Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.     

Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.     

Purine metabolite concentrations in portal and peripheral blood of steers, sheep and rats.

1. The concentration of purine derivatives in portal and peripheral blood of steers, sheep and rats was measured by reverse-phase high performance liquid chromatography. 2. Nucleotides, nucleosides (apart from inosine), adenine and guanine were not found in the plasma samples. Allantoin, uric acid, hypoxanthine and xanthine accounted for virtually all purine metabolites in plasma samples. 3. Non-oxidized derivatives (hypoxanthine and xanthine) were consistently detected in sheep but not in steer or rat plasma samples showing a differential availability of reutilizable purine derivatives between species.     

Specific degradation of histones H1 and H3

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca⁺⁺ or Na⁺⁺ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.     

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.The amino acid/polyamine/organocation (APC)² superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes. These membrane proteins function as solute/cation symporters or solute/solute antiporters (1). One APC subfamily is established by l-amino acid transporters (LATs), which correspond to the light subunits of eukaryotic heteromeric amino acid transporters (2, 3). Heteromeric amino acid transporters are composed of a light subunit that provides transport activity and a disulfide-linked heavy subunit that shows responsibility for plasma membrane targeting. Genetic defects in light and heavy subunits cause a number of inherited human diseases. Mutations in the light as well as the heavy subunit of system b^0,+ lead to cystinuria (4, 5), whereas mutations in the light subunit y⁺LAT1 cause lysinuric protein intolerance (6, 7). Another light subunit, xCT that mediates cysteine uptake and glutamate efflux (8, 9), is involved in vivo in cocaine relapse (10) and maintenance of the plasma redox balance (11). LAT1, the light subunit of system L, is overexpressed in certain primary human tumors. It transports essential neutral amino acids with long, branched, or aromatic side chains required by tumor cells to support their unabated growth (12). Therefore, amino acid transporters like LAT1 are attractive anticancer drug targets.So far a high resolution structure of a eukaryotic LAT family member is not available. However, studies on xCT revealed a membrane topology of 12 transmembrane helices (TMHs) with cytosolic N and C termini and a re-entrant loop structure between TMHs II and III (13). The identified first prokaryotic member of the LAT family, SteT from Bacillus subtilis, is a serine/threonine antiporter, which shows high sequence identity (∼30%) to the light subunits of eukaryotic heteromeric amino acid transporters. Moreover SteT exhibits a similar putative membrane topology and sequential mode of obligate exchange (14). Thus, SteT is an excellent model for studying the structure-function relationship of LAT family members.According to current models, transport proteins undergo functionally related conformational changes. Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15–22). However, prior to conformational changes substrates have to be recognized and bound. If substrates are amino acids, three main features can be used for specific selection and binding: (i) the negatively charged α-carboxyl group, (ii) the positively charged α-amino group, and (iii) the electrostatic, hydrophobic, or spatial properties of the side chain (22–24). α-Carboxyl and α- amino groups of l-amino acids possess similar structural and chemical characteristics (except for proline); however, their side chains differ in shape, size, and electrostatic properties. Combinations of these features are assumed to establish different interactions within the side chain binding pocket, which determines the substrate specificity of the transporter. The two main substrates of SteT, l-serine and l-threonine, differ by only one methylene group in their side chain; thus they have similar properties. Additionally SteT transports aromatic l-amino acids (Trp, Tyr, and Phe) albeit less efficiently (14).Since its invention, the atomic force microscope (AFM) (25) has evolved from a surface imaging device to a versatile tool for studying interactions of manifold biological systems (26–31). Introduced to characterize interactions between receptor-ligand complexes (32, 33) and complementary DNA strands (34), AFM-based single molecule force spectroscopy (SMFS) has been exploited to explore antibody-antigen recognition (35) and unfolding and refolding of soluble proteins (29, 36) and to probe the adhesion of living cells at molecular resolution (37). Applied to membrane proteins, SMFS uses the AFM stylus to exert a mechanical pulling force to the terminal end of a protein that is embedded and anchored by the lipid membrane (see Fig. 1A) (38). Sufficiently high stretching forces initiate sequential unfolding of the membrane protein with each step indicating the unfolding of a structural segment (39). Recording the applied force over pulling distance results in a force-distance (F-D) curve in which individual force peaks represent the rupture of intra- and intermolecular interactions. The height of a force peak measures the strength of an interaction with piconewton accuracy, and the pulling distance, at which the force peak occurs, allows the interaction within the membrane protein structure to be located (38).Open in a separate windowFIGURE 1.SMFS of SteT. A, pushing the AFM stylus onto the proteoliposomes promotes contacting single transporters to the stylus. This molecular link allows exertion of a mechanical pulling force that initiates stepwise unfolding of SteT. During the experiments, sample and cantilever are immersed in buffer solution. B, F-D curves recorded while unfolding single substrate-free SteT molecules. C, superimpositions of F-D curves recorded while unfolding SteT in buffer lacking any substrate (top) and supplemented with 5 mm l-serine (middle) or 5 mm l-threonine (bottom). Superimpositions are represented as density plots, each calculated from 60 F-D curves. Gray lines represent WLC curves with a persistence length of 0.4 nm and contour length (in amino acids) as indicated by the numbers next to the lines. The contour lengths were obtained from the Gaussian fits shown in D. F-D curves were obtained at room temperature at a pulling velocity of 654 nm/s in buffer solution (150 mm NaCl, 20 mm Tris-HCl, pH 8.0, substrate as indicated). D, frequency of force peaks detected at different positions of the stretched polypeptide. Every force peak detected in individual F-D curves (B) was fitted using the WLC model with the contour length of the stretched polypeptide as the only fitting parameter. The frequency at which the force peaks appeared is plotted in the histogram: substrate-free, n = 132; 5 mm l-serine, n = 128; and 5 mm l-threonine, n = 127. The bin size of the histograms is 3 aa and reflects the accuracy of fitting the WLC model (55) to individual force peaks. Error bars representing the S.E. were calculated using S.E. = (p(1 − p)/n)^0.5 where p is the probability and n is the total number of F-D curves. The width of each force peak distribution is given by the experimental noise, conformational variability of the structural segments, and fitting accuracy of the force peaks (53, 99–102). The gray solid curve represents the sum of seven Gaussian fits to the seven main peaks from the histograms and superimpositions (C). Numbers next to peaks denote peak positions (measured in amino acids) obtained from Gaussian fits.Besides quantification and localization of molecular interactions in membrane proteins, SMFS provides information about their energy landscape. For that purpose, the interactions of membrane proteins are probed over a range of different time scales by dynamic force spectroscopy (DFS). Bell (40) and Evans and co-worker (41, 42) provided the most commonly used theoretical framework to analyze DFS data. Their model describes the deformation of the energy landscape by an externally applied force, F. Such force-induced deformations reduce the energy barriers that separate bound and unbound states (see Fig. 2). Consequently transition rates over such energy barriers are force-dependent. Probing the interactions at different pulling velocities and thus at different force loading rates, r_f, leads to a so-called dynamic force spectrum in which the most probable force, F*, of rupture is plotted versus the logarithm of r_f. In these dynamic force spectra, each linear regime represents an energy barrier. Energy barriers located closer to the bound state are probed at higher pulling velocities because the energy barriers located further from the bound state are suppressed by increasingly applied forces (see Fig. 2) (41). The slope of each linear regime measures the distance from the ground state to the transition state, whereas extrapolation of a linear regime to zero force provides the rate constant of crossing the corresponding barrier in the absence of any load. These two parameters allow an estimate of the rigidity of the probed structure (43, 44).Open in a separate windowFIGURE 2.Energy landscape tilted by force. Schematic representation of the free energy profile along the reaction coordinate and applied force according to the Bell-Evans theory (40–42). The potential along the reaction coordinate (vector of force) in the absence of force (black curve) exhibits two energy barriers separating the folded from the unfolded state. Application of an external force, F, changes the thermal likelihood of reaching the top of the energy barrier(s). Although for a sharp barrier the position, x_u, of the energy barrier relative to the folded state is not changed, the thermally averaged projection of the energy profile along the pulling direction is tilted by the mechanical energy (−F·cos θ)x (long-dashed line). This tilt decreases the energy barriers (short-dashed curve). Consequently the relevant energy barrier that has to be overcome is the outermost barrier. At slow pulling velocities, the thermal contribution is higher, and therefore, the mechanical energy required to overcome the barrier is smaller. With increasing pulling velocities, the barriers are further lowered. At some velocity, the height of the outer barrier will be lower than that of the inner barrier (short-dashed curve), which then becomes the relevant energy barrier to be overcome. Each energy barrier manifests as a linear regime in dynamic force spectra (Fig. 3).In this study, we applied SMFS to characterize molecular interactions that stabilize SteT in the absence and in the presence of its substrates, l-serine and l-threonine. We used DFS to characterize how substrate binding changes the energy landscape and the mechanical properties of the antiporter. It was observed that the structural regions stabilized within SteT did not depend on substrate binding. However, substrate binding dynamically changed the energy landscape of these structures. In the absence of substrate all structural regions within SteT were stabilized by a narrow inner energy barrier and co-stabilized by a second outer energy barrier. The unique properties of these energy barriers restricted the conformation of SteT thereby trapping the antiporter in a kinetically instable and mechanically rigid conformation. In contrast, substrate binding sets SteT into a different energy minimum that significantly increased the kinetic stability and conformational flexibility of the antiporter.     

Analysis of candidate genes underlying two epistatic quantitative trait loci on SSC12 affecting litter size in pig

The previous results from a genome scan for total number of piglets born and number of piglets born alive in a F₂ Iberian by Meishan intercross showed several single and epistatic QTL. One of the most interesting results was obtained for SSC12, where two QTL affecting both traits showed epistatic interaction. In this study, we proposed two genes ( SLC9A3R1 and NOS2 ) as biological and potentially positional candidates underlying these QTL. Both cDNAs were characterized and 23 polymorphisms were detected. A chromosome scan was conducted with 12 markers, plus one SNP in SLC9A3R1 and one in NOS2, covering 110 cM of SSC12. The epistatic QTL (QTL1 at 15 cM and QTL2 at 97 cM) were confirmed, and SLC9A3R1 and NOS2 were mapped around the QTL1 and QTL2 regions respectively. Several SNPs in both genes were tested with standard animal model and marker assisted association tests. The most significant results were obtained with the NOS2 haplotype defined by one missense SNP c.2192C > T (Val to Ala) and a 15 bp duplication at the 3'UTR. This duplication seems to include AU-rich elements, and could be a target site for miRNA, therefore there are statistical and biological indications to consider this haplotype as the potential causal mutation underlying QTL2. SLC9A3R1 results were not conclusive. Although the interaction between the SNPs was not significant, we cannot reject the possibility of interaction of the NOS2 haplotype with other polymorphisms closely linked to the SL9A3R1 SNPs analysed.     

Sequencing and gene expression of the porcine ITIH SSC13 cluster and its effect on litter size in an Iberian × Meishan F2 population

The aim of the present study was to identify polymorphisms and to analyze endometrial gene expression of the porcine SSC13 ITIH cluster that could explain differences in prolificacy of 255 F(2) sows derived from an Iberian (Ib)×Meishan (Me) intercross in which QTL for the number of piglets born alive (NBA) and total number of piglets born (TNB) were previously detected on this chromosome. Sequencing of ITIH-1, -3, and -4 mRNAs was done and several polymorphisms segregating within the Ib×Me population were found in all three genes. Significant associations with NBA were found for two SNPs from ITIH-1, four from ITIH-3, and four SNPs from ITIH-4 (p<0.05). Haplotypes for the significant SNPs were calculated by segregation analysis and a marker assisted association test indicated that the alleles coming from the Meishan breed had a favorable effect on NBA for all three genes. Interestingly, some of the significant SNPs were located within the von Willebrand domain of the ITIH proteins, the binding site of molecules essential for the synthesis of the extracellular matrix during cumulus expansion. Gene expression analyses also revealed differences in the expression level of the ITIH-3 gene regarding the prolificacy performance (high or low) and the uterus sample (apical or basal).     

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM.