bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of skeletal ryanodine receptors by dihydropyridine receptor II–III loop C-region peptides: relief of Mg2+ inhibition
Authors: HAARMANN Claudia S. , DULHUNTY Angela F. , LAVER Derek R. 
Published: April 2005

The aim of the present study was to explore interactions between surface-membrane DHPR (dihydropyridine receptor) Ca2+ channels and RyR (ryanodine receptor) Ca2+ channels in skeletal-muscle sarcoplasmic reticulum. The C region (725Phe-Pro742) of the linker between the 2nd and 3rd repeats (II–III loop) of the α1 subunit of skeletal DHPRs is essential for skeletal excitation–contraction coupling, which requires a physical interaction between the DHPR and RyR and is independent of external Ca2+. Little is known about the regulatory processes that might take place when the two Ca2+ channels interact. Indeed, interactions between C fragments of the DHPR (C peptides) and RyR have different reported effects on Ca2+ release from the sarcoplasmic reticulum and on RyR channels in lipid bilayers. To gain insight into functional interactions between the proteins and to explore different reported effects, we examined the actions of C peptides on RyR1 channels in lipid bilayers with three key RyR regulators, Ca2+, Mg2+ and ATP. We identified four discrete actions: two novel, low-affinity (&gt;10 μM), rapidly reversible effects (fast inhibition and decreased sensitivity to Mg2+ inhibition) and two slowly reversible effects (high-affinity activation and a slow-onset, low-affinity inhibition). Fast inhibition and high-affinity activation were decreased by ATP. Therefore peptide activation in the presence of ATP and Mg2+, used with Ca2+ release assays, depends on a mechanism different from that seen when Ca2+ is the sole agonist. The relief of Mg2+ inhibition was particularly important since RyR activation during excitation–contraction coupling depends on a similar decrease in Mg2+ inhibition.
Pharmacokinetics of 1-nitrosomelatonin and detection by EPR using iron dithiocarbamate complex in mice
Authors: PEYROT Fabienne , GRILLON Catherine , VERGELY Catherine , ROCHETTE Luc , DUCROCQ Claire 
Published: April 2005

The N-nitroso-derivative of melatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NO• [oxidonitrogen(•)] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug, is its ability to release both melatonin and NO•, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-3H]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)2–Fe2+ (iron–N-methyl-D-glucamine dithiocarbamate) complex as a spin trap. The behaviour of NOM was compared with that of GSNO (S-nitrosoglutathione), a hydrophilic NO• donor. In the first minutes following [O-methyl-3H]NOM intraperitoneal injection, the radioactivity was found in organs (6% in the liver, 1% in the kidney and 0.6% in the brain), but not in the blood. In both liver and brain, the radioactivity content decreased over time with similar kinetics reflecting the diffusion and metabolism of NOM and of its metabolites. Based on the characterization and the quantification of the EPR signal in vitro with NOM or GSNO using (MGD)2–Fe2+ complex in phosphate-buffered solutions, the detection of these nitroso compounds was realized ex vivo in mouse tissue extracts. (MGD)2–Fe2+–NO was observed in the brain of NOM-treated mice in the first 10 min following injection, revealing that NOM was able to cross the blood–brain barrier, while GSNO was not.
Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA
Authors: MAESTRO Beatriz , SANZ Jesús M. 
Published: April 2005

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed β-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 °C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)→I1⇆I2. The N→I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1⇆I2 transition is co-operative and reversible, with an associated freeenergy change (ΔG0) of 30.9±0.8 kJ·mol−1. The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2⇆[I2]2, ΔG0=50.1±0.8 kJ·mol−1). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal β-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.
Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression
Authors: ROGER Thierry , MICONNET Isabelle , SCHIESSER Anne-Laure , KAI Hirofumi , MIYAKE Kensuke , CALANDRA Thierry 
Published: April 2005

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.
Cleavage at the stem region releases an active ectodomain of the membrane type 1 matrix metalloproteinase
Authors: TOTH Marta , OSENKOWSKI Pamela , HESEK Dusan , BROWN Stephen , MEROUEH Samy , SAKR Wael , MOBASHERY Shahriar , FRIDMAN Rafael 
Published: April 2005

MT1-MMP (membrane type 1 matrix metalloproteinase) is a membrane-anchored MMP that can be shed to the extracellular milieu. In the present study we report the primary structure and activity of the major soluble form of MT1-MMP. MS analysis of the purified 50-kDa soluble MT1-MMP form shows that the enzyme extends from Tyr112 to Val524, indicating that formation of this species requires a proteolytic cleavage within the stem region. In agreement, deletion of the entire stem region of MT1-MMP inhibited shedding of the 50-kDa species. A recombinant 50-kDa species (Tyr112–Val524) expressed in cells exhibited enzymatic activity against pro-MMP-2 and galectin-3, and thus this species is a competent protease. The recombinant 50-kDa soluble form also decreased the level of surface-associated TIMP-2 (tissue inhibitor of metalloproteinase 2) when administered to cells expressing wild-type membrane-anchored MT1-MMP, suggesting that ectodomain shedding of MT1-MMP can alter the MMP/TIMP balance on the cell surface. A ∼53-kDa species of MT1-MMP was also isolated from a non-detergent extract of human breast carcinoma tissue and was found to lack the cytosolic tail, as determined with specific MT1-MMP domain antibodies. Together, these data show that MT1-MMP ectodomain shedding is a physiological process that may broaden MT1-MMP activity to the pericellular space.
Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen
Authors: JONSSON Magnus , LINSE Sara , FROHM Birgitta , LUNDWALL Åke , MALM Johan 
Published: April 2005

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)−1 and with an average dissociation constant of approx. 5 μM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.
Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants
Authors: SCHWARZ Alexandra , PIERFEDERICI Francesco Maria , NIDETZKY Bernd 
Published: April 2005

Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 °C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334→Gln) and H334N mutants, and that of H334A was unchanged. In the direction of α-glucan synthesis, selectivity for the reaction with G1P (α-D-glucose 1-phosphate) compared with the selectivity for reaction with α-D-xylose 1-phosphate decreased from a wild-type value of ∼20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and α-glucan was not affected. Quenching of fluorescence of the pyridoxal 5′-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme–phosphate complex occurred in a single step. The 50-fold higher constant (Kd) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal Kd value established a linear free-energy relationship (slope=1.19±0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.
Identification of adipocyte adhesion molecule (ACAM), a novel CTX gene family, implicated in adipocyte maturation and development of obesity
Authors: EGUCHI Jun , WADA Jun , HIDA Kazuyuki , ZHANG Hong , MATSUOKA Takashi , BABA Masako , HASHIMOTO Izumi , SHIKATA Kenichi , OGAWA Norio , MAKINO Hirofumi 
Published: April 2005

Few cell adhesion molecules have been reported to be expressed in mature adipocytes, and the significance of cell adhesion process in adipocyte biology is also unknown. In the present study, we identified ACAM (adipocyte adhesion molecule), a novel homologue of the CTX (cortical thymocyte marker in Xenopus) gene family. ACAM cDNA was isolated during PCR-based cDNA subtraction, and its mRNA was shown to be up-regulated in WATs (white adipose tissues) of OLETF (Otsuka Long–Evans Tokushima fatty) rats, an animal model for Type II diabetes and obesity. ACAM, 372 amino acids in total, has a signal peptide, V-type (variable) and C2-type (constant) Ig domains, a single transmembrane segment and a cytoplasmic tail. The amino acid sequence in rat is highly homologous to mouse (94%) and human (87%). ACAM mRNA was predominantly expressed in WATs in OLETF rats, and increased with the development of obesity until 30 weeks of age, which is when the peak of body mass is reached. Western blot analysis revealed that ACAM protein, approx. 45 kDa, was associated with plasma membrane fractions of mature adipocytes isolated from mesenteric and subdermal adipose deposits of OLETF rats. Up-regulation of ACAM mRNAs in obesity was also shown in WATs of genetically obese db/db mice, diet-induced obese ICR mice and human obese subjects. In primary cultured mouse and human adipocytes, ACAM mRNA expression was progressively up-regulated during differentiation. Several stably transfected Chinese-hamster ovary K1 cell lines were established, and the quantification of ACAM mRNA and cell aggregation assay revealed that the degree of homophilic aggregation correlated well with ACAM mRNA expression. In summary, ACAM may be the critical adhesion molecule in adipocyte differentiation and development of obesity.
Vanishin is a novel ubiquitinylated death-effector domain protein that blocks ERK activation
Authors: SUR Runa , RAMOS Joe W. 
Published: April 2005

The ERK (extracellular-signal regulated-kinase)/MAPK (mitogen-activated protein kinase) pathway can regulate transcription, proliferation, migration and apoptosis. The small DED (death-effector domain) protein PEA-15 (phosphoprotein enriched in astrocytes-15) binds ERK and targets it to the cytoplasm. Other DED-containing proteins including cFLIP and DEDD can also regulate signal transduction events and transcription in addition to apoptosis. In the present study, we report the identification of a novel DED-containing protein called Vanishin. The amino acid sequence of Vanishin is closest in similarly to PEA-15 (61% identical). Vanishin mRNA is expressed in several mouse tissues and in both mouse and human cell lines. Interestingly, Vanishin is regulated by ubiquitinylation and subsequent degradation by the 26 S proteasome. The ubiquitinylation is complex and occurs at both the internal lysine residues and the N-terminus. We further show that Vanishin binds ERK/MAPK but not the DED proteins Fas-associated death domain, caspase 8 or PEA-15. Vanishin is present in both the nucleus and Golgi on overexpression and forces increased ERK accumulation in the nucleus in the absence of ERK stimulation. Moreover, Vanishin expression inhibits ERK activation and ERK-dependent transcription in cells, but does not alter MAPK/ERK activity. Therefore Vanishin is a novel regulator of ERK that is controlled by ubiquitinylation.
Microsatellite markers for heart of palm –Euterpe edulis and E. oleracea Mart. (Arecaceae)
Authors: Gaiotto F. A. , Brondani R. P. V. , Grattapaglia D. 
Published: April 2005

Abstract Euterpe edulis is the species of palm found in the Atlantic Forest and Cerrado gallery forest that yields the best heart of palm. A battery of 18 highly polymorphic microsatellite markers was developed from an enriched genomic library. Using fluorescence automated detection an average of 10.6 alleles per locus were found on a sample of 66 individuals sampled from a natural population. These loci allow extremely precise paternity testing, estimation of gene flow and of parentage coefficients among trees in the wild.
Information and communication technology (ICT) education in The City School, Pakistan
Authors: Ali Iftikhar , Proctor John 
Published: April 2005

PurposeIn an age of information technology some developing countries are more vulnerable than others to international competition through failure to utilize fully the benefits of an ICT culture. The authors suggest that the strategies in response must include a radical review of attitudes and methods of delivery of ICT in schools and give as an example the recent experience of The City School, the nationwide schools’ organisation in Pakistan with whom they are employed.Design/methodology/approachThe authors review the current position of ICT in schools in Pakistan and suggest as a model of development, for schools of a corresponding standing, that of The City School. They describe how The City School responded to ICT, ensuring that all its pupils would have access to the most modern of ICT courses. Discusses how the decision to implement a complete change, or revolution, in teaching ICT was brought about in a relatively short time. It discusses the nature of the programme, how it was organised, the materials required and the outcomes of its implementation including its outstanding success with pupils and their parents.FindingsThe authors chronicle the historical developments within The City School that brought about radical change within a comparatively short period and identify careful planning, training, and the motivation of stakeholders, i.e. pupils, teachers and parents, as key elements in its successful implementation.Originality/valueThe authors suggest that The City School experience provides a model that may be emulated by schools elsewhere in both developing and industrially developed countries.
How to manage the big bang: evolution or revolution in the introduction of an MLE?
Authors: Quinsee Susannah , Sumner Neal 
Published: April 2005

PurposeTo examine how introducing an institution‐wide managed learning environment impacts on the processes of organisational change using City University, London as a case study.Design/methodology/approachLiterature‐based discussion of current issues around the introduction of online learning to provide theoretical framework. Action research methodology used for interviews with leading members of the institution.FindingsThere is a significant amount of literature available on institutional change and managed learning environments; however, how the introduction of such systems operates in practice depends on the context of the institution. In the interviews with key stakeholders six significant themes are identified for the management of change in this area: pedagogic direction; operational connections and development; organisational structure and change; system process; professional development; strategic vision and perception. Any implementation project regarding the introduction of managed learning environments should encompass these key themes.Research limitations/implicationsBased on interviews with a small number of stakeholders at the institution. Further research could compare the experience at City with other institutions and revisit a wider selection of stakeholders at City to assess their views at a later stage in the implementation.Practical implicationsProvides guidance after the experiences encountered at the institution which could assist other universities both during the planning phases of such a project or during the implementation itself.Originality/valueIdentifies a number of key areas to shape and formulate project management. Combines empirical evidence with theoretical context.
The use of the internet in higher education
Authors: Eynon Rebecca 
Published: April 2005

PurposeTo explore academics’ experiences of using information and communication technologies (ICTs) for teaching and learning.Design/methodology/approachAnalysis of three discipline‐specific focus group discussions held with academics based in Higher Education Institutions (HEIs) that use ICTs for teaching their students.FindingsThe most common use of ICTs in all subjects was to provide students with access to a range of online resources. Academics’ motivations for using ICTs included: enhancing the educational experience for their students; to compensate for some of the changes occurring in higher education, such as the rise in student numbers and demand for flexible learning opportunities; and personal interest and enjoyment. The difficulties academics encountered when using these technologies for teaching included: a lack of time; dissatisfaction with the software available; and copyright.Research limitations/implicationsThis is a small scale, exploratory study. Further research is required that is sampled in such a way as to ensure that the findings can be generalized to all academics in all institutions in the UK.Practical implicationsThe institutional, middle managerial, staff and student level all need to be considered when encouraging the further adoption of new technologies for teaching and learning in higher education. Institutional level strategies must also account for the diversity of ways ICTs may be used in teaching in different contexts across the institution.Originality/valueResearch exploring academics’ experiences of using ICTs for teaching and learning is scarce. Further work is required to ensure the successful development and implementation of future technological and policy developments in this area.