Optimum induction of recombinant thymidine phosphorylase and its application

Recombinant E. coli pDEOA was constructed and lactose can be used instead of IPTG to induce the expression of thymidine phosphorylase by pDEOA. The use of lactose at concentrations higher than 0.5 mmol/L had an induction effect similar to that of IPTG but resulted in a longer initial induction time and better cell growth. The thymidine phosphorylase induced by lactose was very stable at 50°C. Intact pDEOA cells induced by lactose can be used as a source of thymidine phosphorylase. Under standard reaction conditions, several deoxynucleosides were effectively produced from thymidine.     

Proteome dynamics and proteome function of cardiac 19S proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.     

DeltAMT: a statistical algorithm for fast detection of protein modifications from LC-MS/MS data

Identification of proteins and their modifications via liquid chromatography-tandem mass spectrometry is an important task for the field of proteomics. However, because of the complexity of tandem mass spectra, the majority of the spectra cannot be identified. The presence of unanticipated protein modifications is among the major reasons for the low spectral identification rate. The conventional database search approach to protein identification has inherent difficulties in comprehensive detection of protein modifications. In recent years, increasing efforts have been devoted to developing unrestrictive approaches to modification identification, but they often suffer from their lack of speed. This paper presents a statistical algorithm named DeltAMT (Delta Accurate Mass and Time) for fast detection of abundant protein modifications from tandem mass spectra with high-accuracy precursor masses. The algorithm is based on the fact that the modified and unmodified versions of a peptide are usually present simultaneously in a sample and their spectra are correlated with each other in precursor masses and retention times. By representing each pair of spectra as a delta mass and time vector, bivariate Gaussian mixture models are used to detect modification-related spectral pairs. Unlike previous approaches to unrestrictive modification identification that mainly rely upon the fragment information and the mass dimension in liquid chromatography-tandem mass spectrometry, the proposed algorithm makes the most of precursor information. Thus, it is highly efficient while being accurate and sensitive. On two published data sets, the algorithm effectively detected various modifications and other interesting events, yielding deep insights into the data. Based on these discoveries, the spectral identification rates were significantly increased and many modified peptides were identified.     

Metabolomic study of interactive effects of phenol, furfural, and acetic acid on Saccharomyces cerevisiae

Metabolic profiling was carried out to investigate the interactive effects of three representative inhibitors (furfural, phenol, and acetic acid) in lignocellulosic hydrolysate on Saccharomyces cerevisiae during ethanol fermentation. Our results revealed that three inhibitors exhibited significantly synergistic effects on the growth, fermentation, and some metabolites of yeast. Acetic acid exerted the most severe effects on yeast in the combination of three inhibitors, enhancing amino acids metabolism and inhibiting central carbon metabolism. The effects on yeast cells by acetic acid were enhanced by the presence of phenol and furfural, which might be owing to the loss of membrane integrity and the inhibition on metabolism. Further investigation indicated that the combination of inhibitors also exhibited antagonistic effects mainly on threonine, cadaverine, inositol, and tryptophan, weakening or reversing the effects of individual inhibitor. It might be due to the more severe damage by the combined inhibitors, and different repairing mechanism of cells in the presence of individual and combined inhibitors. Better understanding of the synergistic and antagonistic effects of the inhibitors will be helpful for the improvement of tolerant strains and the optimization of lignocellulosic fermentation.     

The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH

Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)(2)D(3) or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR-deficient (CaR(-/-)) mice to those of double homozygous CaR- and 1α(OH)ase-deficient [CaR(-/-)1α(OH)ase(-/-)] mice or those of double homozygous CaR- and PTH-deficient [CaR(-/-)PTH(-/-)] mice at 2 weeks of age. Compared to wild-type littermates, CaR(-/-) mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR(-/-) mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR(-/-) mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR(-/-) mice and that defects in endochondral bone formation in CaR(-/-) mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.     

A missense mutation in PPARD causes a major QTL effect on ear size in pigs

Chinese Erhualian is the most prolific pig breed in the world. The breed exhibits exceptionally large and floppy ears. To identify genes underlying this typical feature, we previously performed a genome scan in a large scale White Duroc × Erhualian cross and mapped a major QTL for ear size to a 2-cM region on chromosome 7. We herein performed an identical-by-descent analysis that defined the QTL within a 750-kb region. Historically, the large-ear feature has been selected for the ancient sacrificial culture in Erhualian pigs. By using a selective sweep analysis, we then refined the critical region to a 630-kb interval containing 9 annotated genes. Four of the 9 genes are expressed in ear tissues of piglets. Of the 4 genes, PPARD stood out as the strongest candidate gene for its established role in skin homeostasis, cartilage development, and fat metabolism. No differential expression of PPARD was found in ear tissues at different growth stages between large-eared Erhualian and small-eared Duroc pigs. We further screened coding sequence variants in the PPARD gene and identified only one missense mutation (G32E) in a conserved functionally important domain. The protein-altering mutation showed perfect concordance (100%) with the QTL genotypes of all 19 founder animals segregating in the White Duroc × Erhualian cross and occurred at high frequencies exclusively in Chinese large-eared breeds. Moreover, the mutation is of functional significance; it mediates down-regulation of β-catenin and its target gene expression that is crucial for fat deposition in skin. Furthermore, the mutation was significantly associated with ear size across the experimental cross and diverse outbred populations. A worldwide survey of haplotype diversity revealed that the mutation event is of Chinese origin, likely after domestication. Taken together, we provide evidence that PPARD G32E is the variation underlying this major QTL.     

Transportin-SR is required for proper splicing of resistance genes and plant immunity

Transportin-SR (TRN-SR) is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR) proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14), a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R) protein snc1 (suppressor of npr1-1, constitutive 1). MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

E2F1 is involved in DNA single-strand break repair through cell-cycle-dependent upregulation of XRCC1 expression

The X-ray repair cross complementing group 1 (XRCC1) protein is involved in DNA base excision repair and its expression varies during the cell cycle. Although studies have demonstrated that rapid XRCC1-dependent single-strand break repair (SSBR) takes place specifically during S/G(2) phases, it remains unclear how it is regulated during the cell cycle. We found that XRCC1 is a direct regulatory target of E2F1 and further investigated the role of XRCC1 in DNA repair during the cell cycle. Saos2 primary osteosarcoma cells stably transfected with inducible E2F1-wt or mutant E2F1-132E were treated with hydroxurea (HU) for 36h and were subsequently withdrawn HU for 2-24h to test whether cell-cycle-dependent DNA SSBR requires E2F1-mediated upregulation of XRCC1. We found that SSBR activity, as determined using a qPCR-base method, was correlated with E2F1 levels at different phases of the cell cycle. XRCC1-positive (AA8) and negative (EM9) CHO cells were used to demonstrate that the alterations in SSBR were mediated by XRCC1. The results indicate that E2F1-mediated regulation of XRCC1 is required for cell-cycle-dependent SSBR predominantly in G(1)/S phases. Our observations have provided new mechanistic insight for understanding the role of E2F1 in the maintenance of genomic stability and cell survival during the cell cycle. The regulation of XRCC1 by E2F1 during cell-cycle-dependent SSBR might be an important aspect for practical consideration for resolving the problem of drug resistance in tumor chemotherapies.     

Determination of unbound vismodegib (GDC-0449) concentration in human plasma using rapid equilibrium dialysis followed by solid phase extraction and high-performance liquid chromatography coupled to mass spectrometry

A rapid equilibrium dialysis (RED) assay followed by a solid phase extraction (SPE) high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of unbound vismodegib in human plasma was developed and validated. The equilibrium dialysis was carried out using 0.3 mL plasma samples in the single-use plate RED system at 37°C for 6h. The dialysis samples (0.1 mL) were extracted using a Strata-X-C 33u Polymeric Strong Cation SPE plate and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization (ESI) mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL for vismodegib, was fitted to a 1/x(2) weighted linear regression model. The lower limit of quantitation (LLOQ, 0.100 ng/mL) was sufficient to quantify unbound concentrations of vismodegib after dialysis. The intra-assay precision of the LC-MS/MS assay, based on the four analytical QC levels (LLOQ, low, medium and high), was within 7.7% CV and inter-assay precision was within 5.5% CV. The assay accuracy, expressed as %Bias, was within ±4.0% of the nominal concentration values. Extraction recovery of vismodegib was between 77.9 and 84.0%. The assay provides a means for accurate assessment of unbound vismodegib plasma concentrations in clinical studies.