Biological properties of the phage-tail-like particles from Myxococcus coralloides D

Abstract Electron microscopy of preparations of the Myxocococcus coralloides D autolytic supernatants revealed the presence of phage-tail-like particles. The particles consisted of a core, a contractile sheath and a baseplate with fibers. The induction of the defective prophage occurred when the cultures reached the stationary phase, but the events occurring during induction did not lead to cell lysis. Particles nerver appeared during exponential growth. They were not observed when M. coralloides D grew on solid media, either. Purified samples of phage tails were able to inhibit most of the myxobacterial strains which were tested when the particles were in the extended state, but they were unable to multiply on the sensitive strains.     

The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.     

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis.To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.     

Biological properties of selenobiotin.

Selenobiotin is an excellent growth factor, as efficient as biotin in supporting the growth of biotin requiring microorganisms. It is incorporated in carboxylases leading to active “selenocarboxylases”. With E. Coli, the activity of the selenoacetyl CoA carboxylase is very similar to that of the normal enzyme.     

Biological responses to diesel exhaust particles (DEPs) depend on the physicochemical properties of the DEPs

Diesel exhaust particles (DEPs) are the main components of ambient particulate materials, including polyaromatic hydrocarbons (PAHs), n-PAHs, heavy metals, and gaseous materials. Many epidemiological, clinical, and toxicological studies have shown that ambient particles, including DEPs, are associated with respiratory disorders, such as asthma, allergic rhinitis, and lung cancer. However, the relationship between the biological response to DEPs and their chemical composition remains unclear. In this study, we investigated the physicochemical properties of DEPs before toxicological studies, and then administered a single intratracheal instillation of DEPs to mice. The mice were then killed 1, 7, 14 and 28 days after DEP exposure to observe the biological responses induced by DEPs over time. Our findings suggest that DEPs engulfed into cells induced a Th2-type inflammatory response followed by DNA damage, whereas DEPs not engulfed into cells induced a Th1-type inflammatory response. Further, the physicochemical properties, including surface charge, particle size, and chemical composition, of DEPs play a crucial role in determining the biological responses to DEPs. Consequently, we suggest that the biological response to DEPs depend on cell-particle interaction and the physicochemical properties of the particles.     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

Hysteresis of plant cell-wall beta-glucosidase.

A transient-kinetic study of beta-glucosidase from soyabean cell walls was performed with the use of a stopped-flow apparatus. The progress curve of the reaction exhibits a 'slow' burst of about 1 s before the steady state is reached. In the time scale investigated this burst may be accounted for by only one exponential, whose time constant varies with the substrate concentration. As this concentration is increased the value of the time constant increases at first, then decreases. Premixing the enzyme with glucose, the last product of the reaction sequence, reverses the 'slow' burst into a 'slow' lag. Taken together, these results are only compatible with a model that involves the existence of a 'slow' conformational transition of the enzyme.