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In this paper, the immune algorithm (IA) is proposed to derive the rephasing strategy arrangement of laterals and distribution transformers to enhance three phase balancing of distribution systems. The multi-objective function is formulated by considering the unbalance of phasing currents, the customer service interruption cost and labor cost to perform the optimal rephasing strategy. To demonstrate the effectiveness of the proposed methodology to enhance three-phase balancing of distribution systems, a practical distribution feeder in Taipower with 523 customers is selected for computer simulation. By minimizing the objective function subjected to the operation constraints, the rephasing strategy has been derived by selecting the laterals and distribution transformers for phasing adjustment. The phase currents and neutral currents of test feeder before and after the execution of proposed rephasing strategy have been measured and collected by the SCADA system in Taipower. It is found that the neutral current of test feeder has been reduced to be less than the over neutral current limit by executing the rephasing of laterals and distribution transformers.

The main focus of this study is to develop a poultry litter decision support system (PLDSS) integrated in a geographical information system (GIS) for land application and off-farm usage of poultry litter in the Appalachian Plateau and Black Belt regions of Alabama. The PLDSS is a comprehensive user-friendly system that consists of transportation and assessment tools for poultry litter management in Alabama. The system is developed by programming the Visual Basic for Applications (VBA) with ArcObjects in ArcGIS. Important functional components and analytical capability of ArcGIS 9.1 are implemented in several sets of customized and user friendly tools. The system works on three levels. The first develops a comprehensive nutrient management plan (CNMP), the second performs the transport network analysis and the third evaluates travel cost associated with transportation. The CNMP plan is based on the methodology developed by Alabama Cooperative Extension System (ACES). The transportation analysis allows estimation of the best route, the closest route to fields within certain distance, and Origin -Destination matrix by using Network Model in ArcGIS. The assessment module performs the estimation of travel cost and other transportation analyses. Models are integrated in a GIS environment, which serves as the repository of the data as well as the user interface. Overall, the PLDSS will help provide an optimal, state-wide management of poultry litter, which is crucial for sustaining the poultry industry in Alabama. It will also help row crop agriculture and beef cattle industry to grow in the Black Belt region.

A systematic procedure is proposed to study the effect to temperature change to distribution feeder load profiles and losses by using the typical load patterns of customer classes. The database of an automated mapping/facility management (AM/FM) system is used to retrieve the component attributes and the topology process is executed to determine the electrical network configuration and the customers served by each distribution transformer. By using the monthly energy consumption of customers in customer information system (CIS) and the typical daily load patterns of customer classes, the hourly loading profiles of distribution transformers and service zones can be derived to solve the loadings of each primary feeder and lateral. The sensitivity analysis of load demand with respect to the temperature change for each customer class is performed by statistic regression according to the actual customer power consumption and temperature data. The load contribution by each customer class is updated by the corresponding temperature sensitivity and integrated together to form the new load profile of a service district with temperature change. To investigate the temperature effect to the distribution feeder, two of the Taipower distribution feeders are selected for computer simulation. The power demand at each load bus of the distribution feeder is calculated by applying the temperature sensitivity and the three- phase load flow analysis is then executed to find the new feeder loading and power loss with the temperature change.

The objective of this study was to isolate and identify lactic acid bacteria able to inhibit the in vitro formation of Streptococcus mutans biofilm as well as the in vivo formation of oral biofilm. Two strains, CMS1 and CMS3, exhibiting profound inhibitory effects on the formation of S. mutans biofilm and the proliferation of S. mutans, were isolated from children's saliva and identified as Weissella cibaria by 16S rDNA sequencing. The water-soluble polymers produced from sucrose by the W. cibaria isolates also inhibited the formation of S. mutans biofilm. According to the results of thin-layer chromatographic analysis, the hydrolysates of water-soluble polymers produced by the isolates were identical to those of dextran, forming mostly alpha-(1-6) glucose linkages. In the clinical study, the subjects mouthrinsed with a solution containing W. cibaria CMS1 evidenced plaque index reduction of approximately 20.7% (p0.001). These results indicate that the W. cibaria isolates possess the ability to inhibit biofilm formation, both in vitro and in vivo.

Highway culverts are installed to safely drain the design peak runoff and the dimensions are to be determined based on the estimated design peak runoff and hydraulic computations. The critical storm duration and peak discharge according to the rainfall distribution type, the position of peak rainfall intensity, and frequency were evaluated by seven hydrological models. The hydrological models were tested with field data measured from two small watersheds. The hydrological parameters were defined using the GIS data of the watersheds. And the results from different peak runoff equations and hydrological models were found that the simulated for runoff hydrographs were comparative to the observed hydrographs. Hydraulic designs of highway culverts, thirty five drainage watersheds along renovating segments of Highway No. 39 in Korea, were studied to define the optimum dimensions and hydraulic variables for the design peak runoff. This study may also be applicable to the design of highway drainage systems for areas with future and postulated land uses and new development.

Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets ofmicrosatellite markers of flounder. Optimal growth temperature was 20 degrees C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses.

To evaluate the degree of competitive exclusion against Salmonella gallinarum(S. gallinarum) of Salmonella enteritidis(S. enteritidis) infected chickens, fifty-six, 4-week old Hyline layer suspected of S. enteritidis infection were challenged with S. gallinarum. All chickens were tested for S. enteritidis isolation using cloacal swabs and serum plate agglutination test using S. enteritidis Ag. before challenge and classified into four groups(SE isolated, SE nonisolated, SE seropositive and SE seronegative). None of the SE isolated and the SE seropositive groups died after challenge and the average weight gains were 245.5g and 254.6g, respectively. But in the SE nonisolated and the SE seronegative groups, mortality was 18.2% and 20.6% and the average weight gains were 150.1g and 111.2g. The incidence of reisolation of S. gallinarum of the SE isolated and the SE seropositive groups were 41.7% and 47.6% from liver, 33.3% and 47.6% from spleen and 8.3% and 14.3% from cecum, respectively, and the SE nonisolated and the SE seronegative group were 63.6% and 64.7% from liver, 84.1% and 88.2% from spleen and 47.7% and 52.9% from cecum. The serological response of the SE isolated and the SE seropositive groups hardly changed from 75.0 and 81.8% before challenge to 75.0 and 85.7% after. But, the other two groups were found to be significantly higher after challenge and increased from 0 and 18.2% to 100%. Consequently, S. enteritidis preinfected chickens were found to be significant different in terms of mortality, weight gain, reisolation of S. gallinarum and serological response compared to noninfected chickens. Moreover, our study shows that S. enteritidis infected chickens appear strong competitive exclusion against the colonization of S. gallinarum.

It has been reported that abnormal steps associated with an ankle hyper-extension during walking were observed in adult rats after 2 weeks of hindlimb suspension (Canu and Falempin, 19971998). But such phenomena were normalized after 7 days of reambulation recovery. Canu and Falempin (1996) suggested that the spinal cord has a capacity to generate a well-organized pattern of locomotion even after a period of muscle disuse. There are, however, no reports about the effects of more prolonged suspension on motor performances. In the present study, 7 weeks old male rats were hindlimb-unloaded by tail suspension for 9 consecutive weeks and landing performances in response to drop from head-down, head-up, or supine position were investigated during 8 weeks of recovery. Posture maintenance during right-left translation was also checked.

The novel alleles DRB1*12:03:03 and DRB1*13:143 differ from DRB1*12:03:02 and DRB1*13:02:01 by one nucleotide in exon 2, respectively.

This paper investigates the load transfer function of the distribution automation project to be implemented in Taipower distribution system. During the summer peak season, the main transformers of distribution substations in Taipower become overloaded due to the usage of air conditioners in the commercial and residential sections. The noninterruptible load transfer among distribution main transformers has to be executed for power reallocation to solve the problem. In this paper, the hourly loading of each service zone in the distribution system is determined by the load composition of the customers served within the service zone. The hourly current flows of line switches are derived by simplified three-phase load flow analysis. Applying the binary integer programming (BIP) to solve the distribution system reconfiguration so that the load transfer among main transformers can be obtained derives the optimal switching operation. With the execution of the load transfer for Taipower distribution system by the proposed BIP method, the load balance among distribution main transformers during the summer peak hours is obtained. A Taipower distribution system with six feeders is selected for computer simulation to demonstrate the effectiveness of the proposed methodology.

Three cereal crops – rice, wheat and maize – feed the world. These crops supply 49% of the calories consumed by the world population; 23% come from rice, 17% from wheat and 9% from maize. Rice is the primary food source for more than a third of the world’s population. It is planted on almost 150 million ha annually or 11% of the world’s cultivated land. More than 90% of rice is produced and consumed in Asia. It is also an important staple in Latin America, Africa and the Middle East. Rice is grown under a wide range of agroclimatic conditions. Four major ecosystems are generally recognized: irrigated (55%), rain-fed lowland (25%), upland (12%) and floodprone (8%).

Introduction Local anesthetics combined with corticosteroids are commonly used for management of back pain in interventional spinal procedures. Several recent studies suggest cytotoxicity of bupivacaine, whereas others report protective and cytotoxic effects of corticosteroids on chondrocytes and intervertebral disc cells. Considering the frequent use of these agents in spinal interventions, it is meaningful to know how they affect intervertebral disc cells. This study was conducted to assess the effects of bupivacaine and triamcinolone, both alone and in combination, on viability of intervertebral disc cells in vitro. Materials and Methods Nucleus pulposus cells were isolated from human disc specimens from patients undergoing surgery due to disc herniation or degenerative disc disease. They were grown in three-dimensional alginate beads for 1 week to maintain their differentiated phenotypes and to allow for matrix formation before analysis. After 1 week of culture, the cells were exposed to bupivacaine (0.1, 0.25, 0.5, and 1%) or bupivacaine (0.1, 0.25, 0.5, and 1%) with 1 mg triamcinolone for 1, 3, or 6 hours. Cell viability was measured using trypan blue exclusion assay and flow cytometry. Live-cell/dead-cell fluorescent imaging was assessed using confocal microscopy. Results Trypan blue exclusion assays demonstrated dose- and time-dependent cytotoxic effects of bupivacaine on human nucleus pulposus cells. Similar but reduced cytotoxicity was observed after exposure to the combination of bupivacaine and 1 mg of triamcinolone. Flow cytometry showed a dose-dependent cytotoxic effect of bupivacaine on nucleus pulposus cells after 3 hours of exposure. The reduced cytotoxicity of bupivacaine combined with 1 mg triamcinolone was also confirmed in flow cytometry. Confocal images showed that the increase in dead cells correlated with the concentration of bupivacaine. Nevertheless, fewer cells died after exposure to several different concentrations of bupivacaine combined with 1 mg triamcinolone than did after exposure to bupivacaine alone. Conclusion The combination of bupivacaine and triamcinolone induced dose- and time-dependent cytotoxicity on human intervertebral disc cells in vitro, but the cytotoxicity was much weaker than that of bupivacaine alone. This study shows a potential protective influence of triamcinolone on intervertebral disc cells. Disclosure of Interest None declared References Hsiao CJ, Cherry DK, Beatty PC, Rechtsteiner EA. National Ambulatory Medical Care Survey: 2007 summary. Natl Health Stat Rep 2010; (27):1–32 Boswell MV, Trescot AM, Datta S, et al; American Society of Interventional Pain Physicians. Interventional techniques: evidence-based practice guidelines in the management of chronic spinal pain. Pain Physician 2007;10(1):7–111 Bogduk N. The lumbar disc and low back pain. Neurosurg Clin N Am 1991;2(4):791–806 Sehgal N, Fortin JD. Internal disc disruption and low back pain. Pain Physician 2000;3(2):143–157 Wetzel FT, LaRocca SH, Lowery GL, Aprill CN. The treatment of lumbar spinal pain syndromes diagnosed by discography. Lumbar arthrodesis. Spine 1994;19(7):792–800 Zucherman J, Hsu K, Picetti G III, White A, Wynne G, Taylor L. Clinical efficacy of spinal instrumentation in lumbar degenerative disc disease. Spine 1992;17(7):834–837 Choy DS, Ascher PW, Ranu HS, et al. Percutaneous laser disc decompression. A new therapeutic modality. Spine 1992;17(8):949–956 Onik G, Helms CA, Ginsburg L, Hoaglund FT, Morris J. Percutaneous lumbar diskectomy using a new aspiration probe. AJR Am J Roentgenol 1985;144(6):1137–1140 Wittenberg RH, Oppel S, Rubenthaler FA, Steffen R. Five-year results from chemonucleolysis with chymopapain or collagenase: a prospective randomized study. Spine 2001;26(17):1835–1841 Zhou Y, Abdi S. Diagnosis and minimally invasive treatment of lumbar discogenic pain—a review of the literature. Clin J Pain 2006;22(5):468–481

Introduction Ioxitalamate (Telebrix 300) is an ionic iodinated contrast medium (CM) commonly used for discography or PELD (percutaneous endoscopic lumbar discectomy), though it has side effects such as anaphylactic shock and renal toxicity. Indigocarmine is an organic compound dye with a distinctive blue color that is commonly used during PELD to stain the acidic, degenerated nucleus pulposus. Although ioxitalamate and indigocarmine are widely used in spinal surgery, there have been no reports on their effects on nucleus pulposus (NP) cells. We studied the toxicity of both ioxitalamate and indigocarmine to NP cells. Materials and Methods NP cells were obtained via discectomy from lumbar disc patients and isolated. NP cells were cultured in three-dimensional (3D) alginate beads with 0.001, 0.1, 10, and 100 mg/mL ioxitalamate, 0.00001, 0.001, 0.1 and 10 mg/mL indigocarmine, or a mixture of both for 1, 2, or 3 days. The living cells were analyzed with trypan blue staining. FACS analysis using Annexin V and propidium iodide (PI) and 3D alginate bead immunostaining was performed to identify live, apoptotic, and necrotic cells. Results Ioxitalamate, indigocarmine, and their combination induced statistically significant NP cell injury that was both time and dose dependent ( p < 0.05). Also, at the same concentration, ioxitalamate was more cytotoxic than was indigocarmine or the combination ( p < 0.05). All three treatments also showed dose-dependent cytotoxicity according to flow cytometry and immunostaining. Conclusion Ioxitalamate and indigocarmine are toxic to human NP cells in vitro in a time- and dose-dependent manner. We assume that ioxitalamate and indigocarmine may have similar effects in patients undergoing discography and PELD. Thus, we suggest that ioxitalamate and indigocarmine should be used carefully at low concentrations. Disclosure of Interest None declared References Stout A. Discography. Phys Med Rehabil Clin N Am 2010;21(4):859–867 Manchikanti L, Glaser SE, Wolfer L, Derby R, Cohen SP. Systematic review of lumbar discography as a diagnostic test for chronic low back pain. Pain Physician 2009;12(3):541–559 Carragee EJ, Tanner CM, Yang B, Brito JL, Truong T. False-positive findings on lumbar discography. Reliability of subjective concordance assessment during provocative disc injection. Spine 1999;24(23):2542–2547 Laslett M, Oberg B, Aprill CN, McDonald B. Centralization as a predictor of provocation discography results in chronic low back pain, and the influence of disability and distress on diagnostic power. Spine J 2005;5(4):370–380 Kim D, Wadley R. Variability in techniques and patient safety protocols in discography: a national multispecialty survey of International Spine Intervention Society members. J Spinal Disord Tech 2010;23(6):431–438 Carragee EJ, Tanner CM, Khurana S, et al. The rates of false-positive lumbar discography in select patients without low back symptoms. Spine 2000;25(11):1373-1380, discussion 1381 Paul MD, Tsou M. Posterolateral percutaneous endoscopic lumbar discectomy. Oper Tech Orthop 2003;13:207-213 Yeung AT. Selective endoscopic discectomy. In: Kambin P, ed. Arthroscopic and Endoscopic Spinal Surgery. Totowa, NJ: Humana Press; 2005:205–225 Mayer HM, Brock M. Percutaneous endoscopic discectomy: surgical technique and preliminary results compared to microsurgical discectomy. J Neurosurg 1993;78(2):216–225 Yeung AT, Tsou PM. Posterolateral endoscopic excision for lumbar disc herniation: Surgical technique, outcome, and complications in 307 consecutive cases. Spine 2002;27(7):722–731 Lee H, Sowa G, Vo N, et al. Effect of bupivacaine on intervertebral disc cell viability. Spine J 2010;10(2):159–166 Hsu SP, Tsai TJ, Chien CT. Ioxitalamate induces renal tubular apoptosis via activation of renal efferent nerve-mediated adrenergic signaling, renin activity, and reactive oxygen species production in rats. Toxicol Sci 2010;114(1):149–158 Lee HC, Chang JG, Yen HW, Liu IH, Lai WT, Sheu SH. Ionic contrast media induced more apoptosis in diabetic kidney than nonionic contrast media. J Nephrol 2011;24(3):376–380 Belhadjali H, Bouzgarrou L, Youssef M, Njim L, Zili J. DRESS syndrome induced by sodium meglumine ioxitalamate. Allergy 2008;63(6):786–787 Lacour B, Lacroix C, Jungers P, Bailly M. [Effects of intravenous urography using sodium and meglumine ioxitalamate (Telebrix 38) on blood creatinine and urinary excretion of N-acetyl-beta-D-glucosaminidase]. Ann Biol Clin (Paris) 1984;42(6):407–411

The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP. To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala. None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP. However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S. Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins. These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase.

In contrast to intrinsic drug resistance, induced multidrug resistance in gastric cancer cells has not been well studied. Therefore, two doxorubicin-resistant cell lines, (SNU-1DOX, SNU-16DOX), were derived in vitro from gastric carcinoma cell lines (SNU-1, SNU-16) respectively, and their characteristics were investigated. These resistances were not associated with overexpression of mdrl, multidrug resistance associated protein 1 (MRP1), pi or liver class of glutathione S transferase (GST pi, GSTL), heat shock protein 70 (HSP70), p53 or transglutaminase C (TGC). Levels of p21WAF1 RNA and topoisomerase II protein were decreased in the SNU-16DOX, but not in SNU-1DOX. However, the subsequent enzyme activity of topoisomerase II in SNU-16DOX was not decreased, but rather increased in SNU-16DOX. Furthermore, both resistant cell lines showed lower uptake and higher efflux of doxorubicin and induced cross-resistance to etoposide and vincristine in addition to doxorubicin, indicating a multi-drug resistance phenotype. In summary, we report two gastric carcinoma cell lines exhibiting induced multidrug resistance phenotype and suggest that mdrl, MRP1, GST, TGC, HSP70 and p53 do not play important roles in induced drug resistance in these cell lines. The role of changes in topoisomerase II activity and/or protein is still inconclusive, and p21WAF1 is associated with induced multidrug resistance in the SNU-16DOX gastric carcinoma cell line.

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.

It has been known that the incidence of p53 mutation is very rare in HBX-positive primary human hepatocellular carcinoma (HCC) tissues. The frequency of p53 mutation, however, in established cell lines with integrated HBV DNA and/or HBX has not been well studied. To know p53 mutational frequency, and to investigate whether the presence of HBX DNA sequence correlates with the absence of p53 mutation in the established HCC cell lines, we studied the p53 mutation and the presence of HBX sequence in 12 recently characterized HCC cell lines. As a result, all 12 (100%) lines showed mutation in the p53 gene. Three (25%) cell lines had transversion of codon 215 while no mutation of codon 249 was found. In contrast with previous reports, although HBX DNA was present in 11 cell lines, p53 mutation had occurred, indicating that the presence of HBX viral DNA does not correlate with a lack of p53 mutation in established HCC cell lines. Our results suggest that the frequency of p53 mutation is extremely high even in HBX DNA positive HCC cell lines.

The ATP-dependent protease Ti(Clp) consists of two different multimeric components: ClpA containing ATP-cleaving sites and ClpP, with serine active sites for proteolysis. Here we summarize the most recent results on the structure and function of protease Ti. (1) The clpA gene has dual translational initiation sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa. The abbreviated form of ClpA may play an important role in regulation of the ATP-dependent proteolysis, since it inhibits the ability of the 84-kDa ClpA in supporting the ClpP-mediated protein breakdown and the autodegradation of the 84-kDa ClpA. (2) ClpA contains two highly conserved sequences for ATP-binding: the first site is essential for oligomerization and the second site is responsible for ATP hydrolysis. (3) ATP hydrolysis by ClpA is required not only for assembly of the ClpA/ClpP complex but also for its rapid dissociation.

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

The pharmacokinetics of intravenous recombinant erythropoietin administered during or after dialysis were studied by multicompartmental analysis in eight patients with end-stage renal disease to determine whether significant loss of the drug occurred during the hemodialysis procedure. Each patient had five studies, one in which the erythropoietin was given near the end of a dialysis session (baseline). In the other studies, erythropoietin was administered at the beginning of hemodialysis for 4 hrs using each of four different dialysis membranes. Both two compartment and three compartment models were used. In the two compartment solutions, the mass of the exchange compartment was significantly increased in the studies done during dialysis compared with the baseline. Clearance did not change significantly among the study sessions. When a third compartment, representing the dialysis system, was added, the kinetics of the internal exchange compartment and of the dialysis system could be determined separately. At the end of 4 hrs of dialysis after the erythropoietin dose, the dialysis systems contained an average of 7% of the administered dose. It was concluded that hemodialysis immediately after erythropoietin administration does not affect the rate of erythropoietin loss, except for small losses in the dialysis system itself when it is removed at the termination of the procedure.

Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents. Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease Ci must be a zinc metalloprotease. Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed. Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain. Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate. Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain. However, it shows little or no activity against proteins larger than 15 kDa (e.g. ovalbumin and denatured bovine serum albumin). Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.

The subunit patterns of the proteasomes, that were purified from muscle, liver and brain, were found to be significantly different from one another. Furthermore, the proteasomes from adult and embryonic tissues of the same types also differed from each other in their subunit patterns. In addition, the specific activities of the purified proteasomes for peptide-cleavage, but not for casein-hydrolysis, appeared to be varied among the enzymes isolated from the different tissues. Thus, expression of a large number of proteasome subunits appears to be tissue-specific and under developmental control, although its relation with the multicatalytic activities of the proteasomes remains unclear.

The novel allele A*02:328 showed one nucleotide difference with A*02:06:01 in exon 3 resulting in an amino acid change at codon 120 from Gly to Arg.

308 female students from six State high schools in inner metropolitan Sydney responded to self-administered questionnaires on their sex behavior and contraceptive use. Respondents were in their tenth year of school and of mean age 15.6 years. The survey was conducted with no refusals over the period March-June 1991 upon the sample from four coed and two single-sex schools. 18% of the sample reported ever having sexual intercourse, although substantial interschool variation was observed; less than 5% of girls in one school with a predominantly non-English speaking population were sexually experienced, while almost 45% were experienced in another school with a predominantly Anglo-Saxon population. The mean age at first intercourse for the sexually active group was 14.5 years. 72% used condoms at first intercourse, while 17% used no form of contraception. There were no significant differences in the mean ages of sexually active and nonactive girls, nor between the percentage of sexually active girls in coed schools and in single-sex schools. 25 girls had ever had a total of one sex partner, 27 had 2-5, and two had more than five. The condom is the most commonly used form of contraception, with 38% of sexually active girls using condoms every time. The rest of the girls used them either occasionally or not at all. Oral contraceptive pills (OCP) were used by less than 15% of the sexually active group, with effective contraception overall used by only 43% of the group. Effective contraception is defined as either condom use during every act of sexual intercourse during the preceding six months or daily compliance with the OCP. Eight subjects used no contraception at all during the preceding six months. These findings indicate the need for more education targeted to adolescents and health professionals, especially general practitioners.

Putting is a crucial stroke to determine winning in golf. Putting is more delicate than any other strokes in golf. To keep better putting stroke, psychological factors as well as physical factors are important for golfers. The purpose of this study was to compare differences in kinematic and psychological variables between winner and loser of three skilled levels during competitive golf putting tournament. The participants consisted of 3 groups based on their skill levels: PG (8 professional golfers), RG (8 recreational golfers) and NG (8 novice golfers). Each group performed 8 tournaments by attempting 2.1 m putting from the hole. 3D motion analysis system with 6 high speed cameras and BIOPAC ECG module were used to acquire kinematic data and ECG. To compare differences between winner and loser for each group, time phase of putting stroke, smoothness by jerk cost function (JC), heart rate (HR), ratio of low and high frequency component (LF/HF) in heart rate variability and CSAI-2 scores (competitive state anxiety inventory 2) were used. There was a significant difference in back-swing phase time ratio between PG and other groups and in JC at the putter toe between NG and other groups. There was a significant difference in LF/HF and self-confidence score of CSAI-2 between winner and loser within PG. While PG and RG showed similar kinematic performance, they showed different LF/HF. The tonic states derived from cardiac activity indicated that a higher skilled level was associated with increased LF. Increased LF is associated with increased automaticity and decreased attention demands, which means that increased mental workload reduces LF. For PG, it may reveal that psychological factor is one of the important factors for the better performance during competition. Further studies are necessary to clarify psychological factors of putting by using different measurement techniques such as galvanic skin response.