A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41 800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with K, values of 4.0 pM for folate, 8.0 pM for methotrexate and 34.0 pM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.
Thionins are small cysteine-containing, amphipathic plant proteins found in seeds and vegetative tissues of a number of plant genera. Many of them have been shown to be toxic to microorganisms such as fungi, yeast, and bacteria and also to mammalian cells. It has been suggested that thionins are present in seeds to protect them, and the germinating seedling, from attack by phytopathogenic microorganisms, but the mechanism by which they kill cells remains unclear. Using electrophysiological measurements, we have shown that -purothionin from wheat flour can form cation-selective ion channels in artificial lipid bilayer membranes and in the plasmalemma of rat hippocampal neurons. We suggest that the generalized toxicity of thionins is due to their ability to generate ion channels in cell membranes, resulting in the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.
To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56-2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n=63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing human exposure and disease transmission.
To investigate transport and partitioning processes of
HgT in the Fox River, we coupled detailed time series
data
of total mercury (HgT) at the river mouth with
transect
sampling in the Lower Fox River. Unfiltered HgT
concentrations
in the Fox River during the study period (April 1994−October 1995) ranged from 1.8 to 182 ng L-1
with a median
of 24.8 ng L-1, predominantly (93.6%) in the
particulate
phase. These levels were significantly elevated
compared
with other large tributaries to Lake Michigan (Hurley, J.
P.; Shafer, M. M.; Cowell, S. E.; Overdier, J. T.; Hughes,
P.
E.; Armstrong, D. E. Environ. Sci. Technol.
1996,
30, 2093−2098). Transect sampling revealed progressively
increasing
water column HgT concentrations and HgT
particulate
enrichment downstream, which were consistent with trends
in sediment HgT levels in the river. Resuspended
sediments
are likely the predominant source of Hg from the Fox River
into Green Bay. Despite elevated HgT
concentrations,
methyl mercury (MeHg) concentrations were relatively low,
suggesting limited bioavailability of HgT associated
with
sediments.
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