An easy-to-perform, culture-free Campylobacter point-of-management assay for processing plant applications.

AIMS: Current culture-based methods for detection and determination of Campylobacter levels on processed chickens takes at least 2 days. Here we sought to develop a new complete, low-cost and rapid (approximately 2·5 h) detection system requiring minimal operator input. METHODS AND RESULTS: We observed a strong correlation between culture-based cell counts and our ability to detect either Campylobacter jejuni or Campylobacter coli by loop-mediated isothermal amplification from the same samples. This knowledge was used to develop a rapid and simple five-step assay to quantify Campylobacter, which was subsequently assessed for its specificity, reproducibility and accuracy in quantifying Campylobacter levels from processed chickens. The assay was found to be highly specific for C. jejuni and C. coli and was capable of distinguishing between samples that are either within or exceeding the industry set target of 6000 Campylobacter colony forming units (CFU) per carcass (equivalent to 12 CFU per ml of chicken rinse) with >90% accuracy relative to culture-based methods. CONCLUSIONS: Our method can reliably quantify Campylobacter counts of processed chickens with an accuracy comparable to culture-based assays but provides results within hours as opposed to days. SIGNIFICANCE AND IMPACT OF THE STUDY: The research presented here will help improve food safety by providing fast Campylobacter detection that will enable the implementation of real-time risk management strategies in poultry processing plants to rapidly test processed chickens and identify effective intervention strategies. This technology is a powerful tool that can be easily adapted for other organisms and thus could be highly beneficial for a broad range of industries.

Rotting by radicals--the role of cellobiose oxidoreductase?

Cellobiose oxidoreductase is a flavocytochrome secreted by wood-rotting fungi. The structure and functional role of the enzyme are reviewed, and a mechanism through which the enzyme produces superoxide, ferrous iron and hydrogen peroxide is proposed. The reactions of hydroxyl radicals formed by Fenton chemistry are discussed in the context of lignocellulose biodegradation.

Extracellular heme peroxidases in actinomycetes: a case of mistaken identity.

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.

Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta.

There is increasing evidence that heterotrimeric G-proteins (G-proteins) are involved in many plant processes including phytohormone response, pathogen defence and stomatal control. In animal systems, each of the three G-protein subunits belong to large multigene families; however, few subunits have been isolated from plants. Here we report the cloning of a second plant G-protein gamma-subunit (AGG2) from Arabidopsis thaliana. The predicted AGG2 protein sequence shows 48% identity to the first identified Arabidopsis Ggamma-subunit, AGG1. Furthermore, AGG2 contains all of the conserved characteristics of gamma-subunits including a small size (100 amino acids, 11.1 kDa), C-terminal CAAX box and a N-terminal alpha-helix region capable of forming a coiled-coil interaction with the beta-subunit. A strong interaction between AGG2 and both the tobacco (TGB1) and Arabidopsis (AGB1) beta-subunits was observed in vivo using the yeast two-hybrid system. The strong association between AGG2 and AGB1 was confirmed in vitro. Southern and Northern analyses showed that AGG2 is a single copy gene in Arabidopsis producing two transcripts that are present in all tissues tested. The isolation of a second gamma-subunit from A. thaliana indicates that plant G-proteins, like their mammalian counterparts, may form different heterotrimer combinations that presumably regulate multiple signal transduction pathways.

Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.

Heterotrimeric G proteins consist of three subunits (alpha, beta, and gamma). alpha- and beta- subunits have been previously cloned in plants, but the gamma-subunit has remained elusive. To isolate the gamma-subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta-subunit as the bait protein. One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma-subunits. The predicted AGG1 protein sequence contains all of the typical characteristics of mammalian gamma-subunits such as small size (98 amino acids, 10.8 kDa), presence of a C-terminal CAAX box to direct isoprenyl modification, and an N-terminal alpha-helix region capable of forming a coiled-coil interaction with the beta-subunit. Northern and Southern analyses showed that AGG1 is a single-copy gene in Arabidopsis with a similar expression pattern to the Arabidopsis beta-subunit, AGB1 [Weiss, C. A., Garnaat, C. W., Mukai, K., Hu, Y. & Ma, H. (1994) Proc. Natl. Acad. Sci. USA 91, 9554-9558]. By using the yeast two-hybrid system, we show that AGG1 strongly interacts with tobacco and Arabidopsis beta-subunits. The in vivo results have been confirmed by using in vitro methods to prove the interaction between AGG1 and the Arabidopsis beta-subunit. As previously observed in mammalian systems, both the coiled-coil domain and the WD repeat regions of the beta-subunit are essential for AGG1 interaction. Also in agreement with previous observations, the removal of the N-terminal alpha-helix of the AGG1 greatly reduces but does not completely block the interaction.

Alpha 1-blocker doxazosin improves peripheral insulin sensitivity in diabetic hypertensive patients.

The antihypertensive doxazosin is a selective alpha 1-adrenoceptor-blocking drug whose favorable impact on lipid metabolism is well known. A single-blind placebo-controlled crossover study was designed to determine whether antihypertensive treatment with doxazosin affects insulin sensitivity in diabetic, mildly hypertensive, non-obese patients. Twelve subjects (diastolic blood pressure, 98 +/- 1.5 mm Hg; body mass index, 25 +/- 0.6 kg/m2; hemoglobin A1c [HbA1c], 7.6% +/- 0.4%) who were not taking drugs and were treating diabetes only by diet were randomly assigned to placebo treatment for 6 weeks and then to doxazosin for the same period, or vice versa. The doxazosin dose (maximum, 12 mg/d) was increased to achieve a normotensive blood pressure (final diastolic pressure, 85 +/- 2 mm Hg, P < .05). A euglycemic (100 +/- 4 mg/dL) hyperinsulinemic (61 +/- 6 microU/mL) glucose clamp was performed at baseline and at the end of both placebo and doxazosin administration. Hepatic glucose production was measured by the isotope dilution technique using 3H-glucose. Body weights and HbA1c did not vary during the entire study. The basal mean glucose uptake and the insulin sensitivity index (2.3 +/- 0.3 mg/kg/min and 4 +/- 0.5 mg/kg/min per U/L x 100) remained unchanged during placebo administration (2.5 +/- 0.4 and 4 +/- 0.6, NS), but significantly increased during doxazosin treatment (3.3 +/- 0.4 and 5.6 +/- 0.7, P < .05). Hepatic glucose production showed no modification during both placebo and doxazosin. These data provide evidence that doxazosin improves insulin sensitivity in diabetic hypertensive patients, mainly through peripheral effects.