Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes

Dystropbin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystropbin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (l-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and l-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and l-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.     

Giant aneurysm of an aortocoronary venous bypass graft compressing the right ventricle

A 69-year-old man with a history of coronary artery bypass grafting and a recent inferoposterior myocardial infarction presented to the hospital for diagnostic coronary angiography. Physical examination, laboratory analyses, coronary angiography, echocardiography, and CT scan were performed. A giant aneurysm of the aortocoronary venous bypass graft, associated with compression of the right side of the heart, was revealed. After surgical resection and replacement of the venous graft the patient died due to right ventricular failure. (Neth Heart J 2007;15:252-4.)     

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

VARAN: a web server for variability analysis of DNA microarray experiments

SUMMARY: Here, we describe a tool for VARiability Analysis of DNA microarrays experiments (VARAN), a freely available Web server that performs a signal intensity based analysis of the log2 expression ratio variability deduced from DNA microarray data (one or two channels). Two modules are proposed: VARAN generator to compute a sliding windows analysis of the experimental variability (mean and SD) and VARAN analyzer to compare experimental data with an asymptotic variability model previously built with the generator module from control experiments. Both modules provide normalized intensity signals with five possible methods, log ratio values and a list of genes showing significant variations between conditions. AVAILABILITY: http://www.bionet.espci.fr/varan/ SUPPLEMENTARY INFORMATION: http://www.bionet.espci.fr/varan/help.html     

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

Necrosis- and apoptosis-related Met cleavages have divergent functional consequences

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.Met is a receptor tyrosine kinase expressed predominantly by epithelial cells and activated by its stromal ligand, hepatocyte growth factor/scatter factor (HGF/SF). Met activation stimulates a biological program called invasive growth,¹ involving survival, proliferation, invasion, and morphogenesis of epithelial cells. Ligand-stimulated Met acts, furthermore, as an angiogenic and neurotrophic factor.^{2, 3} HGF/SF and Met are essential to several steps of embryogenesis, experiments on transgenic mice having shown that they are necessary for formation of the placenta, liver, limb muscle, neurons, and lung airspace.^{4, 5, 6, 7, 8} In adults, HGF/SF and Met promote regeneration of several organs, including the liver, kidneys, and thymus.^{9, 10, 11, 12, 13}Aberrant Met and HGF/SF signaling contributes to promoting tumorigenesis and metastasis (for review see Furlan et al.).¹⁴ A direct link between Met and cancer has been evidenced by observation of Met germinal mutations linked to hereditary papillary renal carcinoma.¹⁵ Met and/or HGF/SF are/is also overexpressed in several human cancers.¹⁶ Given its important oncogenic activity, Met is the target of many therapeutic agents currently under clinical investigation.¹⁷Downregulation of Met following its activation by HGF/SF is an important negative regulatory mechanism preventing receptor overactivation. We have previously shown that Met expression and activity are also controlled by proteolytic cleavages. Under steady-state conditions, Met is processed by PS-RIP (presenilin-regulated intramembrane proteolysis).^{18, 19} This process involves cleavage of Met within its extracellular juxtamembrane domain by A disintegrin and metalloproteinase (ADAM)-10,²⁰ generating a soluble N-terminal fragment (Met-NTF), which is released into the extracellular space, and a membrane-anchored C-terminal Met fragment (Met-CTF). The latter is in turn efficiently degraded by the lysosome and by further γ-secretase cleavages. Constitutive degradation of the Met receptor by PS-RIP contributes to regulating its half-life.Under apoptotic conditions, Met is cleaved by caspases²¹ within its C-terminal tail and its intracellular juxtamembrane domain. These cleavages remove the C-terminal tail of Met and separate the extracellular ligand-binding domain from the intracellular kinase domain. The generated 40-kDa intracellular fragment, previously called ‘p40Met'' and here called p40Met^caspase, can increase cell death by promoting mitochondrial permeabilization.^{22, 23} Removal of the C-terminal tail of Met is required for the efficient pro-apoptotic action of the fragment. This pro-apoptotic function of Met makes it a member of the dependence receptor family.²⁴ Met cleavages are illustrated in Figure 6a.Although the mechanisms underlying apoptosis have been studied extensively, necrosis has only recently been described as a regulated cell death mechanism.²⁵ Necrosis is an adenosine triphosphate (ATP)-independent cell death mechanism featuring early plasma membrane and organelle disruption. Many pathways can lead to cell necrosis, including calcium overload. This type of cell stress has been amply described in the nervous system, where an increase in intracellular calcium results in neuronal injury and neurodegenerative diseases. In many other cell types, calcium ionophores such as ionomycin can induce rapid necrosis. An increase in intracellular calcium triggers activation of several proteases, including calpains and cathepsins.^{26, 27, 28} Calpains are calcium-dependent proteases capable of cleaving multiple substrates and involved in regulating various cellular processes, including migration, autophagy, apoptosis, and necrosis. Interestingly, the effector role of calpains during necrosis is reminiscent of the function of caspases during apoptosis. Caspases are directly involved in morphological changes observed during apoptosis, while calpains can cleave cytoskeletal proteins such as spectrin and tubulin, thus favoring dismantling of cell structure during necrosis.^{29, 30, 31}Although apoptosis resistance is a hallmark of many cancer cells,³² some such cells remain sensitive to other cell death processes, including necrosis.³³ Thus, a better understanding of the mechanisms underlying necrosis is important, as it could help to elaborate novel therapeutic strategies. Here we show that calcium stress induced by calcium ionophores triggers Met degradation during necrotic cell death. This loss of Met receptor occurs early during the process and is mediated by Met cleavages: by calpains in its intracellular part and by metalloproteases in its extracellular part. These cleavages generate an extracellular fragment and an intracellular fragment with a molecular weight close to that of p40Met^caspase.     

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.