bluuebunny/crossref_metadata_2025_split huggingface dataset:
Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-β autocrine regulatory loop
Authors: ALLEGRA Séverine , LI Jacques Yuan , SAEZ José Maria , LANGLOIS Dominique 
Published: July 2004

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the βMHC (β-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFβ (transforming growth factor β) on βMHC expression in Sol 8 cells. TGFβ1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited βMHC expression in Sol 8 cells. This inhibition of βMHC expression was progressively lost when TGFβ1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFβ1 did not inhibit βMHC accumulation any longer. Two independent approaches showed that a TGFβ autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFβs released by the cells into DM increased βMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFβ type II receptor was overexpressed in Sol 8 cells under the control of the βMHC promoter. Both the dominant-negative receptor and the βMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFβ signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote βMHC expression. To conclude, TGFβ inhibits Sol 8 cell terminal differentiation within a narrow time interval (24–34 h) that coincides with the onset of βMHC expression.
Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity
Authors: CHOWDARY Tirumala Kumar , RAMAN Bakthisaran , RAMAKRISHNA Tangirala , RAO Chintalagiri Mohan 
Published: July 2004

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.
Regulation of the cerebellar inositol 1,4,5-trisphosphate receptor by univalent cations
Authors: COQUIL Jean-François , BLAZQUEZ Samantha , SOAVE Sabrina , MAUGER Jean-Pierre 
Published: July 2004

In the present study we investigated the effects of K+ and other univalent cations on [3H]InsP3 {[3H]Ins(1,4,5)P3} binding to sheep cerebellar microsomes. In equilibrium binding experiments performed over 4 s at pH 7.1 and 20 °C, the addition of K+ to the binding medium decreased the affinity and increased the total number of binding sites for InsP3 in a dose-dependent manner. At low InsP3 concentration (0.5 nM) these effects resulted in a biphasic dose–response curve, with maximal binding at about 75 mM K+. In contrast, the dose–response curve calculated for InsP3 at the physiological concentration of 5 μM, was linear up to 200 mM K+. Univalent inorganic cations stimulated [3H]InsP3 binding to various extents, with the following descending order of efficiency at 75 mM: Cs+≈Rb+≈K+&gt;Na+&gt;Li+. The effect of K+ on InsP3R affinity was rapidly reversed upon cation removal. We were therefore also able to demonstrate that K+ increased Bmax (maximal specific binding) by pre-treating microsomes with K+ before measuring [3H]InsP3 binding in the absence of that cation. The increase in Bmax was reversible, but this reversal occurred less rapidly than the change in affinity. These results are consistent with a process by which K+ reversibly converted very low-affinity sites into sites with higher affinity, making them detectable in competitive binding experiments. They suggest that interconversion between these two affinity states constitutes the basis of a K+-controlled regulatory mechanism for cerebellar InsP3R.
Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras
Authors: PRASKOVA Maria , KHOKLATCHEV Andrei , ORTIZ-VEGA Sara , AVRUCH Joseph 
Published: July 2004

MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2–5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 μM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12→Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1–MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.
Reduction of anti-leishmanial pentavalent antimonial drugs by a parasite-specific thiol-dependent reductase, TDR1
Authors: DENTON Helen , McGREGOR Joanne C. , COOMBS Graham H. 
Published: July 2004

The reason why Leishmania parasites are susceptible to organic antimonial drugs, the standard chemotherapeutic agents for over 50 years, apparently lies in the fact that the mammalian stage of the parasite reduces the pentavalent form of the administered drug to a trivalent form that causes parasite death. We have identified and characterized a parasite-specific enzyme that can catalyse the reduction of pentavalent antimonials and may therefore be central to the anti-parasite activity of the drug. The unusual protein, a trimer of two-domain monomers in which each domain has some similarity to the Omega class glutathione S-transferases, is a thiol-dependent reductase (designated TDR1) that converts pentavalent antimonials into trivalent antimonials using glutathione as the reductant. The higher abundance of the enzyme in the mammalian stage of the parasite could explain why this parasite form is more susceptible to the drug.
Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5
Authors: GRAHAM Mark E. , RUMA-HAYNES Patricia , CAPES-DAVIS Amanda G. , DUNN Joanne M. , TAN Timothy C. , VALOVA Valentina A. , ROBINSON Phillip J. , JEFFREY Peter L. 
Published: July 2004

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.
Actin filaments play a permissive role in the inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes
Authors: OMATSU-KANBE Mariko , SHIBATA Masaru , YAMAMOTO Takefumi , ISONO Takahiro , MATSUURA Hiroshi 
Published: July 2004

Stimulation of P2 receptors with micromolar concentration of ATP evokes a transient increase in [Ca2+]i (intracellular free Ca2+ concentration), primarily due to release of Ca2+ from intracellular stores; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca2+]i increase mediated through a store-operated Ca2+ entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect of the extracellular ATP on store-operated Ca2+ entry, using fura 2 fluorescence for continuous measurement of [Ca2+]i, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 μM) or latrunculin A (3 μM) prevented the inhibitory effect of ATP (10 μM) on the thapsigargin (100 nM)-evoked store-operated Ca2+ entry, without changing the effect of ATP in increasing [Ca2+]i. In normal cells, bath application of ATP induced a transient [Ca2+]i increase, consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 μM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest that the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca2+ entry in rat brown adipocytes.
Efficacy of the A‐line™ AEP monitor as a tool for predicting successful insertion of a laryngeal mask during sevoflurane anesthesia
Authors: Alpiger S. , Helbo‐Hansen H. S. , Vach W. , Ording H. 
Published: July 2004

Background:  The use of clinical signs for assessing depth of anesthesia is unreliable during periods with little noxious stimulation. A patient may appear adequately anesthetized at one moment at a given level of stimulation, but may later, when facing other more intense stimuli, show signs of insufficient anesthesia. In order to prevent under‐ or overdosing of anesthetics, an anesthesia depth monitor that is able to predict responses to noxious stimulation would therefore be useful. Auditory evoked potentials (AEP) is one of several physiological parameters under investigation. The method has been improved by rapid extraction and conversion of the AEP curve into an index (A‐Line ARX Index™ = AAI). We aimed to determine the clinically required depth of anesthesia, measured by the A‐line™ AEP Monitor, for at least 90% probability of acceptable insertion conditions for a laryngeal mask airway (LMA).Methods:  We studied 112 patients anesthetized by mask with increasing concentration of sevoflurane in oxygen. The monitor was programmed to give an alarm at AAI between 15 and 40 according to randomization. When the alarm sounded, the end‐expiratory sevoflurane concentration was recorded and the LMA inserted. Insertion conditions were assessed by an observer blinded to the AAI.Results:  The ED95 and prediction probability (PK) for AAI were 14.5 (CI 7.4–21.6) and 0.76 (0.66–0.86), respectively, while the ED95 and PK for expiratory sevoflurane concentration were 4.36% (CI 3.73–4.98) and 0.95 (0.91–0.99).Conclusions:  AAI indicates the level of depth of anesthesia necessary for acceptable laryngeal mask insertion conditions. End‐expiratory sevoflurane concentration was the better predictor.
Nitrous oxide increases the incidence of bowel distension in patients undergoing elective colon resection
Authors: AkÇa O. , Lenhardt R. , Fleischmann E. , Treschan T. , Greif R. , Fleischhackl R. , Kimberger O. , Kurz A. , Sessler D. I. 
Published: July 2004

Background:  Nitrous oxide rapidly inflates gas‐filled spaces such as the intestines; but whether the resulting bowel distension is clinically important remains unclear. We therefore tested the hypothesis that nitrous oxide produces clinically important bowel distension.Methods:  Patients scheduled for colon resection were anesthetized with isoflurane and 35% oxygen and randomly assigned to 65% nitrous oxide (n = 175) or 65% nitrogen in air (n = 169). At the end of surgery, blinded surgeons rated the degree of bowel distension as none, mild, moderate, or severe. Patients reported pain, and nausea and vomiting (PONV) 2 h after surgery. Data are reported as means (SD). P < 0.05 was statistically significant.Results:  Morphometric and demographic data were similar in the groups. The duration of surgery was 3.0 (1.2) h in the nitrous oxide group and 3.4 (1.5) h in the air group (P = 0.017). Postoperative self‐reported pain scores (visual analog scale, 0–100 mm) were greater in the nitrous oxide group (43 [30] mm) than in the air group (35 [31] mm, P = 0.018). Although the incidence of PONV was similar in the groups, VAS scores for nausea were significantly greater in the nitrous oxide group (P = 0.040). Moderate‐to‐severe bowel distension was observed in 23% of nitrous oxide patients, but in only 9% of patients in the air group (P < 0.001). The number‐needed‐to‐harm for moderate or severe bowel distension from nitrous oxide was thus seven.Conclusions:  Our results suggest that avoiding nitrous oxide administration during prolonged bowel operations will minimize bowel distension and possibly reduce postoperative pain related to it.
Intrathecal morphine provides effective and safe analgesia in children after cardiac surgery
Authors: Suominen P. K. , Ragg P. G. , Mckinley D. F. , Frawley G. , But W. W. , Eyres R. L. 
Published: July 2004

Background:  The purpose of this prospective, randomized, blinded to observer study was to assess the analgesic effect and safety of intrathecal morphine (ITM) in post‐operative pain control in children after heart surgery with a sternotomy incision.Methods:  Eighty children, 3–55 kg in body weight, undergoing elective cardiac surgery with opioid‐based anaesthesia were randomly divided into two treatment groups to receive either 20 µg/kg ITM at induction of anaesthesia or control. To standardize the protocol for administration of post‐operative rescue intravenous morphine boluses and infusion (20–60 µg/kg/h), the Cardiac Analgesic Assessment Scale (CAAS) was used.Results:  Nine patients were excluded from the study after randomization. Thirty‐five patients were enrolled to the ITM group and 36 to the control group. The groups were similar for demographics and intra‐operative clinical characteristics. The mean time for the first intravenous morphine dose from ITM administration or equivalent time zero in the control group was significantly longer (P = 0.003) in the ITM group compared with the control group (12.3 vs. 8.7 h). Time from Paediatric Intensive Care Unit (PICU) admission to the start of intravenous morphine was also significantly longer (P = 0.01) in the ITM group (6.0 vs. 3.4 h). The total intravenous morphine consumption over the mean 19 post‐operative hours was significantly lower (P = 0.03) in the ITM group. However, the use of ITM did not result in earlier extubation or earlier discharge from the PICU. Of the 35 patients who received ITM at induction of anesthesia, 20% (n = 7) did not require any additional morphine in the PICU compared with three out of 36 control group patients. This did not reach statistical significance. The incidence of adverse events was low in both groups.Results:  An ITM dose of 20 µg/kg had a significant (P = 0.03) intravenous morphine‐sparing effect after cardiac surgery. Effective analgesia was observed for 12 h after administration of intrathecal morphine.
Early bioavailability of paracetamol after oral or intravenous administration
Authors: Holmér Pettersson P. , Öwall A. , Jakobsson J. 
Published: July 2004

Background:  Paracetamol is a peripherally acting analgesic commonly used in multimodal post‐operative pain management to reduce the need for more potent analgesics with their unwanted side‐effects. The dose and optimal galenical form for achieving analgesic concentrations is not well defined. The primary aim of this pilot project was to study the early bioavailability for two fixed doses of orally administrated paracetamol and one dose of intravenous propacetamol, all of which were given after minor surgery.Methods:  Thirty‐five patients undergoing day surgery were divided into five groups, seven patients each. Groups received either 1 g of an ordinary paracetamol tablet, 2 g of an ordinary paracetamol tablet, 1 g of a bicarbonate paracetamol tablet, 2 g of a bicarbonate paracetamol tablet or 2 g intravenously of prodrug propacetamol. We studied the plasma concentration of paracetamol during the first 80 min after administration.Results:  Within 40 min, intravenous propacetamol gave a median plasma paracetamol concentration of 85 µmol/l (range 65–161) and decreased thereafter. After oral administration, median plasma paracetamol concentration increased with increasing dose and time, but there were huge inter‐individual differences at all time points studied. At 80 min after oral paracetamol the median plasma concentrations were 36 and 129 µmol/l for the 1‐ and 2‐g groups, respectively, with an overall range between 0 and 306 µmol/l.Conclusion:  Oral administration of paracetamol as part of multimodal pain management immediately post‐operatively resulted in a huge and unpredictable variation in plasma concentration compared with the intravenous administration.
Satisfying and Retaining Customers through Independent Service Representatives
Authors: Brown Steven P. , Chin Wynne W. 
Published: July 2004

ABSTRACTThe degree of leverage possessed by manufacturers who outsource their customer service function to channel partners over customer satisfaction and loyalty is assessed empirically. Data provided by independent service representatives are linked with data from their customers. Results indicate that a manufacturer's support of its representatives increases their job satisfaction which, in turn, is indirectly linked to the customer satisfaction through shared perceptions of service performance and quality. However, the strength of the effects is modest, suggesting that manufacturers may need to consider direct customer contact strategies or contractual means of assuring customer satisfaction when independent service representatives “own the customer.” Conceptually, the hypothesized shared mindset model is tested against the competing affect transfer model, resulting in support for the former. Job satisfaction moderates the degree of agreement between representatives' and customers' perceptions of service performance and quality such that greater agreement occurs when job satisfaction is high.
Service Businesses and Productivity*
Authors: Schmenner Roger W. 
Published: July 2004

ABSTRACTThe records of superior performance of selected service firms over many years suggest that they may be more productive than others. This article uses the Theory of Swift, Even Flow to explain why that might be true. In the process, this article improves Schmenner's 1986 service process matrix. The redefinition of the axes of this matrix and of the resulting diagonal leads to enhanced understanding of productivity for service operations and helps to explain how some leading service companies have been able to sustain their competitive positions for decades.
Ability of Experience Design Elements to Elicit Emotions and Loyalty Behaviors
Authors: Pullman Madeleine E. , Gross Michael A. 
Published: July 2004

ABSTRACTExperience design, an approach to create emotional connection with guests or customers through careful planning of tangible and intangible service elements, has gained popularity in many hospitality and retail businesses. With ever‐increasing competition, service providers seek to develop loyalty by aggressively designing, continuously innovating, and managing their customer experiences. This article explores the relationship between different service elements designed to create enhanced experience and customer loyalty. In addition, it looks at emotional responses as mediating factors between the physical and relational elements and loyalty behaviors. A model is proposed and tested with a VIP hospitality tent for an internationally renowned touring circus. Results of the study indicate that while a few design elements directly affect loyalty behavior, the relationship between most design elements and loyalty behavior is strongly mediated by eliciting certain types of emotional behavior. This connection has implications for the focus of service managers' efforts in different environments.