Methane digester effluent from swine excreta as a nutrient and water source for growing and finishing swine.

The experimental objective was to evaluate swine methane digester effluent (SMDE) as a water and nutrient source for swine. The mesophilic methane digester was loaded daily with manure from finishing swine fed a corn-soybean meal diet. Dry diet was mixed with SMDE (3.7% DM) and fed twice daily in troughs. Tap water was provided and consumption measured. Barrows were group fed (3 pigs/pen) and adapted to SMDE by increasing SMDE for 7 d, with the full amount fed from d 8 to the end of the feeding phase (d 21, 14, 23, or 37 for Exp. 1 to 4, respectively). Blood samples were collected on d 0, 10, 21, and 31 to determine plasma concentrations of glucose and plasma urea N (PUN). Barrows were placed in individual metabolism cages for a 5-d acclimation and a 5-d fecal and urine collection to determine apparent N and energy utilization. For Exp. 1, 18 pigs averaging 75 kg BW were allotted to diets with 0, 48.6, or 63.7% SMDE, as-fed basis. For Exp. 2 and 3, 12 pigs/experiment averaged 117 and 70 kg, respectively, and were allotted to diets with 0 or 63.7% SMDE, as-fed basis. At the end of Exp. 2 and 3, pigs were sacrificed and liver samples were collected to determine urea cycle enzyme activity, and loin was saved for taste panel evaluation. For Exp. 4, pigs averaged 40 kg and were allotted to diets with 0 or 57.5% SMDE, as-fed basis. The ADFI, ADG, and G:F of finishing swine (Exp. 1 to 3) were not reduced by feeding diets containing 63.7% SMDE (as-fed basis), whereas ADG and G:F of growing swine (Exp. 4) were reduced (P < 0.01) by feeding a diet containing 57.5% SMDE. Pigs fed diets containing SMDE consumed 31 to 56% less (P < 0.05) water and had greater (P < 0.01) PUN concentrations than pigs fed control diets. Pigs fed diets containing SMDE excreted more (g, P < 0.05) fecal N and absorbed and retained less N (%; P < 0.01) and energy (DE and ME) than pigs fed control diets. Treatment had no effect on urea cycle enzyme activity. In conclusion, finishing swine adapted to diets containing 63.7% SMDE (as-fed basis) based on growth performance, whereas growing swine did not adapt to a diet containing 57.5% SMDE because of the large content of nonprotein N in SMDE. Recycling SMDE to swine greatly reduced fresh water consumption, whereas the protein and energy values of SMDE were approximately 0 for swine. Therefore, SMDE is more appropriately recycled as a source of water and N for ruminant nutrition or crop production.

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization.

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

The trio guanine nucleotide exchange factor is a RhoA target. Binding of RhoA to the trio immunoglobulin-like domain.

Trio is a complex protein containing two guanine nucleotide exchange factor domains each with associated pleckstrin homology domains, a serine/threonine kinase domain, two SH3 domains, an immunoglobulin-like domain, and spectrin-like repeats. Trio was originally identified as a LAR tyrosine phosphatase-binding protein and is involved in actin remodeling, cell migration, and cell growth. Herein we provide evidence that Trio not only activates RhoA but is also a RhoA target. The RhoA-binding site was mapped to the Trio immunoglobulin-like domain. RhoA isoprenylation is necessary for the RhoA-Trio interaction, because mutation of the RhoA carboxyl-terminal cysteine residue blocked binding. The existence of an intramolecular functional link between RhoA activation and RhoA binding is suggested by the finding that Trio exchange activity enhanced RhoA binding to Trio. Furthermore, immunofluorescence studies of HeLa cells showed that although ectopically expressed Trio was evenly distributed within the cell, co-expression of Trio with RhoA resulted in relocalization of Trio into punctate structures. Relocalization was not observed with Trio constructs lacking the immunoglobulin-like domain, indicating that RhoA acts to regulate Trio localization via binding to the immunoglobulin-like domain. We propose that Trio-mediated RhoA activation and subsequent RhoA-mediated relocalization of Trio functions to modulate and coordinate Trio signaling.

Assessment of interlaboratory performance in external proficiency testing programs with a direct HDL-cholesterol assay.

Direct assays for the determination of HDL-cholesterol (HDL-C) have recently become available. The methods are precise, require small sample volume, and appear to be less affected by increased triglycerides than traditional precipitation methods. In this study, we describe the inter- and intralaboratory variability of the Boehringer Mannheim Corporation direct HDL-C assay and its performance in external proficiency testing surveys. A comparison study among three laboratories, using different analyzers and 85 serum specimens, showed a correlation coefficient (r) of 0.99. The direct HDL-C assay also showed good agreement with the ultracentrifugation-dextran sulfate-Mg2+ method (r = 0.98) and the Cholesterol Reference Method Laboratory Network-Designated Comparison Method (a = 0.98x + 4.75 mg/L, r = 0.98). Total error at medical decision levels ranged from -0.8% to +11.1%. Furthermore, this assay performed adequately in the College of American Pathologists and the ALERT surveys as well as the CDC Lipid Standardization Program and met all performance criteria of regulatory agencies.

Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method.

LDL-cholesterol (LDL-C) concentration is currently determined in most clinical laboratories by the Friedewald calculation. This approach has several limitations and may not meet the current total error requirement in LDL-C measurement of < or = 12%. We evaluated the analytical and clinical performance of the direct N-geneous LDL-C assay (Equal Diagnostics). The N-geneous method correlated highly with the modified beta-quantification assay (r = 0.95; y = 0.91x + 70.6 mg/L; n = 199), showed no significant effect of increased triglyceride or other common interferants, and performed adequately in serum samples from nonfasting individuals. This assay demonstrated a mean total error of 6.75% over a wide range of LDL-C concentrations. In addition, at the medical decision cutoff points, this LDL-C assay showed positive predictive values of 78-95% and negative predictive values of 84-99%. We conclude that the N-geneous LDL-C meets the currently established analytical performance goals and appears to have a role in the diagnosis and management of hypercholesterolemic patients.

Three generations of high-density lipoprotein cholesterol assays compared with ultracentrifugation/dextran sulfate-Mg2+ method.

We report on the analytical performance of three generations of HDL-cholesterol assays: phosphotungstic acid/Mg2+, Spinpro, and a homogeneous method, N-geneous. The run-to-run imprecision (CV) of all assays was < or = 4.9%, and all results correlated highly with those of a modified reference procedure (r > or = 0.96). At triglycerides concentrations < 4000 mg/L, these field methods showed an acceptable systematic error (y = 1.12x - 47, 1.05x - 23, and 0.96x + 8 for the phosphotungstate, Spinpro, and N-geneous assays, respectively), and the total error of the field methods met the current National Cholesterol Education Program (NCEP) performance goal of < or = 22%. Regression analyses of results for samples with triglycerides > 4000 mg/L produced the following results for the above respective assays: y = 1.08x - 4.2, 1.02x + 3.6, and 0.85x + 108. In this hypertriglyceridemic group, only the N-geneous assay (at an HDL-cholesterol content of 240 mg/L) had a total error (35%) that exceeded the NCEP limit. Bilirubin and ascorbate produced a negative interference with the phosphotungstate and Spinpro assays but had little effect on the N-geneous assay.

The A1 agonist CCPA reduced bisoxonol-monitored membrane potential depolarization elicited by high K+ in cerebrocortical nerve endings.

In this study the effect of the A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA) on bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol)-monitored membrane potential in cerebrocortical nerve endings was evaluated. CCPA (30, 100 and 300 microM) caused a dose-dependent decrease of high K(+)- and veratridine-induced membrane depolarization. This decrease was counteracted by the A1-specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (30-100 microM). On the contrary, the A2 receptor antagonist 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolol-[1,5-c]quinazol ine-5- imine (CGS 15943) was unable to interfere with the lowering effect exerted by CCPA (100 microM) on K(+)-elicited membrane depolarization. Finally, the A2 receptor agonist 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS 21680) did not induce any modification of K(+)-induced membrane depolarization. The results of the present study suggest that K(+)-induced membrane depolarization in cerebrocortical brain nerve endings may be modulated by A1 receptors.

Appearance of depolarization- and maitotoxin-induced [Ca2+]i elevation in single LAN-1 human neuroblastoma cells on exposure to retinoic acid.

LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage-sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid-treated cells. Both high K(+)- and maitotoxin-induced [Ca2+]i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd3+, an inorganic Ca(2+)-entry blocker, and by the snail toxin omega-conotoxin GVIA, which interacts with the N subtype of voltage-sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel subtype, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K(+)-induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K(+)-induced increase of [Ca2+]i was still present.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between time of activation of phospholipase C-linked plasma membrane receptors and reloading of intracellular Ca2+ stores in LAN-1 human neuroblastoma cells.

The effect of sequential stimulation of different inositol (1,4,5)-trisphosphate (IP3)-linked receptors on the functioning of intracellular Ca2+ stores was evaluated in single LAN-1 human neuroblastoma cells by means of fura-2 microfluorimetry. Homologous restimulation both in the absence and in the presence of extracellular Ca2+ with endothelin-1 (ET-1), Lys-bradykinin (BK), and ATP did not elicit an intracellular Ca2+ increase, whereas a [Ca2+]i elevation after carbachol (CCh) re-exposure was obtained only in the presence of extracellular Ca2+. Since thapsigargin and ionomycin, in the absence of extracellular Ca2+, were still able to release Ca2+ after ET-1, BK, and ATP but not after CCh, it can be argued that in the first case the stores were not completely depleted. This evidence was also confirmed by the fact that LAN-1 cells, sequentially exposed in different order to ET-1, BK, ATP, and upon extracellular Ca2+ removal, showed an increase of [Ca2+]i although progressively reduced in magnitude. By contrast, when CCh was perfused as the first agonist, it completely precluded any further Ca2+ mobilization by the other three agonists. In addition, the lack of potentiation of the Ca2+ response when BK and ET-1 were superfused together and the potentiation of Ca2+ response elicited by ET-1 after BK, when the plasma membrane Ca2+ efflux pathways were blocked by lanthanum during the first agonist exposure, indicated that LAN-1 cells can recycle cytoplasmic Ca2+ when exposed to ET-1, BK, ATP but not when exposed to CCh. This inhibitory effect of CCh (perfused for 90 s) on Ca2+ refilling was strictly dependent on the time of receptor occupancy since the exposure to CCh for a shorter period (15 s) produced the same effect on Ca2+ refilling when ET-1, BK, and ATP were perfused, as first agonist, for 90 s. Furthermore, the entity of Ca2+ refilling after 15 s of BK receptor occupancy was similar to that observed after 90 s. This seems to suggest that the receptors for ET-1, BK, and ATP maintain the transductional mechanisms in an activated state for a time shorter than the time of receptor occupancy. This was confirmed by the fact that IP3 levels during a 90-s BK exposure fell to prestimulated value within 30 s, whereas after CCh they reached a sustained plateau phase, after the peak.

Butanol fermentation liquor production and separation by reverse osmosis.

The problem of dilute solvent concentration in butanol-acetone fermentations can be solved by using reverse osmosis to dewater the fermentation liquor. Polyamide membranes have a potential application in a butanol-acetone fermentation and exhibited rejection rates as high as 98%. Optimum rejection of butanol in the fermentation liquor occurred at recoveries of 20-45%. Flux ranged from 0.05 to 0.6 L m(-2) min(-1).

Successive changes in the epimural bacterial community of young lambs as revealed by scanning electron microscopy.

Scanning electron microscopy was used to determine the time of initial colonization of the rumen epithelium of young lambs and successive changes with time in the morphological composition of the epimural community. Tissue samples were obtained from two groups of lambs at 1, 2, 4, 6, 8, and 10 weeks of age. Comparisons were made with the epimural communities observed at 12 well-distributed sites in the rumen of a mature wether. Epimural bacteria were already present on the epithelium at 1 week of age. The morphological composition of the epimural community changed with age, with the pattern of succession being similar in both groups of lambs. A total of 24 morphotypes were distinguished by scanning electron microscopy; 17 were rod shaped, 4 were cocci, 2 were spiral, and 1 was filamentous. These morphotypes were further subdivided into: (i) those persisting after their initial colonization in young lambs and present in the adult (7 morphotypes), (ii) those seen only in the adult (2 morphotypes), and (iii) those present only in young lambs (15 morphotypes). The seven morphotypes present in both the lamb and the adult could be considered indigenous members of the epimural community. Several morphotypes appeared restricted in their colonization to certain regions of the papillae, suggesting the presence of microhabitats within the epithelial habitat. Two rod-shaped bacteria were repeatedly seen specifically attached to one another, suggesting an interspecific association.

Isolation and identification of adherent epimural bacteria during succession in young lambs.

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.

Effect of dietary fiber on a guinea pig intestinal anaerobe, Bacteroides ovatus.

The purpose of this study was to determine the effect of fibrous plant components on the growth of intestinal bacteria. An anaerobe was isolated from the guinea pig cecum and identified as Bacteroides ovatus. The organism was grown anaerobically in two types of media and shown to require hemin or protoporphyrin IX. Treatment of the media with water-insoluble fractions of alfalfa, cabbage, spinach or wheat bran inhibited growth of the culture. Inhibition occurred whether the residue remained in the medium during culture growth or was removed before inoculation. A chelating resin also removed an essential component of the medium. Both treatments depleted the medium of hemin and addition of hemin restored growth and acid production. Treatment of the water-insoluble residues of alfalfa or cabbage with dilute NaOH decreased their inhibitory effects. This suggests that plant cell wall components possess unique and labile chemical properties. Dietary fiber may inhibit the growth of some anaerobic species in the lower intestinal tract by making hemin unavailable.

Eliminating a severely retarded blind adolescent's tantrums using mild behavioral interruption: a case study.

A blind 19 yr old severely retarded man was referred for behavior therapy because of violent and disruptive tantrums. Previous behavioral strategies had failed for various reasons. A very mild, brief, vestibular oriented physical procedure was designed to provide a low level disruptive effect. Intervention consisted of loud teacher demands to stop and work appropriately as well as guiding him through one 360 degrees turn while standing. The data demonstrated rapid and long term success in eliminating the client's tantrums. The results were interpreted in terms of a behavioral interruptor of a chained sequence allowing refocusing of client attention and increased levels of reinforcement.

Characterization of bacteria from a Swine manure digester.

One-hundred thirty bacteria isolated from a swine manure digester were predominately gram-positive anaerobes which were tentatively classified into the following genera: Peptostreptococcus, Eubacterium, Bacteroides, Lactobacillus, Peptococcus, Clostridium, and Streptococcus plus two unidentified groups. The major fermentation products formed by these organisms included acetate, propionate, succinate, lactate, and ethanol, singly or in various combinations. Acetate was the sole end product of several groups. Few of the isolates (14%) reduced the pH below 6.0. The predominate bacteria appear to differ from the predominate organisms isolated from other anaerobic ecosystems.

Medium for enhanced growth of bacteria from a Swine manure digester.

Most bacterial strains isolated from a swine manure digester grew sufficiently to permit transfer of cultures, but not characterization. Substrates, crude extracts, growth factors, and electron acceptors were evaluated for growth promotion. The growth of all but one group of the isolates was substantially increased with a medium containing glucose, cellobiose, soluble starch, pyruvate, peptone, yeast extract, minerals, volatile acids, vitamins, hemin plus vitamins K(1) and K(3), sodium bicarbonate, cysteine, and digester fluid. The strains require both known and unknown factors (in crude extracts) for maximal growth.

Medium for the enumeration and isolation of bacteria from a Swine waste digester.

A habitat-simulating medium was developed for the enumeration and isolation of bacteria from a swine waste digester. A roll tube medium with growth factors for strict anaerobes from previously studied anaerobic ecosystems was used to evaluate the effects of deletion, addition, or level of digester fluid, digester fluid treated with acid or base, rumen fluid, fecal extract, anaerobic pit extract, tissue extract, carbohydrates, peptones, short-chain fatty acids, minerals, vitamins, N and P sources, reducing and solidifying agents, buffers, and gases on colony counts. Decreasing the agar concentration from 2.5 to 1.0% increased the counts twofold. Blending increased the counts 1.7-fold. With a medium (174) containing digester fluid, peptones, minerals, cysteine, sodium carbonate, and agar, colony counts were 60% of the microscopic count and improved yields 2.5 to 20 times those obtained with media previously used for digesters or developed for other anaerobic ecosystems. Colony counts continued to increase for up to 4 weeks of incubation. Medium 174 permits the enumeration of total, methanogenic, and, with deletion of reducing agent, aerotolerant bacteria. The results suggest that the predominant bacteria grow slowly and have requirements different from those of bacteria from other ecosystems.

Glucose fermentation products in Ruminococcus albus grown in continuous culture with Vibrio succinogenes: changes caused by interspecies transfer of H 2 .

The influence of a H(2)-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H(2)-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H(2) from glucose by R. albus. V. succinogenes used the H(2) produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H(2). CO(2) was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H(2) was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H(2) for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H(2) by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H(2) formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H(2) when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H(2) gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.

An anaerobic chemostat that permits the collection and measurement of fermentation gases.

A chemostat was designed to allow anaerobic growth in the culture vessel in the absence of a continuous stream of O(2)-free gas. Produced gases were collected within the culture and collection vessels, and pressure build-up was prevented by allowing gases to expand into a collapsed football bladder. The culture overflow was collected in a flask, held at 0 C, that was emptied by applying a positive CO(2) pressure to the system. Ruminococcus albus, a H(2) and CO(2)-producing anaerobe, was used to test the operation of the apparatus. H(2) production was measured by sampling the various gas spaces of known volume and measuring H(2) concentration by gas chromatography. Measurement of accumulated fermentation gases and the effects of the accumulation on fermentations can be studied with the apparatus.