Ruminal tryptophan-utilizing bacteria degrade ergovaline from tall fescue seed extract.

The objectives of this study were to evaluate degradation of ergovaline in a tall fescue [ (Schreb.) Darbysh.] seed extract by rumen microbiota ex vivo and to identify specific bacteria capable of ergovaline degradation in vitro. Rumen cell suspensions were prepared by harvesting rumen fluid from fistulated wether goats ( = 3), straining, and differential centrifugation. Suspensions were dispensed into anaerobic tubes with added Trypticase with or without extract (∼10 µg kg ergovaline). Suspensions were incubated for 48 h at 39°C. Samples were collected at 0, 24, and 48 h for ergovaline analysis and enumeration of hyper-ammonia producing (HAB) and tryptophan-utilizing bacteria. Ergovaline values were analyzed by repeated measures using the mixed procedure of SAS. Enumeration data were log transformed for statistical analysis. When suspensions were incubated with extract, 11 to 15% of ergovaline disappearance was observed over 48 h ( = 0.02). After 24 h, suspensions with added extract had 10-fold less HAB than controls ( = 0.04), but treatments were similar by 48 h ( = 1.00). However, after 24 h and 48 h, suspensions with extract had 10-fold more tryptophan-utilizing bacteria ( < 0.01) that were later isolated and identified by their 16S RNA gene sequence as . The isolates and other known rumen pure cultures ( JB1, B159, HD4, B, F, MD1, SR) were evaluated for the ability to degrade ergovaline in vitro. Pure culture cell suspensions were incubated as described above and samples were taken at 0 and 48 h for ergovaline analysis. Data were analyzed using the ANOVA procedure of SAS. All HAB, including the isolates, tested degraded ergovaline (54 to 75%; < 0.05). B14 was also able to degrade ergovaline but to a lesser capacity (12%; < 0.05), but all other bacteria tested did not degrade ergovaline. The results of this study indicate which rumen bacteria may play an important role in ergovaline degradation and that microbiological strategies for controlling their activity could have ramifications for fescue toxicosis and other forms of ergotism in ruminants.

An aberrant protein complex in CSF as a biomarker of Alzheimer disease.

OBJECTIVE: To determine if an aberrant protein complex consisting of prostaglandin-d-synthase (PDS) and transthyretin (TTR) in CSF differentiates between subjects with Alzheimer disease (AD) and normal control (NC) subjects. METHODS: Western blot analysis and a unique sandwich ELISA were used to quantify levels of complexed PDS/TTR in ventricular CSF of subjects with autopsy-verified diagnoses and in lumbar CSF of living subjects with mild to moderate probable AD and age-matched NC subjects. Ventricular CSF was obtained from short postmortem interval autopsies of 7 NC subjects (4 men/3 women), 12 diseased control (DC) subjects (7 men/5 women), 4 subjects with mild cognitive impairment (MCI) (2 men/2 women), and 8 subjects with late-stage AD (LAD) (4 men/4 women). Lumbar CSF was obtained from 15 subjects with probable AD (5 men/10 women) and 14 age-matched NC subjects (10 men/4 women) and was analyzed in a double-blind fashion. RESULTS: A significant increase in complexed PDS/TTR in ventricular CSF was found in MCI and LAD subjects but not DC subjects compared with NC subjects. Double-blind analysis of complexed PDS/TTR in lumbar CSF showed a significant sixfold increase in levels of the PDS/TTR complex in living probable AD subjects compared with age-matched NC subjects and a 100% sensitivity and 93% specificity in the identification of subjects with AD. CONCLUSION: After further study of larger numbers of patients, quantifying prostaglandin-d-synthase/transthyretin complex in CSF may be useful in the diagnosis of Alzheimer disease, possibly in the early stages of the disease.

Proteomic identification of proteins specifically oxidized by intracerebral injection of amyloid beta-peptide (1-42) into rat brain: implications for Alzheimer's disease.

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain.

The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and alpha-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, alpha-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

Evaluation of axial DC offsets during scanning of a quadrupole ion trap for sensitivity improvements.

In the normal operation of quadrupole ion trap mass spectrometers, approximately half of the trapped ions are ejected through the source endcap during a mass-selective instability scan. This reduces the sensitivity of the instrument by approximately 50%. In this preliminary study, a circuit was constructed that produced a dipolar DC offset on the axial modulation waveform to recover this lost ion current. A variable (0 to 10 V DC), positive and negative offset was applied to the source and detector endcap, respectively. This DC offset axially displaced the ion cloud toward the detector endcap increasing the probability of detection. Several compounds, including 11 pesticides, were evaluated. Sensitivity enhancements ranged from 13 to 97% (theoretical 100%). No spectral resolution problems were observed; however, a compound-dependent mass discrimination was observed in several cases. This mass discrimination problem is currently under investigation.

Photooxidative bleaching of chlorophyllin.

Chlorophyllin, a water-soluble, copper-containing porphyrin, can be bleached rapidly in the light or slowly in the dark in a reaction which is oxygen dependent. Both the photo and the dark bleaching reactions are temperature dependent. Cyclic voltammetry measurements show that the copper in the bleached and nonbleached state remains in the +2 redox state and could be readily reduced. This would imply that there is no net oxidative change to the copper during the bleaching process. FT-IR absorption spectroscopy showed vibrations characteristic of a vinyl functionality disappeared upon bleaching. Aqueous solutions of chlorophyllin were not dialyzable through dialysis tubing of molecular weight cut-off, 6000-8000 molecular weight, indicative of an aggregate chlorophyllin micelle. Analysis of products by high performance liquid chromatography showed that the chlorophyllin mixture was more complex than originally anticipated and that two components were lost from the mixture upon photobleaching. One compound that is preferentially lost upon photobleaching has been identified by mass spectral analysis as Cu(II) chlorin e6.

Determination of hydroxylated polychlorinated biphenyls by ion trap gas chromatography-tandem mass spectrometry.

Hydroxylated polychlorinated biphenyls also known as biphenylols, are suspected estrogen mimics found in the environment. Various derivatization schemes were evaluated and a gas chromatography-ion trap mass spectrometry method was developed for the trifluoroacetyl derivative using MS-MS techniques for the analysis of eleven biphenylols. A time-segmented chromatographic method was developed using the respective MS-MS parameters to analyze all the eleven biphenylols in a single chromatographic run. Isomers were differentiated based on the MS-MS data of the trofluoroacetyl-biphenylol derivatives. The method was applied to detect 40 pg on-column of these compounds in a spiked egg sample which simulates a real world sample.

Liver and kidney concentrations of zinc, copper and cadmium in channel catfish (Ictalurus punctatus): variations due to size, season and health status.

Significant differences in liver and kidney concentrations of zinc (Zn), copper (Cu) and cadmium (Cd) were detected in normal Mississippi farm-raised channel catfish (Ictalurus punctatus) collected at different times of the year. These seasonal differences were not solely due to variation in fish size. Comparing the concentration of each metal in liver vs kidney indicated that Cd was lower in liver for all seasons studied, Cu was higher in liver for all seasons studied, and Zn was higher in the liver in the winter-killed (winter mortality syndrome) and the spring fish groups. Metal concentration was associated with body weight, as indicated by significant Pearson correlation coefficients for kidney Cd (all seasons and fall), liver Cu (summer), liver Zn (all seasons and winter), and kidney Zn (all seasons and winter). The adjusted means were not dramatically changed as compared to the raw data. Differences were noted when seasonal values obtained from normal fish were compared to tissues of moribund fish afflicted with winter mortality syndrome. Zinc was reduced in liver and kidney of these moribund fish.