GNE Is Involved in the Early Development of Skeletal and Cardiac Muscle

UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during in vitro embryogenesis by comparing the developmental profile in culture of embryonic stem cells (ES) from wild type and from GNE KO E3.5 mice embryos, during 45 days. Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease

Dental profile of patients with Gaucher disease

BACKGROUND: This study was conducted to determine whether patients with Gaucher disease had significant dental pathology because of abnormal bone structure, pancytopenia, and coagulation abnormalities. METHODS: Each patient received a complete oral and periodontal examination in addition to a routine hematological evaluation. RESULTS: Gaucher patients had significantly fewer carious lesions than otherwise healthy carriers. Despite prevalence of anemia, there was no increase in gingival disease; despite the high incidence of thrombocytopenia, gingival bleeding was not noted; and despite radiological evidence of bone involvement, there was no greater incidence loss of teeth or clinical tooth mobility. CONCLUSIONS: These data represent the first survey of the oral health of a large cohort of patients with Gaucher disease. It is a pilot study of a unique population and the results of the investigation are indications for further research. Based on our findings, we recommend regular oral examinations with appropriate dental treatment for patients with Gaucher disease as for other individuals. Consultation between the dentist and physician, preferably one with experience with Gaucher disease, should be considered when surgical procedures are planned

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease

Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>Heparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells.</p> <p>Methods</p> <p>Three heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The <it>in vitro </it>invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.</p> <p>Results</p> <p>Transfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the <it>in vitro </it>invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the <it>in vitro </it>angiogenesis of cancer cells in a dose-dependent manner.</p> <p>Conclusions</p> <p>These results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells <it>in vitro</it>, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer.</p

Predicting In Vivo Efficacy of Potential Restenosis Therapies by Cell Culture Studies: Species-Dependent Susceptibility of Vascular Smooth Muscle Cells

Although drug-eluting stents (DES) are successfully utilized for restenosis therapy, the development of local and systemic therapeutic means including nanoparticles (NP) continues. Lack of correlation between in vitro and in vivo studies is one of the major drawbacks in developing new drug delivery systems. The present study was designed to examine the applicability of the arterial explant outgrowth model, and of smooth muscle cells (SMC) cultures for prescreening of possible drugs. Elucidation of different species sensitivity (rat, rabbit, porcine and human) to diverse drugs (tyrphostins, heparin and bisphsophonates) and a delivery system (nanoparticles) could provide a valuable screening tool for further in vivo studies. The anticipated sensitivity ranking from the explant outgrowth model and SMC mitotic rates (porcine>rat>>rabbit>human) do not correlate with the observed relative sensitivity of those animals to antiproliferative therapy in restenosis models (rat≥rabbit>porcine>human). Similarly, the inhibitory profile of the various antirestenotic drugs in SMC cultures (rabbit>porcine>rat>>human) do not correlate with animal studies, the rabbit- and porcine-derived SMC being highly sensitive. The validity of in vitro culture studies for the screening of controlled release delivery systems such as nanoparticles is limited. It is suggested that prescreening studies of possible drug candidates for restenosis therapy should include both SMC cell cultures of rat and human, appropriately designed with a suitable serum

Ets-1 Confers Cranial Features on Neural Crest Delamination

Neural crest cells (NCC) have the particularity to invade the environment where they differentiate after separation from the neuroepithelium. This process, called delamination, is strikingly different between cranial and trunk NCCs. If signalings controlling slow trunk delamination start being deciphered, mechanisms leading to massive and rapid cranial outflow are poorly documented. Here, we show that the chick cranial NCCs delamination is the result of two events: a substantial cell mobilization and an epithelium to mesenchyme transition (EMT). We demonstrate that ets-1, a transcription factor specifically expressed in cranial NCCs, is responsible for the former event by recruiting massively cranial premigratory NCCs independently of the S-phase of the cell cycle and by leading the gathered cells to straddle the basal lamina. However, it does not promote the EMT process alone but can cooperate with snail-2 (previously called slug) to this event. Altogether, these data lead us to propose that ets-1 plays a pivotal role in conferring specific cephalic characteristics on NCC delamination

Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

Human neutrophils (PMNs) which have been incubated with lipoteichoic acid (LTA) from group A streptococci generated large amounts of Superoxide (O 2 − chemiluminescence and hydrogen peroxide when challenged with anti-LTA antibodies. Cytochalasin B further enhanced O 2 * generation. The onset of Of generation by the LTA-anti-LTA complexes was much faster than that induced by BSA-anti-BSA complexes. LTA-treated PMNs generated much less O 2 * when challenged with BSA complexes, suggesting that LTA might have blocked, nonspecifically, some of the Fc receptors on PMNs. PMNs treated with LTA-anti-LTA complexes further interacted with bystander nonsensitized PMNs resulting in enhanced Of generation, suggesting that small numbers of LTA-sensitized PMNs might recruit additional PMNs to participate in the generation of toxic oxygen species. Protelolytic enzyme treatment of PMNs further enhanced the generation of O 2 − by PMNs treated with LTA-anti-LTA. Superoxide generation could also be induced when PMNs and anti-LTA antibodies interacted with target cells (fibroblasts, epithelial cells) pretreated with LTA. This effect was also further enhanced by pretreatment of the target cells with proteases. PMNs incubated with LTA released lysosomal enzymes following treatment with anti-LTA antibodies. The amounts of phosphatase, Β -glucoronidase, N -acetylglucosaminidase, mannosidase, and lysozyme release by LTA-anti-LTA complexes were much smaller than those released by antibody or histone-opsonized streptococci, suggesting that opsonized particles are more efficient lysosomal enzyme releasers. However, since the amounts of O 2 − generated by the LTA complexes equaled those generated by the opsonized particles, it is assumed that the signals for triggering a respiratory burst and lysosomal enzyme secretion might be different.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44498/1/10753_2004_Article_BF00914316.pd