Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.     

Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

4-deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-β-D-xylopyranosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-reactive triple bond-containing hydrophobic agents in the presence of Cu(2+) salt/ascorbic acid, a step known as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén conditions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of α:β-isomers of twelve derivatives were then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat various human diseases in which glycosaminoglycans participate.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.     

The origin of polyploid genomes of bluegrasses Poa L. and gene flow between Northern Pacific and sub-Antarctic islands

The involvement of present-day diploid bluegrass species in the formation of polyploid genomes was investigated using comparison of sequences of internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA sequence. It was demonstrated that highly polyploid New Zealand bluegrasses, P. cita (2n = 84; ca. 96 to 100), P. chathamica (2n = 112), and P. litorosa (2n = 263 to 266) formed separate highly supported clade together with tetraploids (2n = 28) P. intrusa, P. anceps, and P. trioides (Austrofestuca littoralis). Among the diploid species (2n = 14), the closest relatives of these species, as well as of the polyploid species of section Poa, are the genomes of Eurasian species P. remota, P. chaixcii (sect. Homalopoa), P densa (Bolbophorum), and P. sibirica (sect. Macropoa). Nuclear genomes of polyploid Stenopoa, Tichopoa, Oreinos, and Secundae are definitely related to the genome of Arctic species P. pseudabbreviata (sect. Abbreviatae). On the contrary, judging by the genes for nuclear 45S rRNA, genomes of diploid P. trivialis (sect. Pandemos), P. annua, and P. supina (sect. Ochlopoa both) are only remotely related to the genomes of highly polyploid species (distances p between them and other bluegrass species from different sections of subgenus Poa constitute 6-10% and 11-15%, respectively). The conclusion on the relationships between highly polyploid and diploid bluegrass species was tested using analysis of synapomorphic mutations in the 5.8S rRNA gene. It was demonstrated that genomes of Poa eminens (2n = 42) and P. schischkinii (2n = 70) (sect. Arctopoa both) were noticeably different in ITS regions from the genomes of the members of the type subgenus Poa. A comparison of the Arctopoa ITS regions showed that the differences between them constituted only 0.2%. At the same time, p distances between the Arctopoa ITS and those from the species belonging to other sections of the genus Poa varied from 5 to 14%. South American species P chonotica (sect. Andinae) (=Ncoraepoa chonotica) (2n = 42) was found to be related to Arctagrostis, Festucella, and Hookerochloa, being at the same time quite distant from the other species of the genus Poa. Polymorphic in chromosome number highly polyploid species of Northern Hemisphere, P. arctica (2n = 42 to 106), P. turneri (2n = 42, 63 to 64), and P. smirnovii (2n = 42, 70) (sect. Malacanthae) are relative to a large group of tetraploid (2n = 28) endemic bluegrass species from New Zealand and sub-Antarctic islands (P. novae-zelandiae and allied species).     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.     

Tug-of-war model for the two-bandit problem: Nonlocally-correlated parallel exploration via resource conservation

We propose a model - the “tug-of-war (TOW) model” - to conduct unique parallel searches using many nonlocally-correlated search agents. The model is based on the property of a single-celled amoeba, the true slime mold Physarum, which maintains a constant intracellular resource volume while collecting environmental information by concurrently expanding and shrinking its branches. The conservation law entails a “nonlocal correlation” among the branches, i.e., volume increment in one branch is immediately compensated by volume decrement(s) in the other branch(es). This nonlocal correlation was shown to be useful for decision making in the case of a dilemma. The multi-armed bandit problem is to determine the optimal strategy for maximizing the total reward sum with incompatible demands, by either exploiting the rewards obtained using the already collected information or exploring new information for acquiring higher payoffs involving risks. Our model can efficiently manage the “exploration-exploitation dilemma” and exhibits good performances. The average accuracy rate of our model is higher than those of well-known algorithms such as the modified ?-greedy algorithm and modified softmax algorithm, especially, for solving relatively difficult problems. Moreover, our model flexibly adapts to changing environments, a property essential for living organisms surviving in uncertain environments.