Are Small GTPases Signal Hubs in Sugar-Mediated Induction of Fructan Biosynthesis?

External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events

Monocytes and their pathophysiological role in Crohn's disease

Our immune system shows a stringent dichotomy, on the one hand displaying tolerance towards commensal bacteria, but on the other hand vigorously combating pathogens. Under normal conditions the balance between flora tolerance and active immunity is maintained via a plethora of dynamic feedback mechanisms. If, however, the balancing act goes faulty, an inappropriate immune reaction towards an otherwise harmless intestinal flora causes disease, Crohn's disease for example. Recent developments in the immunology and genetics of mucosal diseases suggest that monocytes and their derivative cells play an important role in the pathophysiology of Crohn's disease. In our review, we summarize the recent studies to discuss the dual function of monocytes - on the one hand the impaired monocyte function initiating Crohn's disease, and on the other hand the overactivation of monocytes and adaptive immunity maintaining the disease. With a view to developing new therapies, both aspects of monocyte functions need to be taken into account

A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein.

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction

Expression and activation of NF-kappa B in the antrum of the human stomach

Both in vitro studies and experiments in mice suggest a key role for transcription factor NF-kappa B as a mediator of mucosal inflammation. Experiments in vitro show that NF-kappa B activation may be a critical event in the production of proinflammatory molecules in Helicobacter pylori-associated gastritis. This study examines the expression and activity of NF-kappa B in situ in antral biopsies of 69 consecutive patients with immunohistochemical techniques. In the uninflamed stomach, NF-kappa B was highly expressed and active in a subset of epithelial cells, which were identified as predominantly G cells. In accordance with this activity, G cells were shown to express high levels of the NF-kappa B target cytokine TNF-alpha, a well-documented stimulator of gastrin production. In patients with H. pylori-associated gastritis, NF-kappa B activity was markedly enhanced. Activation occurred preferentially in the epithelial cells. The number of cells showing activated NF-kappa B correlated with the activity of gastritis, a measure of neutrophil influx, whereas no correlation was found with the chronicity of inflammation, a measure of the presence of mononuclear inflammatory cells. This correlation is direct evidence of the importance of NF-kappa B-dependent signal transduction for neutrophil influx in H. pylori-associated gastriti