Evaluation of an automated insulated isothermal polymerase chain reaction system for rapid and reliable, on-site detection of African swine fever virus.

Objective: To evaluate the utility of an automated insulated isothermal PCR (iiPCR) system for rapid and reliable on-site detection of African swine fever virus (ASFV) in swine biological samples.

Sample: Lymph node, tissue homogenate, whole blood, serum, spleen, and tonsil samples collected from swine in North and South Vietnam.

Procedures: Analytic sensitivity of the iiPCR system was determined by serial dilution and analysis of 2 samples (swine tissue homogenate and blood) predetermined to be positive for ASFV.

A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage.

Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress.

Architecture and characterization of a thermostable MoxR family AAA(+) ATPase from Thermococcus kodakarensis KOD1.

AAA(+) ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths.

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1.

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa.

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1.

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and L: -proline dehydrogenase activity.

Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis.

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis KOD1.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa.

Oxidized NADH oxidase inhibits activity of an ATP/NAD kinase from a Thermophilic archaeon.

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization.

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1.

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T.