RyR2, phospho-CaMKII, phospho-p38MAPK and phospho-PLB Thr17 were significantly up-regulated in HRC-KD TAC-FH. The cardiac cell death markers such as LC3A/B and active caspase 9 were also increased, consistent with our results of TUNEL assay. Collectively, the increased cytosolic Ca2+ level could activate CaMKII and hence phosphorylation of p38MAPK causing the enhanced mitochondrial death pathway in TAC-FH. Our results show evidence that down-regulation of HRC is linked to deterioration of cardiomyocytes in the pathological state. (Supported by Korea NRF Grant (2010-0002159), GIST Systems Biology Infrastructure Establishment Grant (2010) and KISTI-KREONET (2010).

Purpose: We evaluated the outcome of laparoscopic retroperitoneal ureterolithotomy (LRU) and compared the results with the rigid ureteroscopic removal of stones with the LithoclastR (rigid URS) for the management of large upper ureteral stones (≥10 mm). Materials and Methods: Between July 2002 and March 2008, rigid URS and LRU were performed in 37 and 24 patients, respectively. We evaluated the outcomes of each procedure and compared the success rate according to the location (above and below the L3 level by the third intervertebral disc of the lumbar spine) and size of the stones (10-15 mm and ≥15 mm in diameter). Results: The overall success rate for rigid URS and LRU were 70.3% (26/37) and 91.7% (22/24), respectively (p=0.059). For rigid URS, the success rate was 50.0% (8/16) and 85.7% (18/21) for stones above and below the L3 level (p=0.030), respectively, and 85.7% (23/28) and 33.3% (3/9) for stones 10-15 mm and ≥15 mm in diameter, respectively (p=0.011). For LRU, the success rate was 92.3% (12/13) and 90.9% (10/11) for stones above and below the L3 level, respectively (p=0.902), and 50.0% (1/2) and 95.5% (21/22) for stones 10-15 mm and ≥15 mm in diameter, respectively. Conclusions: LRU demonstrated a high success rate regardless of the location and size of the stones. The outcomes with rigid URS were more varied. These results suggest that LRU is a feasible alternative for large upper ureteral stones that are 15 mm or more in size or located above the intervertebral disc between the third and fourth lumbar vertebrae. (Korean J Urol 2009;50:349-354) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

Byung Hwan Kim, Jae Ho Kim, Woo Jin Park, Dong Soo Ryu, Jun O Kwon, Tae Hee Oh From the Department of Urology, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, Korea From February 2004 to August 2005, 3 patients with muscle invasive bladder adenocarcinoma were identified as candidates for partial cystectomy, and they underwent laparoscopic partial cystectomy. Case 1 and case 2 were primary bladder adenocarcinoma with a pathologic stage of T3aN0M0 and T2bN0M0, respectively, and case 3 was metastatic bladder adenocarcinoma from gastric cancer. The mean surgical time was 213 minutes (range: 140-300). The blood loss was 117cc (range: 60-220), respectively. There were no significant complications after surgery. During a mean follow-up period of 22 months, case 1 and case 2 with primary adenocarcinoma did not have local or systemic recurrence, but case 3 with metastatic adenocarcinoma had intra-abdominal recurrence without local recurrence. Laparoscopic partial cystectomy is a safe, feasible, minimally invasive alternative to open partial cystectomy for treating selected cases of patients with muscle invasive bladder adenocarcinoma. (Korean J Urol 2007;48:990-993)