In silico methods in enzyme screening and gene expression

INTMSAlign is a software to assign consensus residues of target protein utilizing large amount of their family sequences. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL (1). a-Amino-e-caprolactam (ACL) racemase from Achromobacter obae has been shown to be an effective catalyst for the dynamic kinetic resolution of amino acid amide and a-aminonitriles to form chiral amino acids. We searched for ACL racemase in silico with INTMSAlign software. By fixing Lys 241 as one of the key residues, we discovered thirteen ACL racemase genes from 413 fold type-I PLP genes (2). Insolubility of proteins expressed in Escherichia coli expression hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). We discovered a phenomenon of soluble expression of HNL from Manihot esculenta, in E. coli. By random mutagenesis, we found that a single point mutation H103L, and mutation with alterations at three positions (Lys-Pro mutations at positions 176, 199 and 224) cause total solubility in E. coli even when grown at 37°C (3). If a relationship between soluble expression and mutation points could be established, it will become very easy to generate a mutant for correctly folded expression in E. coli. Using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules of thumb to help identify hotspots: one focuses on the hydrophobicity of amino acids in the a-helix structure, and another one focuses the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. Using these two relationships together, we succeeded in developing methods to improve the solubility of expressed proteins in E. coli (4). References: (1) S. Nakano and Y. Asano, Sci. Rep., 5, 8193 (2015). (2) W. Payoungkiattikun, S. Okazaki, S. Nakano, A. Ina, A. H-Kittikun, and Y. Asano, Appl. Biochem. Biotechnol., 176 (5), 1303-1314 (2015). (3) Y. Asano, M. Dadashipour, M. Yamazaki, N. Doi, and H. Komeda. Prot. Eng. Des. Sel., 24 (8), 607-616 (2011). (4) D. Matsui, S. Nakano, M. Dadashipour, and Y. Asano, submitted

The Dry Matter Yield and Nutritive Value of Wet Tolerant Tropical Forage Legumes in Single Cropping or Mixed Cropping with Gramineous Forage Crops in Drained Paddy Field

In Japan the production of rice has been controlled since the 1970\u27s and some parts of the paddy fields have been laid off for forage production. However, in poorly-drained fields or fields with high ground water table, forage species with high tolerance of wet conditions are required. The tropical forage legumes Aeschynomene americana cv. Glenn (Glenn) and Macroptilium lathyroides (L.) Urb. cv. Murray (phasey bean) have a high wet endurance (Bishop et al., 1985; Tobisa et al., 1999) and show high dry matter productivity (Skerman et al., 1988; Tobisa et al., 1999). The objective of this experiment was to evaluate the dry matter yield and nutritive value of Glenn and phasey bean in single cropping or mixed cropping with gramineous forage crops in drained paddy fields

Reconstruction of ancestral L-amino acid oxidases to broaden substrate selectivity

Characteristic functions of enzymes, such as high thermal stability and substrate specificity, are attained during the evolutionary process. Ancestral sequence reconstruction (ASR) is applied to infer the process by designing artificial enzymes which are located on ancestral node of phylogenetic tree; here, the inferred enzymes called ancestral enzymes. Ancestral enzymes often exhibit substrate promiscuity and high thermal stability of which functions are suitable to perform enzyme engineering. In addition, applicability of the ASR is high because the method requires only sequence data to design ancestral enzymes. Thus, we believe that artificial enzymes contributing to progress in enzyme engineering can be designed by ASR. Please click Additional Files below to see the full abstract

Assessment of the intrapulmonary ventilation-perfusion distribution after the Fontan procedure for complex cardiac anomalies: Relation to pulmonary hemodynamics

AbstractIn 12 patients who underwent the Fontan procedure for complex cardiac anomalies, lung scanning with xenon-133 was performed to assess the intrapulmonary ventilation-perfusion distribution, and comparison was made with a control group. All data were then analyzed in relation to either pre- or postoperative pulmonary hemodynamic data. In ventilation scans, the intrapulmonary distribution in the right lung was almost normal.In perfusion scans, an abnormal increased upper to lower lobe perfusion ratio greater than the normal value found in the control group was noted in seven patients (58.3%). There was a significant correlation (p < 0.02) between the upper to lower lobe perfusion ratio and postoperative pulmonary vascular resistance. Furthermore, this perfusion ratio correlated inversely with the preoperative (p < 0.005) and postoperative (p < 0.02) right pulmonary artery area index, defined as the ratio of cross-sectional area to the normal value. Of five patients with < 90% arterial oxygen saturation, four showed an abnormal distribution of pulmonary blood flow greater than the normal perfusion ratio. No patient had evidence of a pulmonary arteriovenous fistula by the echocardiographic contrast study.These results suggest that abnormal distribution of pulmonary blood flow to the upper lung segment may develop in patients after the Fontan procedure, and that insufficient size of the pulmonary artery before operation and the consequent postoperative elevation of pulmonary vascular resistance may be responsible for this perfusion abnormality

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt- misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.info:eu-repo/semantics/publishedVersio

Hypergastrinemia and a duodenal ulcer caused by gastric duplication

BackgroundHypergastrinemia and the resultant peptic ulcer related to an enteric duplication has been quite rarely reported in the literature.Case presentationWe herein report the case of a 4-year-old girl who presented with hypergastrinemia and a duodenal ulcer at 2 years of age. She had been followed up with a proton pump inhibitor, which resulted in resolution of the ulcer; however, unexplained hypergastrinemia had continued. A cystic lesion at the antrum was discovered at 4 years of age, which we suspected to be a gastric duplication. After we resected the lesion, the hypergastrinemia resolved without recurrence of the duodenal ulcer. The histology was compatible with a gastric duplication, and the lumen was lined with antral mucosa that strongly stained positive for gastrin. We presumed that the antral mucosa inside the duplication in our case had no hydrogen ion feedback inhibition of gastrin release from gastrin cells and increased release of gastrin from the mucosa inside the duplication led to the duodenal ulcer. Only two cases have been reported in the literature that had hypergastrinemia related to enteric duplication.ConclusionGastric duplication should be included in the differential diagnosis of sustained hypergastrinemia in children