Expression analysis of recombinant lysyl oxidase (LOX) in myofibroblastlike cells

Lysyl oxidase (LOX), originally known as the enzyme required for initiation of covalent cross-linking in collagens and elastin, is now known to be a member of a family of genetically related proteins. LOX, or a related protein, has also been localized intracellularly, both in association with the cytoskeleton and in the cell nucleus. To determine the structural requirements for secretion, maturation, and nuclear location of LOX in a cellular context, we have devised an homologous cell model for expression of the recombinant protein. Murine recombinant LOX was expressed in 3T6-5 myofibroblast-like cells as a 51-kD precursor, which was observed in the cytoplasm but not in the nucleus. To investigate whether potential alternative translation initiation sites were involved in specifying a nuclear form of LOX, constructs mutated or deleted for ATG(+1) were used, but alternative initiation at CTG(-315) or ATG(+418) did not lead to the expression of intranuclear forms. Residues 23 to 157 of the proregion were essential for export of the precursor, while mutation of the putative site for maturation by procollagen C-proteinase abolished processing to the mature form of the enzyme. Cross-linking of collagen, as measured by pyridinoline analysis, increased twofold with the recombinant cells, compared to non-transfected controls. This shows the specific contribution of LOX, as opposed to other genetic forms of the enzyme, to cross-linking in a cellular context.     

Model-based interpretation of cardiac beats by evolutionary algorithms: signal and model interaction

This paper presents a new approach for cardiac beat interpretation, based on a direct integration between a model and observed ECG signals. Physiological knowledge is represented by means of a semi-quantitative model of the cardiac electrical activity. The interpretation of cardiac beats is formalized as an optimization problem, by minimizing an error function defined between the model's output and the observations. Evolutionary algorithms (EAs) are used as the search technique in order to obtain the set of model parameters reproducing at best the observed phenomena. Examples of model adaptation to three different kinds of cardiac beats are presented. Preliminary results show the potentiality of this approach to reproduce and explain complex pathological disorders and to better localize their origin.     

Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.     

CT quantification of pulmonary emphysema: assessment of lung structure and function

Accurate diagnosis and quantification of pulmonary emphysema in vivo is important to understand the natural history of the disease, to assess the extent of the disease, and to evaluate and follow-up therapeutic interventions. Because pulmonary emphysema is defined by pathology, new diagnostic methods for quantification should be validated by reference to pathological and histological standards. Recent studies have addressed the capability of computed tomography (CT) to accurately quantify pulmonary emphysema. These studies that have been overviewed in this article have been based on CT scans obtained after deep inspiration or expiration, on subjective visual grading, and on objective measurements of attenuation values by using dedicated software providing numerical data on two-dimensional and on three-dimensional approaches, and compared CT data with pulmonary function tests. More recently, fractal and textural analyses were applied to CT scans to assess the presence, extent, and types of emphysema. Quantitative CT has already been used in patient selection for surgical treatment of pulmonary emphysema and in pharmacotherapeutical trials. However, despite numerous and extensive studies already available, this technique has not yet been standardized, and important questions about how to best use CT for the quantification of pulmonary emphysema remain to be addressed.     

Bullous pemphigoid,primary biliary cirrhosis and vitiligo: a multiple autoimmune syndrome?

Bullous pemphigoid is the most frequent autoimmune blistering skin disease. There have been few reports of an association with primary biliary cirrhosis and vitiligo. We report the simultaneous occurrence of bullous pemphigoid and primary biliary cirrhosis in an 86-year-old patient who also suffered from vitiligo. Multiple autoimmune syndrome, proposed by Humbert and Dupond, can be divided into three groups based on preferential associations of autoimmune disorders. The association of bullous pemphigoid, cirrhosis biliary primary and vitiligo has been reported three times in the literature. This association is probably not fortuitous and suggests a pathogenic relationship. This association is not typical of the multiple autoimmune syndrome as defined by Humbert and Dupond but the collection of such observations may contribute to revise the classification of autoimmune disease and provide a better understanding of the pathophysiological mechanisms of autoimmunity.     

Smooth muscle dysfunction in resistance arteries of the staggerer mouse, a mutant of the nuclear receptor RORalpha

Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. The mouse mutant staggerer (sg/sg) carries a deletion within the RORalpha gene. RORalpha plays a major role in cellular differentiation during development and growth. In the present study, we found a lower mean arterial blood pressure in sg/sg than in +/+ mice (80.1+/-1.2 and 87.0+/-0.9 mm Hg, respectively; P<0.0002) and a smaller increase in blood pressure after in vivo injections of phenylephrine. To elucidate the mechanisms responsible for this phenotype, we investigated the vascular reactivity of large vessels (aorta and carotid arteries) and small resistance mesenteric arteries in response to mechanical forces or vasoactive agents. Arteries from sg/sg and +/+ mice were studied in vitro in arteriographs. Vascular responses of large vessels to all stimuli were similar in both groups. However, we found a markedly altered vascular function in mesenteric arteries from sg/sg mice. Flow-induced dilation, pressure-induced myogenic tone, responses to endothelium-dependent or -independent vasodilators, and responses to vasoconstrictors were significantly reduced in sg/sg compared with +/+ mice. We also determined by Western blot analysis the expression of smooth muscle (SM)-myosin, calponin, and heavy (h)-caldesmon, in large and small arteries of sg/sg and +/+ mice, and found a marked decrease in the expression of these contractile proteins in mesenteric arteries of sg/sg mice. Our findings provide the first evidence that functional RORalpha is required for normal contractile phenotype of smooth muscle cells (SMCs) in small resistance arteries and suggest that RORalpha might be involved in the differentiation of SMCs in mesenteric arteries.