A comparative study between human skin substitutes and normal human skin using Raman microspectroscopy.

Research in the field of bioengineered skin substitutes is motivated by the need to find new dressings for people affected by skin injuries (burns, diabetic ulcers), and to develop adequate skin models to test new formulations developed in vitro. Thanks to advances in tissue engineering, it is now possible to produce human skin substitutes without any exogenous material, using the self-assembly method developed by the Laboratoire d'Organogénèse Expérimentale. These human skin substitutes consist of a dermis and a stratified epidermis (stratum corneum and living epidermis). Raman microspectroscopy has been used to characterize and compare the molecular organization of skin substitutes with normal human skin. Our results confirm that the stratum corneum is a layer rich in lipids which are well ordered (trans conformers) in both substitutes and normal human skin. The amount of lipids decreases and more gauche conformers appear in the living epidermis in both cases. However, the results also show that there are fewer lipids in the substitutes and that the lipids are more organized in the normal human skin. Concerning the secondary structure of proteins and protein content, the data show that they are similar in the substitutes and in the normal human skin. In fact, the epidermis is rich in α-keratin, whereas the dermis contains mainly type I collagen.

Structure and membrane interactions of the ß-amyloid fragment 25-35 as viewed using spectroscopic approaches.

The ß-amyloid fragment peptide 25-35 (Aß(25-35)) is recognized as the cytotoxic sequence of the parent peptide Aß. However, it remains unclear whether its neurotoxicity originates from its fibrillar form, how it interacts with lipid membranes, and whether cholesterol modulates these interactions. These questions have been addressed at a molecular level using various microscopic and spectroscopic techniques. The data show that Aß(25-35) forms protofilaments at pH 7.4 at a concentration of 5 mM in the absence and presence of DMPC/DMPG model membranes. The peptide adopts a predominant aggregated ß-sheet conformation under these conditions. However, as the peptide concentration decreases, the ß-sheet structure tends to disappear for the benefit of ß-turns, suggesting that the peptide association is reversible. The ß-sheet structure formed by Aß(25-35) appears to be atypical and characterized by the absence of intermolecular dipolar coupling and by a parallel strand configuration. The data show that Aß(25-35)-phospholipid interactions are characterized by an increase in the conformational order of the lipid acyl chains and a change in the fluidity/elasticity of the bilayers. Concomitantly, the peptide seems to lose a few ß-sheet structures, which suggests that the interactions between Aß(25-35) and DMPC/DMPG membranes are partly driven by peptide concentration. Interactions indeed seem to occur when part of the peptides is not involved in protofilaments and increase as the proportion of the free peptide species increases. The interactions are very similar in the presence of cholesterol, except that the concentration effect of Aß(25-35) is cancelled, suggesting that Chol limits the penetration of the peptide inside the bilayers.

Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs.

Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as (31)P and (2)H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.

Prevalence, impact and attitudes toward lower gastrointestinal dysmotility and sensory symptoms, and their treatment in Canada: A descriptive study.

OBJECTIVES: To investigate the impact of lower gastrointestinal (GI) symptoms in the general Canadian population, and to explore patient satisfaction with traditional therapies and the level of patient interest in new treatments. patients and METHODS: Stage 1: A telephone survey of a weighted sample of 1000 adults (18 years of age or older) was conducted to determine the prevalence of five GI symptoms--abdominal pain, abdominal discomfort, bloating, constipation or constipation with occasional diarrhea- that were present for 12 weeks or more (not necessarily consecutive) over the past year. Respondents with only abdominal pain were excluded. Stage 2: A telephone survey of 689 women (18 to 64 years of age), experiencing the GI symptoms described in stage 1, was conducted to assess symptom impact and treatment satisfaction. RESULTS: Overall, 5.2% of the Canadian population (2.3% men and 7.9% women) experienced one or more lower GI symptoms (excluding those reporting abdominal pain alone). In stage 2, 26.2% of respondents had previously been diagnosed with irritable bowel syndrome. Overall, 78.1% of participants experienced two or more symptoms. Bloating was the most common symptom (75.3%) and abdominal pain the most bothersome and most severe. Over the previous three months, 13.2% of respondents missed work or school and 28.8% were less productive. At least one physician (average of 2.2 physicians) was consulted for symptoms in 80.9% of respondents. Of the 63.8% women receiving treatment, most used nonprescription products. Patients receiving prescription treatments for constipation were most often dissatisfied (75%). CONCLUSIONS: Abdominal pain and discomfort, bloating and constipation are common, frequently occurring symptoms in the Canadian population and have a high burden on work performance and health care seeking. Most patients were dissatisfied with traditional therapies.

Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases.

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efficiently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Interestingly, UGT2B10 and UGT2B11, which are considered as orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, conjugated 12-HETE, 15-HETE, and 13-HODE. In summary, our data showed that several members of UGT1A and UGT2B families are capable of converting LA and AA metabolites into glucuronide derivatives, which is considered an irreversible step to inactivation and elimination of endogenous substances from the body.