Impact of water troughs on cattle use of riparian zones in the Georgia Piedmont in the United States.

Cattle use of riparian areas may lead to stream water contamination with nutrients, pathogens, and sediments. Providing alternative water away from the stream may reduce the amount of time cattle spend near streams and therefore reduce contamination. We conducted this study to 1) evaluate the effect of providing water troughs outside of the riparian zones on the amount of time cattle spend in riparian zones, and 2) evaluate if environmental factors such as temperature and humidity affect the impact of water trough availability on the amount of time cattle spend within riparian and nonriparian locations. Global positioning system (GPS) collars were used to document cow locations every 5 min in 2 mixed tall fescue/common bermuda-grass pastures of the Georgia Piedmont in the United States. We found that when the temperature and humidity index (THI) ranged between 62 and 72, providing cattle with water troughs outside of riparian zones tended to decrease time cattle spent in riparian zones by 63% (52 min x d(-1); P = 0.11). When THI ranged between 72 and 84, nonriparian water availability did not have a significant impact on the amount of time cattle spent in the riparian zone or in riparian shade. These results suggest that water troughs placed away from unfenced streams may improve water quality by reducing the amount of time cattle spend in riparian zones when environmental conditions as evaluated by THI are not stressful.

Proteome-based plasma biomarkers for Alzheimer's disease.

Alzheimer's disease is a common and devastating disease for which there is no readily available biomarker to aid diagnosis or to monitor disease progression. Biomarkers have been sought in CSF but no previous study has used two-dimensional gel electrophoresis coupled with mass spectrometry to seek biomarkers in peripheral tissue. We performed a case-control study of plasma using this proteomics approach to identify proteins that differ in the disease state relative to aged controls. For discovery-phase proteomics analysis, 50 people with Alzheimer's dementia were recruited through secondary services and 50 normal elderly controls through primary care. For validation purposes a total of 511 subjects with Alzheimer's disease and other neurodegenerative diseases and normal elderly controls were examined. Image analysis of the protein distribution of the gels alone identifies disease cases with 56% sensitivity and 80% specificity. Mass spectrometric analysis of the changes observed in two-dimensional electrophoresis identified a number of proteins previously implicated in the disease pathology, including complement factor H (CFH) precursor and alpha-2-macroglobulin (alpha-2M). Using semi-quantitative immunoblotting, the elevation of CFH and alpha-2M was shown to be specific for Alzheimer's disease and to correlate with disease severity although alternative assays would be necessary to improve sensitivity and specificity. These findings suggest that blood may be a rich source for biomarkers of Alzheimer's disease and that CFH, together with other proteins such as alpha-2M may be a specific markers of this illness.

Phosphorus, sediment, and Escherichia coli loads in unfenced streams of the Georgia Piedmont, USA.

Contamination of unfenced streams with P, sediments, and pathogenic bacteria from cattle (Bos taurus) activity may be affected by the availability of shade and alternative water sources. The objectives of this study were to evaluate water quality in two streams draining tall fescue (Festuca arundinacea Schreb.)-common bermudagrass (Cynodon dactylon L.) pastures with different shade distribution, and to quantify the effects of alternative water sources on stream water quality. For 3 yr, loads of dissolved reactive phosphorus (DRP), total phosphorus (TP), and total suspended solids (TSS) were measured during storm flow, and loads of DRP, TP, TSS, and Escherichia coli were measured every 14 d during base flow. We also used GPS collars to determine amount of time cattle spent in riparian areas. Our results showed that cattle-grazed pastures with unfenced streams contributed significant loads of DRP, TP, TSS, and E. coli to surface waters (p < 0.01). Time spent by cattle in riparian areas as well as storm flow loads of DRP, TP, and TSS were larger (p < 0.08) in the pasture with the smaller amount of nonriparian shade. Water trough availability decreased base flow loads of TSS and E. coli in both streams, and decreased time cattle spent in riparian areas in the pasture with the smaller amount of nonriparian shade (p < 0.08). Our results indicate that possible BMPs to reduce contamination from cattle-grazed pastures would be to develop or encourage nonriparian shade and to provide cattle with alternative water sources away from the stream.

Mannosidase production by viridans group streptococci.

The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis, and Streptococcus pneumoniae) and Streptococcus intermedius of the anginosus group. None of the other species within the salivarius and mutans groups or Streptococcus peroris and Streptococcus sanguinis produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man(5) glycan alone was degraded while Man(6) to Man(9), which contain terminal alpha(1-->2) mannose residues in addition to the alpha(1-->3), alpha(1-->6), and beta(1-->4) residues present in Man(5), remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or p-nitrophenol-linked) alpha- or beta-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man(5) glycan of RNase B. No species degraded these alpha-linked mannosides, while degradation of the beta-linked synthetic substrates was restricted to strains within the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.

Detecting mannosidase activities using ribonuclease B and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components-cells and supernates-have been directly analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their specificities and location. Enzymatic cleavage was monitored by observing changes in RNase B glycoform population. Thus a nonspecific alpha-(1 --> 2)-mannosidase activity converts the glycoprotein to its Man(5) form, identifiable by its mass of 14,899 [M + H](+); this species subsequently is converted, by the actions of alpha-(1 --> 3) and alpha-(1 --> 6)-mannosidases, to the Man(1) form via Man(4), Man(3), and Man(2). The Man(1) glycoform (which is readily isolated) has then similarly been used for identifying beta-(1 --> 4)-mannosidase and the derived Man(0) form has served in turn as a natural substrate for beta-(1 --> 4) N-acetylglucosaminidase producing a species possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mannose liberated from the actions of mannosidases can, if desired, be quantified by, for example, chromatography. The actions and specificities of endoglycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetlyglucosaminidases (e.g., endo-F and endo-H), which respectively cleave between the GlcNAc&bond;Asn and GlcNAc&bond;GlcNAc bonds of N-linked glycoproteins, are also demonstrable by MALDI-ToF analysis of RNase B (and derived products). From these digests the completely deglycosylated polypeptide corresponding to RNase A in which Asn has been converted to Asp (mass 13,684) and a species corresponding to RNase A + GlcNAc (mass 13,886) are produced, together with their corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.

Isolation and characterisation of sialidase from a strain of Streptococcus oralis.

Streptococcus oralis, the most virulent of the viridans streptococci, produces a sialidase and this exo-glycosidase has been implicated in the disease process of a number of pathogens. The sialidase of S. oralis strain AR3 was purified in order to understand the characteristics of this putative virulence determinant. The enzyme isolated as a high mol. wt aggregate (c. 325 kDa) was purified 4520-fold from late exponential phase cultures by a combination of ultrafiltration, ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. The sialidase component had a mol.wt of 144 kDa as determined by SDS-PAGE analysis. The purified sialidase released N-acetylneuraminic acid from a range of sialoglycoconjugates including human alpha1-acid glycoprotein, bovine submaxillary mucin, colominic acid and sialyl-alpha2,3- and sialyl-alpha2,6-lactose. Also, N-glycolylneuraminic acid was cleaved from bovine submaxillary mucin. The sialidase had a Km of 11.8 microM for alpha1-acid glycoprotein, was active over a broad pH range with a pH optimum of 6.0 and cleaved alpha2,3-, alpha2,6- and alpha2-8-sialyl glycosidic linkages with a marked preference for alpha2,3-linkages. The enzyme was competitively inhibited by the sialic acid derivative, 2,3-dehydro-N-acetylneuraminic acid, with a K(IC) of 1.2 microM. The characteristics of the purified sialidase would support a nutritional role for this enzyme that may be significant in the proliferation of this organism in the oral cavity and at extra-oral sites in association with life-threatening infections.

N-acetylneuraminic acid transport by Streptococcus oralis strain AR3.

Streptococcus oralis has emerged as one of the most important organisms of the viridans streptococcus group in terms of infections and is recognised as an agent of infective endocarditis and, in immunocompromised patients, septicaemia. The mechanisms by which this organism proliferates in vivo are unknown. However, host-derived sialic acids -- including N-acetylneuraminic acid (NeuNAc) which is present in serum and cell-associated glycoproteins -- are a potential source of fermentable carbohydrate for bacterial proliferation, especially for sialidase-producing bacteria, including S. oralis. To further elucidate the role of NeuNAc in supporting growth, this study determined the ability of S. oralis strain AR3 (isolated from a patient with infective endocarditis) to transport NeuNAc and characterised the transport system. The transport of [14C]-labelled NeuNAc into S. oralis was monitored and this transport system was induced by growth of the bacteria in the presence of the N-acetylated sugars NeuNAc, N-acetylglucosamine and N-acetylmannosamine. The transport system followed typical Michaelis-Menten kinetics, with a Km of 21.0 microM and a Vmax of 2.65 nmoles of NeuNAc transported/min/mg of dry cell mass. NeuNAc transport was inhibited by the presence of exogenous N-glycolylneuraminic acid, a related sialic acid. Chlorhexidine, NaF and 2,4-dinitrophenol were potent inhibitors of the transport system, suggesting that the uptake of NeuNAc occurs via a proton motive force-dependent permease system. This is the first report of the mechanism by which NeuNAc transport occurs in pathogenic streptococci. This transport process may have relevance to the acquisition of a source of fermentable carbohydrate and thus bacterial proliferation in vivo.

Growth of Viridans streptococci on human serum alpha1-acid glycoprotein.

Viridans streptococci have emerged as major opportunistic pathogens. We suggest that for these bacteria to proliferate in vivo and cause disease, they must utilize host tissue components. We have therefore examined the ability of all recognized species of viridans streptococci to liberate and utilize the constituent sugars of the glycans of the extensively sialylated human serum alpha1-acid glycoprotein (AGP) as the sole source of carbohydrate to support in vitro growth. Analysis of residual glycans following bacterial growth was performed by high-pH anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Only those species which produced sialidase-namely, Streptococcus oralis, S. intermedius, and S. defectivus--grew on AGP. The extent of degradation of glycans was dependent on the particular glycosidases produced by the bacteria. S. defectivus produced only a sialidase which released the terminal N-acetylneuraminic acid residues of the glycans, and the liberated sugar was utilized. S. intermedius also produced beta-galactosidase and beta-N-acetylglucosaminidase, which removed galactose and N-acetylglucosamine from desialylated glycans, all of which again were utilized by the organism. S. oralis produced beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-fucosidase and novel alpha- and beta-mannosidases which were apparent only from the analysis of the residual sugars of AGP. S. oralis cleaved all the sugars from AGP except for 22% of the N-acetylglucosamine. The residual N-acetylglucosamine residues remaining were those linked to the asparagine of the peptide backbone. All the monosaccharides released by S. oralis from AGP, with the exception of fucose, were utilized. Sialidase production may be a key factor for growth of these species of viridans streptococci on glycoproteins in vivo, since they are commonly associated with extra-oral diseases, with S. oralis emerging as an important pathogen.

Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis.

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.

Evidence for mannosidase activities in Streptococcus oralis when grown on glycoproteins as carbohydrate source.

Streptococcus oralis when cultured using ribonuclease B as the sole source of carbohydrate, selectively uses the sugars of the Man5 glycoform as shown by HPAEC and MALDI-TOF mass spectrometric analyses. The organism is able to do this by producing novel alpha-(1-->3), alpha-(1-->6) and beta-(1-->4) mannosidase activities and these act in a concerted manner in what appears as a single-step process. The selective utilisation of Man5 is explained by the absence of an alpha-(1-->2) mannosidase which is required to initiate breakdown of the glycan chains present in the other glycoforms which are components of the glycoprotein.

Utilization of sialic acid by viridans streptococci.

The importance of viridans streptococci as agents of serious extra-oral diseases, including endocarditis, is now recognized. We have tested the hypothesis that the ability to utilize sialic acid as a nutrient source may play a role in the proliferation of these organisms. The type strains of the 15 presently recognized species of viridans streptococci and two clinical isolates-S. oralis (AR3), isolated from a patient with infective endocarditis, and S. intermedius (UNS35), a brain abscess isolate-were studied for their ability to utilize sialic acid. Only S. oralis, S. sanguis, S. gordonii, S. mitis ("oralis group") S. intermedius, S. anginosus, S. constellatus ("milleri group"), and S. defectivus ("nutritionally variant group") were able to use sialic acid (N-acetylneuraminic acid) efficiently as a sole carbon source. Formate, acetate, and ethanol were produced as the major metabolic end-products of sialic acid metabolism, while corresponding glucose-grown cultures produced lactate as the major metabolic end-product. Utilization of sialic acid was independent of the production of sialidase. Cell-free extracts of sialic acid-grown cultures expressed elevated levels of N-acetylneuraminate pyruvate-lyase (NPL; the first enzyme in the intracellular catabolism of sialic acid) and N-acetylglucosamine-6-phosphate (GlcNAc-6-P) deacetylase and glucosamine-6-phosphate (GlcN-6-P) deaminase (enzymes involved in the intracellular catabolism of N-acetylglucosamine). These activities were repressed by growth in the presence of glucose. The intracellular fate of sialic acid, after cleavage by NPL into N-acetylmannosamine (ManNAc) and pyruvate, is uncertain, but the elevated levels of GlcNAc-6-P deacetylase and GlcN-6-P deaminase in sialic acid-grown cells suggest that phosphorylation and isomerization are possible steps in the metabolism of ManNAc to generate an intermediate common to the pathway of N-acetylglucosamine metabolism. The species of viridans streptococci that have the ability to utilize sialic acid are those most commonly associated with extra-oral diseases, and this ability is likely to play a role in the persistence and survival of these infecting organisms in vivo.