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Among the different classes of integral membrane proteins, G protein-coupled receptors (GPCR) constitute the largest family. They are involved in most essential physiological functions and particularly play a key role in cell-to-cell communication and sensory signal transduction. They represent targets for approximately 30% of currently marketed drugs. In order to better understand their functioning, define their tridimensional structure and develop novel selective and efficient therapeutic compounds, it is crucial to purify these proteins for a full characterization. However, this biochemical step is not trivial since GPCR are present in membranes at very low levels and they require detergents to be extracted from their natural lipid environment and be handled as functional proteins. No universal strategy for GPCR production, purification and stabilization is currently available; each single GPCR possesses a unique set of physicochemical characteristics, preference for some detergents upon solubilization and specific conditions for purification. During the last decade, major breakthroughs regarding overexpression, purification and above all GPCR stabilization, thanks to amphipols and nanodiscs, opened very exciting perspectives for structural and dynamic investigations of these membrane proteins. The aim of this chapter is to provide an overview of the different aspects of GPCR handling., (© 2012 médecine/sciences – Inserm / SRMS.)

The pathophysiology of pelvic floor disorders still remains not well understood. Increasing age as well as vaginal multiparity are the main commonly accepted factors. The hypothesis of a defect of connective tissues of the pelvic floor with aging due to collagen deficiency and/or elastic fiber degradation is often highlighted. The issue of a potential protective role of HRT is also discussed although the recent results from the WHI would suggest a negative impact of HRT on urinary incontinence, especially when HRT is initiated in elderly women, far from the menopause. Nevertheless, environmental factors cannot explain the full pathogenesis of pelvic organ prolapse (POP) and the contribution of genetic factors to the development of pelvic floor disorders is widely recognized. Support for a genetic influence on POP derives from reports suggesting that heritability is a strong contributing factor and a familial history of POP is considered as a classical risk factor. However, the characterization of the underlying molecular mechanisms remains limited, since POP may be considered the end result of a multifactorial process leading to destruction of vaginal wall connective tissue. Experimental studies in mice with null mutations in the genes encoding different putative factors involved in elastic fibers remodeling and homeostasis are crucial in the understanding of the pathogenesis of POP. Mice with null mutation in the gene encoding lysyl oxidase-like 1 (LOXL1) or fibulin-5, demonstrate signs of elastinopathy including the development of a POP in the postpartum. Likewise, homeobox genes such as HOXA11, which are essential in the embryonic development of the urogenital tract might also be involved in the pathogenesis of POP. The better understanding of the underlying determinants of pelvic floor disorders with a special focus on genetic factors may offer new therapeutic strategies, in addition to or replacement of surgical procedures., (2010 Elsevier Masson SAS. All rights reserved.)

A meta-analysis of recent data from the literature underscores the considerable body of present knowledge concerning prostate carcinogenesis, in part due to the numerous molecular biology tools now at our disposal. As concerns early events, much interest is being paid to modifications in the expression of GSTP1 and NKX3.1 occurring in totipotent stem cell populations. The discovery of fusion genes implicating TMPRSS2 and ERG (and, on rare occasions, other ETS family transcription factors) constitutes a major advance. Under physiological androgenic stimulation, the presence of these fusion genes leads to overexpression of genes involved in cell growth and differentiation. Concomitantly, alterations in numerous signalling pathways (growth factors, Wnt-beta catenine, PI3K/Akt) are responsible for the onset of an aggressive tumor phenotype. Hormono-independence is currently explained by an amplification of, or mutations in, androgenic receptors. These are facilitated by genomic instabilities linked to alterations in proteins which regulate gene expression, such as EZH2, and by the influence of the tumor microenvironment. Disturbances in the interactions between tumor cells and the microenvironment contribute to local extension of the tumor. Changes in the expression of E-cadherin are responsible for modifications in cell adhesion to the extracellular matrix. The expression of metalloproteases and of angiogenic factors favors tumor dissemination. Finally, the bone tropism in prostate metastases is probably linked to osteomimetic properties of prostate tumor cells which are capable of expressing certain proteins involved in bone remodelling, such as Runx-2, BSP (bone sialoprotein) and BMP (bone morphogenetic protein). Numerous studies remain to be carried out in order to correlate the identified genetic profiles and molecular anomalies with tumor prognosis. Nevertheless, the possibility of decrypting these anomalies for use in therapeutic applications is encouraging.

The identification of the gene on which mutations cause cystic fibrosis has opened the way to the concept of gene therapy of this disease. Somatic gene therapy for cystic fibrosis should be directed to the epithelial cells of the bronchi where the biological defect is the most deleterious. In order to have the normal DNA coding sequence penetrate the bronchial epithelial cells of a patient, a gene vector is necessary, and recombinant adenoviruses have been the subject of extensive studies in this regard. Data are available from many laboratories as well as from several phase I clinical trials. Successes and limits of this approach are reviewed here. The advantage of non-viral vectors, in particular cationic liposomes, as an alternative to recombinant adenoviruses is briefly discussed. Significant advances still need to be made before gene therapy becomes a reality in cystic fibrosis. These future improvements should include the construction of more efficient gene vectors, and also the development of better tools for the evaluation of a biological effect of gene therapy in individual patients.

Since forty years, many allergens from different species responsible for allergies, have been purified and sometimes identified using classical methods of protein chemistry. For the first time in 1988, molecular biology technologies were applied to allergens, namely to a major allergen of the mite, Dermatophagoides pteronyssinus. During the recent years many other allergens have been produced as recombinant proteins expressed in bacteria or yeast leading to the growing family of recombinant allergens. This talk presents a general view on the allergen cloning procedure and gives an account on future applications of recombinant allergens in both fields of fundamental and practical allergy.

The search for human Hb variants exhibiting a low oxygen affinity without requiring 2,3-diphosphoglycerate, together with a low oxygation rate, is of an increased interest in the view of producing an artificial oxygen carrier. We have synthesized the recombinant Hb beta 41Phe-->Tyr (rHb beta F41Y) which exhibits a low oxygen affinity due to the stabilization of the deoxy state of tetrameric Hb [1]. Interestingly, the autooxydation rate for this mutant is similar to that for Hb A. We have associated the mutation beta F41Y with the naturally occurring beta 82Lys-->Asp substitution (Hb Providence) known to be responsible for a low oxygen affinity [2]. The second-site mutation further decreases the oxygen affinity of the rHb beta F41Y. The effects of the beta F41Y and K82D mutations are additive, resulting in a four fold decrease in oxygen affinity of the artificial mutant Hb beta F41Y-K82D, compared to Hb A. In spite of the marked decrease in oxygen affinity, the autooxydation rate is 2- to 3 fold larger than that of Hb A. These data show that it is possible to adjust the oxygen binding properties of human Hb by using protein engineering methods. Because of the low oxygen affinity coexisting with a moderately increased autooxydation rate, this variant is a good candidate for the development of a Hb-based oxygen carrier.