Beyond UV radiation: a skin under challenge.

Since ancient times, human beings have been trying to protect their skin against the adverse effects of the sun. From the first mineral sunscreens used by Egyptians, to the current more sophisticated ultraviolet (UVA/UVB) organic sunscreens, progress has been made in terms of sun protection and deeper knowledge of skin physiology has been acquired in the process. The solar spectrum is composed of radiations of various wavelengths having specific, as well as overlapping effects on skin. UVB is mainly responsible for sunburn and DNA dimer formation that can lead to mutation. UVA generates oxidative reactions affecting DNA, proteins and lipids, and is also immunosuppressive. Recently, visible light and infrared radiation (IR) have been associated with oxidative damage and IR has been additionally linked to adverse heat effects on skin. Numerous other extrinsic factors, related to environment and lifestyle, also affect the appearance of skin, precipitating ageing. New molecular mechanisms linking sun and environmental factors to skin ageing have been identified: IR affects mitochondrial integrity and specific heat receptors also mediate some of its effects, tryptophan is a chromophore for UVB, and the aryl hydrocarbon receptor (AhR) is activated by light and xenobiotics to alter skin physiology. Integrating all these new elements is changing the way we think about skin extrinsic ageing. Is UVA/UVB sunscreen protection still enough for our skin?

Persistent polyclonal B-cell lymphocytosis: further evidence for a genetic disorder associated with B-cell abnormalities.

Persistent polyclonal B-cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B-cell population is also evidenced by the presence of multiple bcl-2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first-degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B-cell proliferation was shown in two additional siblings. Multiple bcl-2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first-degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co-factors.

Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions.

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.

Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney.

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.

Regulation of cytoskeletal functions by Rho small GTP-binding proteins in normal and cancer cells.

The actin cytoskeleton is involved in numerous cellular functions such as cell motility, mitogenesis, morphology, muscle contraction, cytokinesis, and establishment of cell polarity. The members of the Rho subfamily of small GTP-binding proteins emerge as key regulators of cytokeleton organization. Rho, Rac, and CDC42 are implicated in the regulation of actin microfilament organization of different cell structures, such as stress fibers linked to focal adhesions and membrane ruffles induced by extracellular stimuli. Rho proteins also regulate the activity of several enzymes involved in the formation of phospholipid derivatives, which could mediate their effect on the cytoskeleton. The activity of Rho proteins is regulated by many nucleotide exchange factors and GTPase-activating proteins, some of which are oncogene products, and other disease-associated proteins. The potential role of these small GTP-binding proteins in carcinogenesis is suggested by the actin reorganization seen in transforming cells and by the need for functional Rho proteins in Ras mitogenic activation.

Immunochemical characterization of L-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues.

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

Importance of subtyping in psychopharmacogenetic studies.

Research on the importance of genetic and environmental risk factors (multifactorial) in the etiology of mental disorders and substance use disorders (alcoholism) is now growing rapidly. Biological and psychological studies in this field should include basic concepts of medical genetics and take heterogeneity (multidimensionality) of mental disorders into account. One of the best means to cope with heterogeneity is to redefine the diagnostic phenotype at different levels: biochemical, behavioral, familial. Data are presented where the introduction of different quantitative definitions of the phenotype and their analysis by multivariate technics are used for better understanding of alcoholic disorders.