Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Development and diversity of lignin patterns

Different patterns of lignified cell walls are associated with diverse functions in a variety of plant tissues. These functions rely on the stiffness and hydrophobicity that lignin polymers impart to the cell wall. The precise pattern of subcellular lignin deposition is critical for the structure–function relationship in each lignified cell type. Here, we describe the role of xylem vessels as water pipes, Casparian strips as apoplastic barriers, and the role of asymmetrically lignified endocarp b cells in exploding seed pods. We highlight similarities and differences in the genetic mechanisms underpinning local lignin deposition in these diverse cell types. By bringing together examples from different developmental contexts and different plant species, we propose that comparative approaches can benefit our understanding of lignin patterning mechanisms.

Diverse lignin patterns underpin distinct functions in different plant tissues.     

A 10-miRNA risk score-based prediction model for pathological complete response to neoadjuvant chemotherapy in hormone receptor-positive breast cancer

Patients with hormone receptor(HR)-positive tumors breast cancer usually experience a relatively low pathological complete response(p CR) to neoadjuvant chemotherapy(NAC). Here, we derived a 10-micro RNA risk score(10-mi RNA RS)-based model with better performance in the prediction of p CR and validated its relation with the disease-free survival(DFS) in 755 HRpositive breast cancer patients(273, 265, and 217 in the training, internal, and external validation sets, respectively). This model,pres...     

Brain Transcriptional and Epigenetic Associations with Autism

BackgroundAutism is a common neurodevelopmental syndrome. Numerous rare genetic etiologies are reported; most cases are idiopathic.Conclusions/SignificanceThis work highlights two largely unrecognized molecular pathophysiological themes in autism and suggests differing molecular bases for autism behavioral endophenotypes.     

Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10⁶ cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385. © 2012 Wiley Periodicals, Inc.     

Arylarnine N-acetyltransferase as a potential biornarker in bladder cancer: fluorescent in situ hybridization and irnmunohistochernistry studies

Arylamine N-acetyltransferase isoenzymes NAT1 and NAT2 are encoded at two polymorphic loci on human chromosome 8p22. The two loci have previously been identified using chimeric Yeast Artificial Chromosome (YAC) clones encoding either NAT1 or NAT2 as probes for metaphase chromosomes using fluorescent in situ hybridization. The 8p22 region has been demonstrated to be deleted in highly invasive bladder tumours and since NAT isoenzymes participate in the metabolism of arylamine bladder carcinogens, it is important to determine whether NAT1 and NAT2 gene loci are included in the region of deletion. We describe here the application of a cosmid clone for NAT2 as a biomarker for Fluorescent In Situ Hybridization (FISH) on interphase nuclei of exfoliated bladder cells. We also describe a 70kb probe for NAT1 which is a candidate for a suitable biomarker for use in similar FISH studies. lmmunohistochemical staining of bladder tumour sections with a polyclonal anti-peptide antibody specific for the NATl isoenzyme as a biomarker for NAT1 protein expression is also shown.     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.