R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

BACKGROUND: The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. EXPERIMENTAL APPROACH: Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. KEY RESULTS: In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. CONCLUSION AND IMPLICATIONS: R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

Changes in muscle gene expression related to metabolism according to growth potential in young bulls.

To analyse the effects of genetic selection in favour of high muscle development on muscle gene expression, oligonucleotide microarrays were used to compare the transcriptome of Longissimusthoracis muscle from 15- and 19-month-old Charolais bull calves divergently selected for high (H) or low (L) muscle growth. Transcriptome data revealed that about two thirds of the genes involved in glycolysis were up-regulated at 15 and at 19months of age in H animals. Lastly, some differentially expressed genes were associated with muscle mass in the carcass (FGF6, PLD2) independently of fat deposition and meat quality. Selection for muscle growth potential is associated with modified expression of some genes involved in growth, and also with increased expression of genes involved in glycolysis. Furthermore, this change in muscle metabolism is likely to be dissociated from fat deposition and beef quality, providing new criteria for genetic selection in favour of muscle growth.

New indicators of beef sensory quality revealed by expression of specific genes.

To identify new molecular markers of beef sensory quality, the transcriptomes of Longissimus thoracis muscle from 25 Charolais bull calves were analyzed using microarrays and compared between high and low meat quality groups; 215 genes were differentially expressed according to tenderness, juiciness, and/or flavor. Among these, 23 were up-regulated in the tenderest, juiciest, and tastiest meats, and 18 were highly correlated with both flavor and juiciness (e.g., PRKAG1), explaining up to 60% of their variability. Nine were down-regulated in the same meats, but only DNAJA1 [the results relating to DNAJA1 and its relationship with tenderness have been patented (Genomic marker for meat tenderness; Patent EP06300943.5, September 12, 2006)], which encodes a heat shock protein, showed a strong negative correlation with tenderness that alone explained 63% of its variability. This protein, known for its anti-apoptotic role, could be involved in meat aging. Thus, DNAJA1 could constitute a new marker of beef sensory quality.

Target genes of myostatin loss-of-function in muscles of late bovine fetuses.

BACKGROUND: Myostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM) cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM) animals (n = 3 per group) in the semitendinosus muscle (hypertrophied in DM animals) at 260 days of fetal development (when the biochemical differentiation of muscle is intensive). A heterologous microarray (human and murine oligonucleotide sequences) of around 6,000 genes expressed in muscle was used. RESULTS: Many genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change), and according to the presence of one or two functional myostatin allele(s). They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2) and decreased adipocyte differentiation (down-regulation of C1QTNF3). The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue) is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B). CONCLUSION: Thus, the myostatin loss-of-function mutation affected several physiological processes involved in the development and determination of the functional characteristics of muscle tissue.

Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis.

The recent development of microarray technology has led statisticians and bioinformaticians to develop new statistical methodologies for comparing different biological samples. The objective is to identify a small number of differentially expressed genes from among thousands. In quantitative proteomics, analysis of protein expression using two-dimensional gel electrophoresis shows some similarities with transcriptomic studies. Thus, the goal of this study was to evaluate different data analysis methodologies widely used in array analysis using different proteomic data sets of hundreds of proteins. Even with few replications, the significance analysis of microarrays method appeared to be more powerful than the Student's t test in truly declaring differentially expressed proteins. This procedure will avoid wasting time due to false positives and losing information with false negatives.