Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.     

Expression of Arabidopsis APETALA1 in tomato reduces its vegetative cycle without affecting plant production

Important agronomic traits such as fruit quality, harvesting efficiency or production largely depend on flowering time. We have analysed the effect of the overexpression of the Arabidopsis APETALA1 MADS-box gene on vegetative and reproductive growth of tomato. Constitutive expression of APETALA1 in tomato plants has major effects on the length of their growth cycle as well as on their growth habit. Transgenic tomato plants initiated flowering after the production of 6 vegetative nodes as compared to 11 nodes for the wild type plants. Most of tomato 35S:AP1 plants also showed determinate growth habit, similar to the phenotype of self pruning tomato mutants, as well as an initial reduction of their axillary growth. Moreover, development and fertility of flowers were not affected in plants expressing AP1. Consequently, fruit formation in transgenic plants grown under greenhouse conditions occurred normally, which permitted a similar fruit yield compared to control plants. Since traits conferred by AP1 expression are dominant, its expression in tomato breeding lines could provide advantages for the development of new hybrid varieties with shorter generation time, determinate growth, and reduced pruning requirements.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Protease-activated receptor (PAR)-induced interleukin-8 production in airway epithelial cells requires activation of MAP kinases p44/42 and JNK

The mechanism underlying protease-activated receptor (PAR)-activation and subsequent interleukin (IL)-8 production in airway epithelial cells is not yet understood. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 airway epithelial cells. We studied the consequence of activation of PARs with simultaneous exposure to LPS. Thrombin, PAR-2-activating peptide and LPS, were tested alone and in combination. They induced significant synthesis of IL-8. However, only activation of PAR triggered phosphorylation of ERK1/2 and JNK. The application of the inhibitors of these two MAPKs resulted in reduction of IL-8 production. Thus, activation of PARs but not stimulation with LPS leads to ERK1/2 and JNK-mediated production of IL-8.     

Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells

Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase both of IFN-gamma- (250 U/mL) or TNF-alpha- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.     

GM2 activator protein inhibits platelet activating factor signaling in rats

Platelet activating factor (PAF), an endogenous bioactive phospholipid, has been documented as a pivotal mediator in the inflammatory cascade underlying the pathogenesis of many diseases including necrotizing enterocolitis. Much effort has been directed towards finding an effective in vivo inhibitor of PAF signaling. Here, we report that a small, highly stable, lysosomal lipid transport protein, the GM2 activator protein (GM2AP) is able to inhibit the inflammatory processes otherwise initiated by PAF in a rat model of necrotizing enterocolitis. Based on behavioral observations, gross anatomical observations at necropsy, histopathology and immunocytochemistry, the administration of recombinant GM2AP inhibits the devastating gastrointestinal necrosis resulting from the injection of rats with LPS and PAF. Recombinant GM2AP treatment not only markedly decrease tissue destruction, but also helped to maintain tight junction integrity at the gastrointestinal level as judged by contiguous Zonula Occludens-1 staining of the epithelial layer lining the crypts.     

The role of interleukin-18 in pancreatitis and pancreatic cancer

Originally described as an interferon (IFN)-γ-inducing factor, interleukin (IL)-18 has been reported to be involved in Th1 and Th2 immune responses, as well as in activation of NK cells and macrophages. There is convincing evidence that IL-18 plays an important role in various pathologies (i.e. inflammatory diseases, cancer, chronic obstructive pulmonary disease, Crohn's disease and others). Recently, IL-18 has also been shown to execute specific effects in pancreatic diseases, including acute and chronic pancreatitis, as well as pancreatic cancer. The aim of this study was to give a profound review of recent data on the role of IL-18 and its potential as a therapeutic target in pancreatic diseases. The existing data on this topic are in part controversial and will be discussed in detail. Future studies should aim to confirm and clarify the role of IL-18 in pancreatic diseases and unravel their molecular mechanisms.