Influence of a novel bm3 corn silage hybrid with floury kernel genetics on lactational performance and feed efficiency of Holstein cows.

Dry matter intake, lactation performance, and chewing behavior of multiparous Holstein cows (n = 15) fed diets containing a novel bm3 corn silage hybrid with floury kernel genetics were compared with cows fed diets containing commercially available conventional and bm3 hybrids using a replicated 3 × 3 Latin square design with 28-d periods. Cows were housed in tiestalls, milked 3 times/d, and fed a total mixed ration containing 49.0% (dry matter basis) of (1) a conventional corn silage hybrid (CONV); (2) a brown midrib bm3 hybrid (BMR); or (3) a bm3 hybrid with floury kernel genetics (BMRFL). All diets contained 6.3% hay crop silage and 44.7% concentrate. Dietary nutrient composition averaged 32.7% neutral detergent fiber (NDF) and 26.3 starch (% of dry matter). Data were analyzed by ANOVA using the MIXED procedure in SAS (SAS Institute Inc., Cary, NC). The dry matter intake was greater for cows fed BMR (28.0 kg/d) compared with CONV (26.8 kg/d), whereas dry matter intake for cows fed BMRFL was intermediate (27.6 kg/d). Energy-corrected milk (ECM) yield was greater for cows fed BMR (50.3 kg/d) and BMRFL (51.8 kg/d) compared with CONV (47.2 kg/d). Milk fat yield was higher for cows fed BMRFL (1.87 kg/d) compared with CONV (1.74 kg/d) and BMR (1.80 kg/d). Milk protein yield was greater for cows fed BMR (1.49 kg/d) and BMRFL (1.54 kg/d) compared with CONV (1.36 kg/d). Milk urea-N was reduced for cows fed BMR (11.61 mg/dL) and BMRFL (11.16 mg/dL) compared with CONV (13.60 mg/dL). Feed efficiency (ECM/dry matter intake) was higher for cows fed BMRFL (1.87) compared with CONV (1.76) and BMR (1.79). Milk N efficiency was greatest for cows fed BMRFL (40.4%) followed by BMR (38.1%) and finally CONV (35.3%). Cows fed CONV chewed 5 min more per kilograms of NDF consumed than cows fed either of the BMR hybrids. No differences were observed among diets in apparent total-tract digestibility of NDF (58.1%) or starch (99.3%). Overall lactational performance was enhanced for cows fed diets containing both BMR and BMRFL hybrids versus CONV. In addition, feeding the BMRFL corn silage improved efficiency of component-corrected milk production and milk N efficiency compared with the CONV and BMR silages.

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13.

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

The tyrosine phosphatase STEP: implications in schizophrenia and the molecular mechanism underlying antipsychotic medications.

Glutamatergic signaling through N-methyl-D-aspartate receptors (NMDARs) is required for synaptic plasticity. Disruptions in glutamatergic signaling are proposed to contribute to the behavioral and cognitive deficits observed in schizophrenia (SZ). One possible source of compromised glutamatergic function in SZ is decreased surface expression of GluN2B-containing NMDARs. STEP(61) is a brain-enriched protein tyrosine phosphatase that dephosphorylates a regulatory tyrosine on GluN2B, thereby promoting its internalization. Here, we report that STEP(61) levels are significantly higher in the postmortem anterior cingulate cortex and dorsolateral prefrontal cortex of SZ patients, as well as in mice treated with the psychotomimetics MK-801 and phencyclidine (PCP). Accumulation of STEP(61) after MK-801 treatment is due to a disruption in the ubiquitin proteasome system that normally degrades STEP(61). STEP knockout mice are less sensitive to both the locomotor and cognitive effects of acute and chronic administration of PCP, supporting the functional relevance of increased STEP(61) levels in SZ. In addition, chronic treatment of mice with both typical and atypical antipsychotic medications results in a protein kinase A-mediated phosphorylation and inactivation of STEP(61) and, consequently, increased surface expression of GluN1/GluN2B receptors. Taken together, our findings suggest that STEP(61) accumulation may contribute to the pathophysiology of SZ. Moreover, we show a mechanistic link between neuroleptic treatment, STEP(61) inactivation and increased surface expression of NMDARs, consistent with the glutamate hypothesis of SZ.

Loss of medial septum cholinergic neurons in THY-Tau22 mouse model: what links with tau pathology?

Alzheimer's disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the relationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum. Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.

Within-breed heterozygosity of canine single nucleotide polymorphisms identified by across-breed comparison.

Identification of single nucleotide polymorphisms (SNPs) by DNA sequence comparison across breeds is a strategy for developing genetic markers that are useful for many breeds. However, the heterozygosity of SNPs identified in this way might be severely reduced within breeds by inbreeding or genetic drift in the small effective population size of a breed (population subdivision). The effect of inbreeding and population subdivision on heterozygosity of SNPs in dog breeds has never been investigated in a systematic way. We determined the genotypes of dogs from three divergent breeds for SNPs in four canine genes (ACTC, LMNA, SCGB, and TYMS) identified by across-breed DNA sequence comparison, and compared the genotype frequencies to those expected under Hardy-Weinberg equilibrium (HWE). Although population subdivision significantly skewed allele frequencies across breeds for two of the SNPs, the deviations of observed heterozygosities compared with those expected within breeds were minimal. These results indicate that across-breed DNA sequence comparison is a reasonable strategy for identifying SNPs that are useful within many canine breeds.

Estimate of nucleotide diversity in dogs with a pool-and-sequence method.

Nucleotide diversity (pi), the average number of base differences per site for two homologous sequences randomly selected from a population, is an important parameter used to understand the structure and history of populations. It is also important for determining the feasibility of developing a genetic map for a species from single nucleotide polymorphisms (SNPs). Nucleotide diversity has never been estimated for dogs. Segments of twelve canine genes from ten diverse dog breeds were examined for nucleotide variation by using a pool-and-sequence method. We identified three SNPs in the coding regions (2501 bp) and 11 SNPs in the introns (2953 bp). Each of these putative SNPs was tested by restriction enzyme analysis, and all were verified. Six additional SNPs were identified in a single SINE contained in one gene. Using these data, canine sequence diversity across breeds was estimated to be 0.001 and 0.0004 in intronic and coding regions, respectively, with SNPs spaced every 400 bp on average. Discovery of useful SNPs in 7 of the 12 genes suggests that construction of a canine SNP-based map can be accomplished with current technology. Thirteen polymorphic SNPs were also found in 5847 bp in the cat, horse, ox, and pig, by using four of the same genes from which canine nucleotide diversity was estimated. These results suggest that these species may have similar amounts of nucleotide diversity.

Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase.

A tonoplast-type vesicle preparation, substantially free from other subcellular membranes, was obtained from corn roots by equilibrium sucrose density gradient centrifugation. At pH 6.5 and in the presence of chloride ions, the tonoplast-type ATPase activity as measured by Pi release, was inhibited by nitrate ions. The ATPase activity was insensitive to molybdate and vanadate, indicating a minimum nonspecific phosphatase and plasma membrane contamination. The vesicles exhibited an ATP hydrolysis-supported proton uptake which was measured by the absorption change of acridine orange. The ATP hydrolysis supported uptake and the subsequent perturbant-induced release of protons (decay) was described by a kinetic model which was previously developed to evaluate the coupling between proton pumping and the primary energy yielding process for other biomembranes. The proton pumping activity was more sensitive to nitrate ions then was ATP hydrolysis. The differential effect and the kinetic analysis of nitrate inhibition led us to suggest that (i) the coupling between Pi release and proton pumping was indirect in nature and (ii) the primary inhibitory effect of nitrate ion was originated from an interaction with a protogenic protein domain which is functionally linked to the ATPase in the tonoplast-type membrane.

Evaluation of sexual dysfunction in the female following rectal resection and intestinal stoma.

Problems of sexual dysfunction were surveyed by questionnaire mailed to women who had undergone rectal resection and ostomy surgery over a ten-year period. Completed questionnaires were returned by 30 respondents, 19 to 54 years of age, who indicated both physical and psychological problems. Many of the problems were solved by the patients themselves, but the way can be made easier with the help of an understanding surgeon and knowledgeable stoma therapist who can create a climate in which the patient may feel at ease asking for guidance concerning sexual matters.

Initial experience of a nephrologist-operated vascular access center.

A nephrology practice in Alabama did not feel in control of vascular access management. Scheduling delays, as well as variable techniques and outcomes, leading to high morbidity and mortality, caused frustration with the existing care system for vascular access. Our objective was to develop an integrative system of vascular access care, involving nephrologists along with the other caregivers, and to demonstrate an improvement in outcomes. Nephrology Vascular Labs (NVL), a recent RMS-Lifeline acquisition, opened a vascular access center (VAC) as an extension of the nephrology practice. Both pre-ESRD and ESRD patients are evaluated and treated in the VAC. Treatment is rendered in a timely fashion, to the benefit of the patients. Nephrologists serve as the interventionists. More than 90% of vascular access problems detected at dialysis are treated at the VAC. More than 2000 procedures have been performed over 2 years. Procedures carried out include thrombolysis with angioplasty, fluoroscopy alone or with angioplasty, placement of cuffed and noncuffed catheters, removal of cuffed catheters, and minor surgeries. Success rates have been high. Minor and major complications have been relatively low. Referrals to both surgeons and radiologists are shown to emphasize the role of the VAC as part of an integrative system of vascular access care. Results of a patient satisfaction survey were excellent. The VAC has fulfilled the vision of creating a seamless integration of care for vascular access. Hospitalization rate has been reduced and it is suspected that the global cost of access care is markedly lower than prior to the VAC. Multiple nephrologists can rotate as the VAC's interventionist and jointly obtain good outcomes and have little variability among them. Several reasons for using a nephrologists as the interventionist are discussed.

Effects of multivalent cations on cell wall-associated Acid phosphatase activity.

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu(2+), Mg(2+), Zn(2+), and Mn(2+); unaffected by Ba(2+), Cd(2+), and Pb(2+); and inhibited by Al(3+). The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg(2+). On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al(3+) and Hg(2+) was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg(2+). In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca(2+) significantly reduced the effects of Hg(2+) or Al(3+), but not Mg(2+), to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg(2+) or Al(3+) which caused a Ca(2+)-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase.

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.

Gene-specific universal mammalian sequence-tagged sites: application to the canine genome.

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, and RB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as "anchored reference loci" for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets "universal mammalian sequence-tagged sites" because they should be useful for many mammalian genome projects.

Carbon metabolism in spores of the arbuscular mycorrhizal fungus Glomus intraradices as revealed by nuclear magnetic resonance spectroscopy.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO(2) is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state.

Method for simultaneous epicardial and endocardial mapping of in vivo canine heart: application to atrial conduction properties and arrhythmia mechanisms.

INTRODUCTION: It has been suggested that the three-dimensional structure of the atria may be crucial in arrhythmogenesis; however, previous in vivo atrial activation mapping studies have been limited to either endocardial or epicardial approaches. METHODS AND RESULTS: To investigate the role of endocardial and epicardial structures and their interaction in atrial conduction and arrhythmias, we used five epicardial plaques and two intra-atrial balloon arrays to record a total of 368 unipolar electrograms from the entire epicardial and endocardial surface of both atria. During regular 1:1 pacing from the right atrial appendage, right atrial endocardial activation spread considerably faster than epicardial (total activation time 45+/-12 msec vs 60+/-19 msec, respectively [mean +/- SD]; P < 0.05), pointing to preferential conduction over structures like the crista terminalis and pectinate muscles. No such differences were noted in the left atrium. Transseptal spread occurred via discrete anterior and posterior pathways, causing separate breakthroughs in anterior and posterior atrial regions, respectively. Dissociation between septal pathways played a role in reentry during vagal atrial fibrillation. In 2 of 4 dogs with atrial fibrillation associated with congestive heart failure, single macroreentrant circuits involving endocardial and epicardial components were revealed during the arrhythmia. CONCLUSION: We conclude that activation mapping using simultaneous recording from both epicardial and endocardial surfaces provides potentially important insights into the mechanisms of atrial conduction and arrhythmogenesis.

Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3- exchanger.

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.