Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

Microsatellite DNA Population Structure and Stock Identification of Steelhead Trout (<Emphasis Type="BoldItalic">Oncorhynchus mykiss</Emphasis>) in the Nass and Skeena Rivers in Northern British Columbia

Population structure and the application to genetic stock identification for steelhead (Oncorhynchus mykiss) in the Nass and Skeena Rivers in northern British Columbia was examined using microsatellite markers. Variation at 8 microsatellite loci (Oki200, Omy77, Ots1, Ots3, Ssa85, Ots100, Ots103, and Ots108) was surveyed for approximately 930 steelhead from 7 populations in the Skeena River drainage and 850 steelhead from 10 populations in the Nass River drainage, as well as 1550 steelhead from test fisheries near the mouth of each river. Differentiation among populations within rivers accounted for about 1.9 times the variation observed among years within populations, with differences between drainages less than variation among populations within drainages. In the Nass River, winter-run populations formed a distinct group from the summer-run populations. Winter-run populations were not assessed in the Skeena River watershed. Simulated mixed-stock samples suggested that variation at the 8 microsatellite loci surveyed should provide relatively accurate and precise estimates of stock composition for fishery management applications within drainages. In the Skeena River drainage in 1998, Babine River (27%) and Bulkley drainage populations (31%) comprised the main components of the returns. For the Nass River in 1998 steelhead returning to Bell-Irving River were estimated to have comprised 39% of the fish sampled in the test fishery, with another 27% of the returns estimated to be derived from Cranberry River. The survey of microsatellite variation did not reveal enough differentiation between Nass River and Skeena River populations to be applied confidently in estimation of stock composition in marine fisheries at this time. Received January 14, 2000; accepted July 13, 2000.     

Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.     

An all-atom model of the pore-like structure of hexameric VP40 from Ebola: structural insights into the monomer-hexamer transition

The matrix protein VP40 is an indispensable component of viral assembly and budding by the Ebola virus. VP40 is a monomer in solution, but can fold into hexameric and octameric states, two oligomeric conformations that play central roles in the Ebola viral life cycle. While the X-ray structures of monomeric and octameric VP40 have been determined, the structure of hexameric VP40 has only been solved by three-dimensional electron microscopy (EM) to a resolution of approximately 30 A. In this paper, we present the refinement of the EM reconstruction of truncated hexameric VP40 to approximately 20 A and the construction of an all-atom model (residues 44-212) using the EM model at approximately 20 A and the X-ray structure of monomeric VP40 as templates. The hexamer model suggests that the monomer-hexamer transition involves a conformational change in the N-terminal domain that is not evident during octamerization and therefore, may provide the basis for elucidating the biological function of VP40.     

Mediated microbial biosensor using a novel yeast strain for wastewater BOD measurement

Two new yeast strains (SPT1 and SPT2) were isolated and immobilized on glassy carbon electrodes to form microbial biosensors for estimation of biochemical oxygen demand (BOD). Ferricyanide was proven to be the most efficient mediator to shuttle electrons from the redox center of reduced microbial enzymes to the electrode in the presence of excess glucose/glutamic acid (GGA). With a 3-fold greater metabolic assimilation capability and greater responses to various effluent samples, SPT1 was selected for sensor-BOD measurements. BOD estimations for the GGA standard resulted in an extended linear range: 2-100 mg/l. Response reproducibility was +/-10% for a GGA standard containing 10 mg BOD/l. For analysis of pulp mill effluents, the BOD detection limit was 2 mg/l with a response time of 5 min.     

Otolith morphology and periodicity of increment formation on the sagitta of manybar goatfish Parupeneus multifasciatus (Mullidae)

Otolith morphology and microstructure of the sagitta are described in the mass tropical species manybar goatfish Parupeneus multifasciatus. The hatch check is absent on the sagitta, and the appearance of the first contrasting increment is connected with the transition of the larva to exogenous feeding. The increments are distributed within several concentric zones, and each zone is separated by more pronounced dark areas (in transmitted light) of the increments (checks). The number of increments within a zone is variable; and the modal group (with the exception of the first zone from the primordium to the first check) includes from six to ten increments. In the juveniles, the periodicity of increment formation on the sagitta ranges from <1 to >3 in a day, but it is close to one increment per day in the individuals larger than 86 mm FL and 11 g in weight. The number of increments deposited in a day does not depend on the growth rate or feeding frequency of the juveniles.     

Oral ingestion of insulin liposomes: Effects of the administration route

We prepared an insulin liposome suspension by hot dispersion (50 °C) of a lipid mixture comprising dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (7:2 molar ratio) in an 80 UI/ml acid bovine insulin solution, followed by two minutes of cold sonification (4 °C). Free insulin was removed by ultracentrifugation and the washed insulin liposomes were resuspended in a 1% aqueous saline solution (pH 3). Administration of these liposomes in the buccal cavity of normal rats caused clear hypoglycemia (?37% of the initial glycemia after one hour and ?44% after $\text{2}\text{1}\text{2}$ hours), but the solution was inactive when introduced by a strictly intragastric route. Hypoglycemic effects were also obtained when a mixture containing a liposome suspension devoid of insulin and 10 UI/100 g b.w. of free insulin was given by the buccal route (?56% of initial glycemia one hour later and ?55% after $\text{2}\text{1}\text{2}$ hours). These results show that the route of liposomal insulin administration strongly influences its biological effects.     

Growth Inhibitory,Bactericidal, and Morphostructural Effects of Dehydrocostus Lactone from Magnolia sieboldii Leaves on Antibiotic-Susceptible and -Resistant Strains of Helicobacter pylori

Helicobacter pylori is associated with various diseases of the upper gastrointestinal tract, such as gastric inflammation and duodenal and gastric ulcers. The aim of the study was to assess anti-H. pylori effects of the sesquiterpene lactone dehydrocostus lactone (DCL) from Magnolia sieboldii leaves, compared to commercial pure DCL, two previously known sesquiterpene lactones (costunolide and parthenolide), (–)-epigallocatechin gallate, and four antibiotics. The antibacterial activity of natural DCL toward antibiotic-susceptible H. pylori ATCC 700392 and H. pylori ATCC 700824 strains (MIC, 4.9 and 4.4 mg/L) was similar to that of commercial DCL and was more effective than costunolide, parthenolide, and EGCG. The activity of DCL was slightly lower than that of metronidazole (MIC, 1.10 and 1.07 mg/L). The antibacterial activity of DCL was virtually identical toward susceptible and resistant strains, even though resistance to amoxicillin (MIC, 11.1 mg/L for PED 503G strain), clarithromycin (49.8 mg/L for PED 3582GA strain), metronidazole (21.6 mg/L for H. pylori ATCC 43504 strain; 71.1 mg/L for 221 strain), or tetracycline (14.2 mg/L for B strain) was observed. This finding indicates that DCL and the antibiotics do not share a common mode of action. The bactericidal activity of DCL toward H. pylori ATCC 43504 was not affected by pH values examined (4.0–7.0). DCL caused considerable conversion to coccoid form (94 versus 49% at 8 and 4 mg/L of DCL for 48 h). The Western blot analysis revealed that urease subunits (UreA and UreB) of H. pylori ATCC 43504 were not affected by 10 mM of DCL, whereas UreA monomer band completely disappeared at 0.1 mM of (–)-epigallocatechin gallate. Global efforts to reduce the level of antibiotics justify further studies on M. sieboldii leaf-derived materials containing DCL as potential antibacterial products or a lead molecule for the prevention or eradication of drug-resistant H. pylori.