bluuebunny/crossref_metadata_2025 huggingface dataset:
Calmodulin conformational changes in the activation of protein kinases
Authors: Török K.
Published: January 2005

The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/ calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s−1). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and αcCaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of αCaMKII, ATP binding and Thr286 auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to αCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/ calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.
Sequential extracts of human bone show differing collagen synthetic rates
Authors: Babraj J. , Cuthbertson D.J. , Rickhuss P. , Meier-Augenstein W. , Smith K. , Bohé J. , Wolfe R. R. , Gibson J. N. A. , Adams C. , Rennie M. J.
Published: January 2005

Type I collagen is the major bone protein. Little is known quantitatively about human bone collagen synthesis in vivo, despite its importance for the understanding of bone formation and turnover. Our aim was to develop a method that could be used for the physiological and pathophysiological investigation of human bone collagen synthesis. We have carried out preliminary studies in patients undergoing hip replacement and in pigs to validate the use of the flooding dose method using 13C- or 15N-labelled proline and we have now refined our techniques to allow them to be used in a normal clinical or physiological setting. The results show that the application of a flooding dose causes bone free-proline labelling to equilibrate with that of blood in pigs and human beings, so that only 150 mg of bone will provide enough sample to prepare and measure the labelling of three fractions of bone collagen (dissolved in NaCl, acetic acid and pepsin/acetic acid) which have the same relative labelling (1.0:0.43:0.1) as measured by GC-combustion-isotope ratio MS. The rates of incorporation were substantially faster than in skeletal muscle samples taken at the same time. The results suggest that different fractions of human bone collagen turnover at markedly higher rates than had been previously considered. This approach should allow us to discover how growth and development, food, activity and drugs affect bone collagen turnover and to measure the effects on it of ageing and bone disease.
The ProteinChip® Biomarker System from Ciphergen Biosystems: a novel proteomics platform for rapid biomarker discovery and validation
Authors: Chapman K.
Published: January 2005

Protein biochips are emerging in two distinct formats. The first involves high-density immobilized arrays of recognition molecules (e.g. antibodies), where target binding is monitored indirectly (e.g. via fluorescence). This technology is in its infancy, being limited by the availability of suitable binding molecules that can cope effectively with protein diversity. The second format involves the capture of proteins by biochemical or intermolecular interaction, coupled with direct detection by MS. This technology is available as the ProteinChip® Biomarker System. ProteinChip technology uses surface-enhanced laser desorption/ionization processes to analyse proteins directly from biological samples. Chromatographic surfaces are placed on to ProteinChip Arrays and used to capture subclasses of proteins, dependent on their physical properties. Time-of-flight MS then assigns native molecular masses to the captured proteins. Reproducible protein profiles can be generated from crude biological fluids (e.g. cell lysates, urine or serum). The technology is being applied to a wide range of disciplines, from plant sciences to cancer research, and will be reviewed here.
Gene regulation of the serine proteinase inhibitors α1-antitrypsin and α1-antichymotrypsin
Authors: Kalsheker N. , Morley S. , Morgan K.
Published: January 2005

The serine proteinase inhibitors (serpins) are a superfamily of proteins with a diverse set of functions, including the control of blood coagulation, complement activation, programmed cell death and development. The most abundant serpins in human plasma are α1-antitrypsin (AAT) and α1-antichymotrypsin (ACT). During inflammation, circulating levels can increase by up to 3-fold for the former and by 4–5-fold for the latter. The major site for increased synthesis is the liver. Other tissues, such as the lung, are also capable of synthesizing AAT and ACT, and expression can be increased by up to 100-fold by cytokines. There is a tissue-specific promoter for the liver, and alternative promoters for other tissues that express AAT. Basal AAT expression is regulated by the synergistic action of the tissue-specific transcription factors hepatocyte nuclear factors 1α and 4. An enhancer positioned approx. 1.2 kb from the end of the last exon in the 3′ flanking sequence modulates cytokine-induced expression by interleukin-6 and oncostatin M. Microcell hybrid transfection studies have shown that a sequence containing 15 kb of 5′ flanking sequence is sufficient to allow stable expression of AAT in a position-independent manner. There is probably a single promoter for ACT. Oncostatin M-inducible elements have been identified in the 5′ flanking sequence approx. 100 bp upstream from the transcription initiation site, and a further interleukin-1-responsive enhancer has been identified approx. 13 kb upstream. The pathways for a humoral response are being mapped at high resolution.
Proteinases in chronic obstructive pulmonary disease
Authors: Shapiro S. D.
Published: January 2005

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide, and we have little specific therapy to offer these patients. One potential strategy to limit loss of lung function in COPD would be to inhibit matrix-degrading proteinases. Several serine proteinases and matrix metalloproteinases are expressed in association with COPD in humans. Application of genetargeted macrophage elastase and neutrophil elastase to a mouse model of cigarette-smoke-induced emphysema has uncovered roles for these proteinases in airspace enlargement, and has identified many interactions between these proteolytic systems.
Novel roles of protease inhibitors in infection and inflammation
Authors: Hiemstra P. S.
Published: January 2005

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. α1-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.
Role of plasminogen activators in peritoneal adhesion formation
Authors: Sulaiman H. , Dawson L. , Laurent G. J. , Bellingan G. J. , Herrick S. E.
Published: January 2005

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.
The Escherichia coli RNA degradosome: structure, function and relationship to other ribonucleolytic multienyzme complexes
Authors: Carpousis A. J.
Published: January 2005

mRNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript. The RNase E of Escherichia coli is a single-strand-specific endonuclease involved in the processing of rRNA and the degradation of mRNA. A nucleolytic multienzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E. Two other components are a 3′ exoribonuclease (polynucleotide phosphorylase, PNPase) and a DEAD-box RNA helicase (RNA helicase B, RhlB). RNase E is a large multidomain protein with N-terminal ribonucleolytic activity, an RNA-binding domain and a C-terminal ‘scaffold’ that binds PNPase, enolase and RhlB. RhlB by itself has little activity but is strongly stimulated by its interaction with RNase E. RhlB in vitro can facilitate the degradation of structured RNA by PNPase. Since the discovery of the RNA degradosome in E. coli, related complexes have been described in other organisms.
Translation initiation and surface plasmon resonance: new technology applied to old questions
Authors: von der Haar T. , Hughes J. M. X. , Karim M. Manjurul , Ptushkina M. , McCarthy J. E. G.
Published: January 2005

Translation initiation in eukaryotes is a complicated process involving some of the largest cellular structures, the ribosomes, together with approx. 11 initiation factors, and a poorly characterized set of other proteins. The concerted action of all these components ultimately results in the formation of an 80 S ribosomal complex on the AUG codon of an mRNA, which is competent to start polypeptide production. In this brief overview, we describe the strategies developed by our laboratory to apply surface plasmon resonance (SPR)-based technology to the problem of elucidating kinetic aspects of substeps within the translation-initiation reaction. We then review how other groups have used similar SPR-based techniques to study related interactions.
Extracellular proteolysis in the development and progression of atherosclerosis
Authors: Lijnen H. R.
Published: January 2005

Clinical complications of atherosclerosis are often triggered by the rupture of unstable plaques, while thinning of the atherosclerotic vessel wall owing to elastin and collagen degradation and media necrosis may result in aneurysm formation and bleeding. Proteolysis, mediated via the plasminogen/ plasmin and/or matrix metalloproteinase (MMP) systems may contribute to neovascularization and rupture of plaques, or to ulceration and rupture of aneurysms. In an in vivo model of atherosclerosis, using mice that had a combined deficiency of apolipoprotein E (ApoE) and urokinase-type plasminogen activator (u-PA) and that were maintained on a cholesterol-rich diet, it was observed that u-PA deficiency protects against aneurysm formation. This was explained by the findings that plasmin, generated from plasminogen by u-PA, activates several macrophage-secreted proMMPs (e.g. proMMP-3, −9, −12 and −13), which in turn cause extracellular matrix degradation. A potential role for MMP-3 (stromelysin-1) was confirmed in a subsequent study using mice with a combined deficiency of ApoE and MMP-3, that were kept on a cholesterol-rich diet. The results suggest that MMP-3 contributes to plaque destabilization, possibly by degrading extracellular matrix components, but also promotes aneurysm formation by degrading the elastic lamina. These effects may be mediated by MMP-3 directly or by activation of other proMMPs or other (proteolytic) systems. A functional role of MMPs is further supported by the finding that deficiency in TIMP-1 (tissue inhibitor of MMPs type 1) reduces atherosclerotic plaque size but enhances aneurysm formation. Taken together, these results suggest that u-PA has an important role in the structural integrity of the atherosclerotic vessel wall, which is likely to involve triggering the activation of MMPs and, furthermore, they suggest that increased u-PA levels are a risk factor for aneurysm formation.
Haemostatic factors occupy new territory: the role of the urokinase receptor system and kininogen in inflammation
Authors: Chavakis T. , Kanse S. M. , May A. E. , Preissner K. T.
Published: January 2005

Vascular cell adhesion and migration, proliferation or differentiation are cellular responses that are induced by haemostatic factors of the urokinase/plasminogen activation complex, but the respective underlying mechanisms are largely undefined. The direct and indirect contributions of the urokinase-type plasminogen activator receptor (uPAR) system in inflammatory processes, as they relate to recruitment of leukocytes, define novel functions and could serve as therapeutic targets for related vasculopathies. The presence of uPAR plays a crucial role in β2-integrin-mediated adhesion of leukocytes; uPAR also directly mediates leukocyte adhesion to vitronectin, a multifunctional adhesion protein that is associated with the extracellular matrix. The latter process is inhibited by plasminogen activator inhibitor-1. Both β2-integrin- and uPAR-dependent processes are activated by Zn2+ and are blocked by high-molecular-mass kininogen. Domain 5 of kininogen was identified, in particular, as an anti-adhesive component with a potent antiinflammatory action in a peritonitis mouse model. In patients with acute myocardial infarction, elevated expression of uPAR on monocytes resulted in their increased adherence to the endothelium, which indicates a possible role of the uPAR system in monocyte recruitment to the infarcted area. Urokinase-type plasminogen activator was identified as a potent mitogen for vascular smooth muscle cells, an observation that was independent of the presence of uPAR and its proteolytic activity. Taken together, these results strongly suggest an essential role for the uPAR system in acute inflammation as well as in chronic degenerative vascular processes such as atherosclerosis. Targeting the uPAR system may allow specific therapeutic intervention in vascular pathologies.
Mechanism of thrombin-induced angiogenesis
Authors: Maragoudakis M. E. , Tsopanoglou N. E. , Andriopoulou P.
Published: January 2005

Clinical, laboratory, histopathological and pharmacological evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. Additionally, thrombin was shown to promote tumour progression and metastasis in animals, and epidemiological studies suggest an increased risk of cancer diagnosis after primary thromboembolism. We have proposed that the aforementioned results may be related to our finding that thrombin is a potent activator of angiogenesis. This is a thrombin receptor-mediated event (the receptor is referred to as protease-activate receptor) and is independent of fibrin formation. Many cellular effects of thrombin on endothelial cells can contribute to the angiogenic action of thrombin. (i) Exposure of endothelial cells to thrombin cause a time- and dose-dependent decrease in the attachment of these cells to basement membrane components, with a concomitant increase in matrix metallo-proteinase 2 activation, (ii) Thrombin upregulates the expression of integrin αvβ3, the marker of the angiogenic phenotype of endothelial cells. (iii) Thrombin has chemotactic and aptotactic effects on endothelial cells and upregulates the expression of the vascular endothelial growth factor (VEGF) receptors (KDR and Flt1). Thus, thrombin synergizes with the key angiogenic factor VEGF in endothelial cell proliferation. Furthermore, thrombin enhances the secretion of VEGF and matrix metalloproteinase 9 of PC3 prostate cancer cells. These results can explain the angiogenic and tumour-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.
Cellular mechanisms regulating non-haemostatic plasmin generation
Authors: Bass R. , Ellis V.
Published: January 2005

A variety of proteases have the potential to degrade the extracellular matrix (ECM), thereby influencing the behaviour of cells by removing physical barriers to cell migration, altering cell-ECM interactions or releasing ECM-associated growth factors. The plasminogen activation system of serine proteases is particularly implicated in this pericellular proteolysis and is involved in pathologies ranging from cancer invasion and metastasis to fibroproliferative vascular disorders and neurodegeneration. A central mechanism for regulating plasmin generation is through the binding of the two plasminogen activators to specific cellular receptors: urokinase-type plasminogen activator to the glycolipid-anchored membrane protein uPAR, and tissue plasminogen activator to a type-II transmembrane protein recently identified on vascular smooth muscle cells. These binary complexes interact with membrane-associated plasminogen to form higher order activation complexes that greatly reduce the Km for plasminogen activation and, in some cases, protect the proteases from their cognate serpin inhibitors. Various other proteins that are involved in cell adhesion and migration also interact with these complexes, modulating the activity of this efficient and spatially restricted proteolytic system. Recent observations demonstrate that certain forms of the prion protein can stimulate tissue plasminogen activator-catalysed plasminogen activation, which raises the possibility that these proteases may also have a role in the pathogenesis of the transmissible spongiform encephalopathies.
Urokinase-type plasminogen activator: a potent marker of metastatic potential in human cancers
Authors: Duffy M. J.
Published: January 2005

Urokinase-type plasminogen activator (uPA) is a serine protease that is causally involved in cancer progression, especially invasion and metastasis. Multiple studies have shown that breast cancer patients whose primary cancer contains high levels of uPA have a significantly worse outcome than patients with low levels. As a prognostic marker for breast cancer the information supplied by uPA is both independent of traditionally used factors and significant in the important subgroup of axillary-node patients. Paradoxically, high levels of plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of uPA, also predict for aggressive disease. Recently, the prognostic impact of both uPA and PAI-1 in axillary node-negative breast cancer was confirmed using two different Level 1 Evidence studies, i.e. in both a randomized prospective trial and a pooled analysis. Therefore, uPA and PAI-1 appear to have fulfilled all the criteria for the routine assessment of prognosis in newly diagnosed breast cancer patients
Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis
Authors: James N. J. , Ettelaie C. , Bruckdorfer K. R.
Published: January 2005

Tissue factor (TF) is a transmembrane glycoprotein that was originally recognized for its ability to initiate the extrinsic pathway of coagulation. More recently, additional functions of TF in cellular signalling have emerged, notably the role of TF in vasculogenesis and angiogenesis. We have described previously the ability of a peptide derived from the apolipoprotein B100 (apoB100) moiety of low-density lipoproteins (KRAD14) to inhibit the procoagulant function of TF. In this study, we demonstrate the ability of the KRAD14 peptide to attenuate the density of cellular network structures of T24 cells grown on specialized matrix (MatrigelTM). In addition, an alternative inhibitor of TF activity, the TF8 5G9 antibody, also reduces the density of cellular network formation. Targeted use of a stable structural equivalent of the KRAD14 peptide may thus prove useful in the prophylactic treatment of diseases whose pathologies feature the formation of neovascular tissue, e.g. tumour growth and metastasis, rupture of atherosclerotic plaques and retinopathy secondary to diabetes.
Tissue plasminogen activator as a modulator of neuronal survival and function
Authors: Tsirka S. E.
Published: January 2005

The tissue plasminogen activator (tPA)/plasmin proteolytic system has been implicated in both physiological and pathological processes in the mammalian brain. The physiological roles include facilitating neurite outgrowth and pathfinding. The pathological role involves mediating a critical step in the progression of excitotoxin-induced neurodegeneration. Mechanistically, tPA appears to function through two pathways. The first pathway proceeds via its well established ability to convert plasminogen into plasmin. Plasmin then either promotes neuronal death via both the degradation of the extracellular matrix and the establishment of chemoattractant gradients for microglia, or facilitates neurite outgrowth through the processing of extracellular matrix proteoglycans. The second pathway for tPA does not involve its proteolytic activity: rather tPA functions as an agonist to stimulate a cell-surface receptor on microglia (the macrophage-like immunocompetent cells of the central nervous system) and results in their activation. Once activated after neuronal injury, microglia contribute to the ensuing neurodegeneration. Using tPA as a link between neurons and microglia, we are focusing on understanding their communication and interactions in the normal and diseased central nervous system.
Exertional oxygen uptake kinetics: a stamen of stamina?
Authors: Whipp B. J. , Rossiter H. B. , Ward S. A.
Published: January 2005

The fundamental pulmonary O2 uptake (o2) response to moderate, constant-load exercise can be characterized as (do2/dt)(τ) + Δo2 (t) = Δo2SS where Δo2SS is the steady-state response, and τ is the time constant, with the o2 kinetics reflecting intramuscular O2 uptake (o2) kinetics, to within 10%. The role of phosphocreatine (FCr) turnover in o2 control can be explored using 31P-MR spectroscopy, simultaneously with o2. Although τo2 and τPCr vary widely among subjects (approx. 20–65 s), they are not significantly different from each other, either at the on- or off-transient. A caveat to interpreting the ‘well-fit’ exponential is that numerous units of similar Δo2SS but with a wide τ distribution can also yield a o2 response with an apparent single τ. This τ is, significantly, inversely correlated with lactate threshold and o2max (but is poorly predictive; a frail stamen, therefore), consistent with τ not characterizing a compartment with uniform kinetics. At higher intensities, the fundamental kinetics become supplemented with a slowly-developing phase, setting o2 on a trajectory towards maximum o2. This slow component is also demonstrable in Δ[PCr]: the decreased efficiency thereby reflecting a predominantly high phosphate-cost of force production rather than a high O2-cost of phosphate production. We also propose that the O2-deficit for the slow-component is more likely to reflect shifting Δo2SS rather than a single one with a single τ.
Measuring real levels of oxygen in vivo: opportunities and challenges
Authors: Swartz H. M.
Published: January 2005

Molecular oxygen has multiple roles in cells, which can have different dependencies on the amount of oxygen at particular sites. Therefore, in order to relate oxygen to the processes under consideration, one must know the amount of oxygen present at the site of interest. The use of the various techniques generally is complementary rather than competitive. The key to their successful use requires an understanding of the capabilities of the techniques and of the nature of the process that is being investigated. This paper provides a framework for obtaining such an understanding.
Mitochondrial respiration at low levels of oxygen and cytochrome c
Authors: Gnaiger E. , Kuznetsov A. V.
Published: January 2005

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 μM O2 with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N, N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) + ascorbate] respectively at 30 °C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 μM cytochrome c, which was nearly identical to the high-affinity K'm for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.
Different modes of activating phosphofructokinase, a key regulatory enzyme of glycolysis, in working vertebrate muscle
Authors: Wegener G. , Krause U.
Published: January 2005

Glycolytic flux in white muscle can be increased several-hundredfold by exercise. Phosphofructokinase (PFK; EC 2.7.1.11) is a key regulatory enzyme of glycolysis, but how its activity in muscle is controlled is not fully understood. In order not to neglect integrative aspects of metabolic regulation, we have studied in frogs (Rana temporaria) a physiological form of muscle work (swimming) that can be triggered like a reflex. We analysed swimming to fatigue in well rested frogs, recovery from exercise, and repeated exercise after 2 h of recovery. At various times, gastrocnemius muscles were tested for glycolytic intermediates and effectors of PFK. All metabolites responded similarly to the two periods of exercise, with the notable exception of fructose 2,6-bisphosphate (F2,6P2), which we proved to be a most potent activator of frog muscle PFK. The first bout of exercise triggered a more than 10-fold increase in F2,6P2; PFK activity and the content of F2,6P2 in muscle were well correlated. F2,6P2 decreased to pre-exercise levels in fatigued frogs and it virtually disappeared during recovery. Varying by a factor of 70, F2,6P2 was the most dynamic of all metabolites in muscle. Even more surprisingly, F2,6P2 did not respond at all to a second bout of exercise. Other activators of PFK, such as Pi, AMP and ADP, are increased as a consequence of increased ATP turnover in contracting muscle cells. This does not apply to F2,6P2, which is likely to respond to extracellular signals and could be involved in mechanisms by which muscle metabolism is integrated into the metabolism of the whole body. Whether this phenomenon exists in vertebrates other than the frog, and maybe even in humans, and how the content of F2,6P2 in muscle is controlled are intriguing open questions.
Genetic dissection of plant cell-wall biosynthesis
Authors: Bonetta D. T. , Facette M. , Raab T. K. , Somerville C. R.
Published: January 2005

The plant cell wall is a complex structure consisting of a variety of polymers including cellulose, xyloglucan, xylan and polygalacturonan. Biochemical and genetic analysis has made it possible to clone genes encoding cellulose synthases (CesA). A comparison of the predicted protein sequences in the Arabidopsis genome indicates that 30 divergent genes with similarity to CesAs exist. It is possible that these cellulose synthase-like (Csl) proteins do not contribute to cellulose synthesis, but rather to the synthesis of other wall polymers. A major challenge is, therefore, to assign biological function to these genes. In an effort to address this issue we have systematically identified T-DNA or transposon insertions in 17 Arabidopsis Csls. Phenotypic characterization of ‘knock-out’ mutants includes the determination of spectroscopic profile differences in mutant cell walls from wild-type plants by Fourier-transform IR microscopy. A more precise characterization includes cell wall fractionation followed by neutral sugar composition analysis by anionic exchange chromatography.
A multigene family of glycosyltransferases in a model plant, Arabidopsis thaliana
Authors: Bowles D.
Published: January 2005

Glycosyltransferases transfer sugars from NDP-sugar donors to acceptors. The multigene family of transferases described in this paper typically transfer glucose from UDP-glucose to low-molecular-mass acceptors in the cytosol of plant cells. There are 107 sequences in the genome of Arabidopsis thaliana that contain a consensus, suggesting they belong to this Group 1 multigene family. The family has been analysed phylogenetically, and a functional genomics approach has been applied to explore the relatedness of sequence similarity to catalytic specificity and stereoselectivity. Enzymes belonging to this class of transferases glycosylate a vast array of acceptors, including natural products such as secondary metabolites and hormones, as well as xenobiotics absorbed by the plant, such as herbicides and pesticides. Conjugation to glucose potentially changes the activity of the acceptor molecule and invariably changes its location within the plant cell. Using the genomics approach described, a platform of knowledge has been constructed that will enable an understanding to be gained on the role of these enzymes in cellular homoeostasis, as well as their activity in biotransformations in vitro that require strict regioselectivity of glycosylation.
Role of AMP-activated protein kinase in the regulation of gene transcription
Authors: Leclerc I. , Viollet B. , da Silva Xavier G. , Kahn A. , Rutter G. A.
Published: January 2005

AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism in response to changes in the energy status of the cells. AMPK was known to shut down energy-consuming pathways in response to a fall in the ATP/AMP ratio by phosphorylating key enzymes of intermediate metabolism. Here we will discuss the recent evidence implicating AMPK in the regulation of gene expression in mammals, mainly in the liver and in the pancreatic β-cells.
Structure-function relationships in the β-cell KATP channel
Authors: Mikhailov M. V. , Mikhailova E. A. , Ashcroft S. J. H.
Published: January 2005

The ATP-sensitive potassium (KATP) channel plays a key role in controlling β-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by KATP channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the KATP channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1 -NBD1 and NBD2 - are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.
Grain yield, dry matter, and nitrogen accumulation in the grains of durum wheat and spring triticale cultivars grown in a Mediterranean environment
Authors: Giunta Francesco , Motzo Rosella
Published: January 2005

Comparisons among species can be a valuable approach to identifying traits important for plant breeding. Differences between 2 durum wheat (Duilio and Creso) and 1 triticale (Antares) cultivar have been analysed in a 2-year field trial in Sardinia (Italy), in order to define a more productive durum wheat ideotype for Mediterranean-type environments. The greater grain yield (569 v. 447 g/m2) and the lower protein percentage (9.2 v. 10.6%) of triticale cv. Antares compared with the durum wheat cultivars, at a similar level of biomass produced at heading, were analysed in terms of number of grains per unit surface and rate and duration of dry matter (DM) and nitrogen (N) accumulation, calculated from a logistic curve. When the single grains were considered, Antares showed a lower rate but a longer duration of DM and N accumulation in the more favourable season, resulting in lower DM (40 v. 54 mg) and N (0.7 v. 1.0 mg) contents in the grain. On the other hand, when data were expressed on a per unit surface basis, the greater spike fertility of Antares (53 v. 39 grains per spike) and its longer duration of accumulation, were responsible for similar or even greater amounts of DM and N accumulated in the grains per m2. Growth rate of single grains, although able to explain differences in single grain weight, cannot explain differences in grain weight per m2 and hence in yield, which mainly result from variation in the number of grains per spike.
Nitrogen percentage of the grains decreased from the maximum values observed at the beginning of grain filling, until a constant final value attained before the end of DM and N accumulation. Rate is more important than duration in determining the quality characteristics of grains, as higher grain weights and protein percentages correspond to higher rates of DM and N accumulation.
Orthopaedic measurements with computed radiography: Methodological development, accuracy, and radiation dose with special reference to the weight-bearing lower extremity and the dislocating patella
Authors: Sanfridsson Jan
Published: January 2005

Sanfridsson J.: Orthopaedic measurements with computed radiography. Methodological development, accuracy, and radiation dose with special reference to the weight-bearing lower extremity and the dislocating patella. Lund 2001. ISBN 87-16-16449-0.

The overall aim of this study was to develop and evaluate a measurement system for computed radiography (CR) and Picture Archiving and Communication Systems (PACS), permitting measurements of long distances and angles in and between related images. The developed measurement system, which was based on the QUESTOR Precision Radiography (QPR) system, was applied to the weight-bearing knee with special reference to the dislocating patella.

The QPR system modified for CR fulfilled the criteria for measuring the weight-bearing knee.

The special measuring assistance tools that were developed were important for the implementation of CR and PACS, particularly in workstations programmed for musculoskeletal radiology.

The energy imparted to the patient was reduced by 98% at the lowest exposure of the CR-system, compared with our conventional analogue method, without loss of diagnostic accuracy. The CR technique creates a possibility, to an extent not previously feasible, to differentiate the exposure parametres (and thus minimise the radiation dose to the patient) by carefully considering the purpose of the examination.

A radiographic method for measuring the rotation of the femur and the tibia, the, and the patellar translation was developed and applied to healthy volunteers. The introduced patellar variables have yielded new insights into the complex sequence of motions between the femur, tibia, and patella.

The patients with a dislocating patella were subdivided into one "clean" group of spontaneous dislocations and one group with various traumas in the history, which thus resulted in two groups with distinct radiographic differences. The Q-angle was decreased in knees that had suffered dislocations, and the traditional surgical treatment with a further reduction of the Q-angle must be challenged.

The use of clinical measurements of the Q-angle was not an optimal way to evaluate the mechanical alignment in the patellofemoral joint under physiological conditions.

In this study, we have proved that the developed method for CR and PACS is a useful technique for measurements in and between related images, and is superior to the conventional analogue technique .
Regulation of α5β1 integrin conformation and function by urokinase receptor binding
Authors: Wei Ying , Czekay Ralf-Peter , Robillard Liliane , Kugler Matthias C. , Zhang Feng , Kim Kevin K. , Xiong Jian-ping , Humphries Martin J. , Chapman Harold A.
Published: January 2005

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with β1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the β-propeller of α3β1 empowers vitronectin adhesion by this integrin. How uPAR modifies other β1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5β1 and rather than blocking, renders fibronectin (Fn) binding by α5β1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5β1–uPAR to Fn type III repeats 12–15 in addition to type III repeats 9–11 bound by α5β1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5β1 conformation. A β1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a β1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5β1. Direct binding and regulation of α5β1 by uPAR implies a modified “bent” integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.
Molecular mechanisms of invadopodium formation
Authors: Yamaguchi Hideki , Lorenz Mike , Kempiak Stephan , Sarmiento Corina , Coniglio Salvatore , Symons Marc , Segall Jeffrey , Eddy Robert , Miki Hiroaki , Takenawa Tadaomi , Condeelis John
Published: January 2005

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.
The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex
Authors: LaGrassa Tracy J. , Ungermann Christian
Published: January 2005

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.
Myosin V attachment to cargo requires the tight association of two functional subdomains
Authors: Pashkova Natasha , Catlett Natalie L. , Novak Jennifer L. , Wu Guanming , Lu Renne , Cohen Robert E. , Weisman Lois S.
Published: January 2005

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle–specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.
Concentric zones of active RhoA and Cdc42 around single cell wounds
Authors: Benink Hélène A. , Bement William M.
Published: January 2005

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.
The CD19 Molecule is Crucial for MID‐Dependent Activation of Tonsillar B Cells from Children
Authors: Hadzic R. , Forsgren A. , Cardell L.‐O. , Riesbeck K. , Wingren A. G.
Published: January 2005

The Moraxella immunoglobulin (Ig) D‐binding protein (MID) induces a strong proliferative response in human peripheral blood IgD+ B cells from adults isolated by positive selection using anti‐CD19‐conjugated microbeads. Here, we show that tonsillar B cells from children isolated with positive selection are unable to respond to MID stimulation. The proliferative response was very low or absent at various concentrations of MID tested and at different time points analysed, whereas the MID response of tonsillar B cells from adults isolated with positive selection was considerably higher. Tonsillar B cells from children isolated with positive selection responded to formalin‐fixed preparations of Moraxella catarrhalis and Staphylococcus aureus Cowan strain I. In comparison to cells isolated with positive selection, a much higher proliferative response was recorded in tonsillar B cells from children isolated with negative selection, indicating that occupation of the CD19 molecule (i.e. positive selection) inhibited the response. Indeed, the addition of anti‐CD19 monoclonal antibodies (MoAb) to MID‐activated tonsillar B cells from children isolated with negative selection strongly inhibited the proliferative response. In contrast, anti‐CD21 MoAb at the same concentration did only show a minor inhibition on the MID‐induced response. Pre‐incubation of tonsillar B cells isolated from children with anti‐CD19 or anti‐CD21 MoAb did not affect the binding of biotin‐conjugated MID as analysed by flow cytometry. These results suggest that MID‐activated tonsillar B cells from children have a strong requirement for signalling through the CD19 molecule. Future experiments will further reveal the importance of CD19 and possibly other molecules for optimal activation of tonsillar B cells isolated from both children and adults.
Diminished Th1‐Like Response to Autoantigens in Children with a High Risk of Developing Type 1 Diabetes
Authors: Karlsson Faresjö M. G. E. , Ludvigsson J.
Published: January 2005

The exact role of T‐helper (Th) cells that precede the clinical manifestation of type 1 diabetes remains unclear. The aim of this investigation was to study the Th1‐ and Th2‐like profile in children and adults with high risk of developing the disease. Peripheral blood mononuclear cells were collected from high‐risk children and adults and from healthy individuals matched for age and gender. Using the sensitive enzyme‐linked immunospot (ELISPOT) technique to divide Th1‐ from Th2‐like lymphocytes, secretion of interferon‐γ (IFN‐γ) and interleukin‐4 was analysed from lymphocytes spontaneously and after in vitro stimulation with different antigens, based on present paradigms regarding the pathogenesis of type 1 diabetes. Compared to the response observed in healthy individuals, we found that individuals with a high risk of developing type 1 diabetes, especially children, responded with less IFN‐γ secretion to the three autoantigens glutamic acid decarboxylase 65 (GAD65), insulin and tyrosinphosphatase (IA‐2). Thus, a diminished Th1‐like response by in vitro autoantigen stimulation was observed in especially children with a high risk of developing type 1 diabetes. Reduced Th1/Th2 response was related to signs of β cell exhaustion.