IRMPD Spectroscopy Sheds New (Infrared) Light on the Sulfate Pattern of Carbohydrates.

IR spectroscopy of gas-phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas-phase vibrational spectra in the near and mid-IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well-established DFT (Density Functionnal Theory) modeling. The proposed approach was used to elucidate the sulfate patterns in glycosaminoglycans, a ubiquitous class of mammalian carbohydrates, which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharides from heparin and chondroitin sources were resolved, highlighting the potential of infrared multiple photon dissociation spectroscopy as a novel structural tool for carbohydrates.

Self-organizing behaviour of glycosteroidal bolaphiles: insights into lipidic microsegregation.

In this article we describe work on the synthesis of bolaphile biomimics composed of glucose head groups and steroidal units linked together by a methylene chain of varying length. The condensed phases formed by self-organization of the products as a function of temperature were characterized by differential scanning calorimetry and thermal polarized light microscopy. The results of these studies show that the thermal stabilities of the lamellar mesophases formed vary linearly as a function of increasing aliphatic composition, which reflects a linear hydrophobic-hydrophilic balance with respect to transition temperatures.

Cirsimarin, a potent antilipogenic flavonoid, decreases fat deposition in mice intra-abdominal adipose tissue.

OBJECTIVE: We previously reported that the flavonoid cirsimarin exerts in vitro a strong lipolytic activity on isolated adipocytes. This study was therefore designed to evaluate in vivo the effects of cirsimarin on white adipose tissue (WAT) accretion in mice. METHODS: Male CD1 mice were injected daily with either vehicle (intraperitoneal (i.p.)) or cirsimarin (25 or 50 mg kg(-1) per day, i.p.) for 18 days. Mice were killed and fat pads weighted. Epididymal fat pads were used for cellularity measurement. Effects of cirsimarin treatment on lipolysis and lipogenesis in WAT were assessed. RESULTS: Mice treated with 25 or 50 mg kg(-1) per day cirsimarin showed a decrease in retroperitoneal (-29 and -37% respectively, P<0.005) and epididymal (-25 and -28% respectively, P<0.005) fat pad weights compared with controls. This effect was restricted to intra-abdominal WAT as no difference was noticed for subcutaneous inguinal WAT. The decrease in intra-abdominal WAT accretion was due to a decrease in adipose cell diameter (-5 and -8% for 25 and 50 mg kg(-1) per day cirsimarin, respectively) resulting in a 14 and 35% decrease in adipose cell volume while no change was noticed in total adipocyte number. Direct injection of cirsimarin (50 mg kg(-1)) to rats did not trigger lipolysis. In contrast, cirsimarin showed in vivo as well as in vitro a strong antilipogenic activity, which may be the critical aspect of its effects on fat accretion in mice. The inhibitory concentration 50% of cirsimarin on lipogenic activity in isolated adipocytes was found to be 1.28±0.04 µM. Cirsimarin given orally reduced intra-abdominal fat accretion in mice. CONCLUSION: Cirsimarin exerts potent antilipogenic effect and decreases adipose tissue deposition in mice. Cirsimarin could therefore be a potential candidate for the treatment of obesity.

Thermotropic liquid crystalline glycolipids.

Are the liquid crystalline properties of the materials of living systems important in biological structures, functions, diseases and treatments? There is a growing consciousness that the observed lyotropic, and often thermotropic liquid crystallinity, of many biological materials that possess key biological functionality might be more than curious coincidence. Rather, as the survival of living systems depends on the flexibility and reformability of structures, it seems more likely that it is the combination of softness and structure of the liquid-crystalline state that determines the functionality of biological materials. The richest sources of liquid crystals derived from living systems are found in cell membranes, of these glycolipids are a particularly important class of components. In this critical review, we will examine the relationship between chemical structure and the self-assembling and self-organising properties of glycolipids that ultimately lead to mesophase formation.

Survey of the frequency of USH1 gene mutations in a cohort of Usher patients shows the importance of cadherin 23 and protocadherin 15 genes and establishes a detection rate of above 90%.

BACKGROUND: Usher syndrome, a devastating recessive disorder which combines hearing loss with retinitis pigmentosa, is clinically and genetically heterogeneous. Usher syndrome type 1 (USH1) is the most severe form, characterised by profound congenital hearing loss and vestibular dysfunction. OBJECTIVE: To describe an efficient protocol which has identified the mutated gene in more than 90% of a cohort of patients currently living in France. RESULTS: The five genes currently known to cause USH1 (MYO7A, USH1C, CDH23, PCDH15, and USH1G) were tested for. Disease causing mutations were identified in 31 of the 34 families referred: 17 in MYO7A, 6 in CDH23, 6 in PCDH15, and 2 in USH1C. As mutations in genes other than myosin VIIA form nearly 50% of the total, this shows that a comprehensive approach to sequencing is required. Twenty nine of the 46 identified mutations were novel. In view of the complexity of the genes involved, and to minimise sequencing, a protocol for efficient testing of samples was developed. This includes a preliminary linkage and haplotype analysis to indicate which genes to target. It proved very useful and demonstrated consanguinity in several unsuspected cases. In contrast to CDH23 and PCDH15, where most of the changes are truncating mutations, myosin VIIA has both nonsense and missense mutations. Methods for deciding whether a missense mutation is pathogenic are discussed. CONCLUSIONS: Diagnostic testing for USH1 is feasible with a high rate of detection and can be made more efficient by selecting a candidate gene by preliminary linkage and haplotype analysis.

Thionucleotides as inhibitors of ribonucleotide reductase.

Ribonucleosides and xylonucleosides bearing a disulfide function on the sugar ring were synthesized. Ribonucleosides belonging to the cytidine series were found to efficiently reduce dNTP pools in the human lymphoblastoid CEM/SS cell line.

[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.

Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects.

Ten different mutations have been identified in patients with Becker (n = 1) or Duchenne (n = 9) muscular dystrophy using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and protein truncation test (PTT) analysis. Seven mutations had not been reported previously, and these consist in three nonsense mutations (Q2522X, E2726X, R3381X), three frameshifting deletions (3686-3687delGT, 5126delA, 5759delC), and four splicing defects of which the effects on the muscle dystrophin mRNA transcripts have been analyzed. In one case, a 3' splice-site mutation (IVS74-2A-->G) resulted in a complex pattern of exon skipping involving exons of the C-terminal domain. In the three other cases, nucleotide substitutions in splice donor (IVS26+2T-->A, IVS65+1G-->A) or acceptor (IVS8-15A-->G) recognition sequences led to the use of cryptic splice sites, with consequent insertions of intronic sequences in the processed mRNA. Up to 34% (70/203) of the point mutations reported to date in the dystrophin database (http://www.dmd.nl) affect splice sites of the dystrophin gene. However, altered mRNA splicing has been confirmed experimentally in only 23% of cases (16/70). Combined with PTT, the transcript analysis protocol defined in this study permits direct determination of the impact of intronic variations on the structure of dystrophin mRNA and of the resulting consequences on the translational reading frame. We present evidence for a frequent use of cryptic splice sites as a result of splicing defects.

[Genotypic diagnosis of Duchenne and Becker muscular dystrophies]. / Le diagnostic génotypique des myopathies de Duchenne et de Becker.

Duchenne (DMD) and Becker (BMD) are allelic forms of a X-linked neuromuscular disorder. Both are caused by mutations arising in the gene encoding dystrophin, a cytoskeletal protein. Two-thirds of DMD/BMD patients have large deletions localised in two hot spots, and the remaining cases are presumed to be caused by point mutations. Since Duchenne muscular dystrophy is a serious disorder for which at present there is no effective treatment, much emphasis has been given to prevention. This involves the ascertainment of women likely to have an affected son, and the provision of genetic counselling and prenatal diagnosis for such women. Accurate carrier detection and genetic counselling depend upon identifying the mutation itself in the proband. Large deletions are easily identified using multiplex polymerase chain reaction (PCR) whereas detection of point mutations is restricted to a few number of specialized laboratories. Hence carrier and prenatal diagnosis in 40% of families rely heavily on indirect approaches which presents major drawbacks and are not applicable in sporadic cases of DMD or BMD. We developped a strategy for searching small alterations in the dystrophin gene. As most of the non-deletion mutations cause a premature termination of translation, we have used the Protein Truncation Test to scan specifically the dystrophin transcripts isolated from muscle biopsies. This approach allowed to detect the disease-causing mutations in more than 90% of the patients who have been investigated; his efficiency is thus significantly higher than DNA-based strategies. The identification of the mutation in non deleted sporadic cases allows the at-risks females to benefit from an accurate diagnosis. Also, the characterization of the molecular defects provides a better understanding of the molecular pathology of the dystrophin gene.

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.

Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.