Impact of acute water and feed deprivation events on growth performance, intestinal characteristics, and serum stress markers in weaned pigs.

The impact of acute stressors (24-h feed or water deprivation) on growth performance, intestinal characteristics, and serum stress markers in weaned pigs was evaluated. Pigs (6.21 ± 0.29 kg) were allotted in a randomized complete block design to 4 treatments on the basis of BW at the time of weaning. There were 8 mixed-sex pigs in each of 12 pens per treatment. Treatments were arranged as a 2 × 2 factorial and consisted of a feed or water stressor that included a 0- or 24-h deprivation period postweaning, and pigs were subsequently allowed access to feed and water. Growth performance was measured 1, 7, 14, and 28 d postweaning. Serum and intestinal samples were taken 1 and 7 d postweaning. Serum was analyzed for cortisol and corticotrophin-releasing factor, and villus height, crypt depth, and mast cell density were measured in the jejunum and the ileum. Expression of mucin (MUC2), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), claudin 1 (CL-1), occludin (OC), and zonula occludens 1 (ZO-1) genes were measured on d 1 and 7 postweaning in the jejunum and ileum by real-time PCR. There was a decrease (P < 0.05) in ADG with the water stressor 1 d postweaning, although subsequently, there were improvements (P < 0.05) in ADG and feed efficiency. Furthermore, the water stressor reduced ADFI during the last 14 d of the trial and cumulatively (P < 0.05). Seven days postweaning there was an increase (P < 0.05) in jejunal villous height to depth ratio due to the feed stressor and a decrease (P < 0.05) in the ileal villous height to depth ratio due to the water stressor. There was an increase (P < 0.05) in serum cortisol levels due to the water stressor both 1 and 7 d postweaning. Furthermore, there was an increase in serum corticotrophin-releasing factor 1 d but not 7 d postweaning due to the water stressor (P < 0.05). The feed stressor reduced (P < 0.05) TNF-α gene expression, and the water stressor reduced (P < 0.05) OC gene expression in the jejunum 1 d postweaning. In the ileum, there was a reduction in CL-1 and ZO-1 gene expression (P < 0.05) due to the water stressor 7 d postweaning. The results from the current investigation showed that a 24-h feed or water deprivation at the time of weaning has negative impacts on growth performance, intestinal characteristics, and serum stress responses immediately following the stress event and throughout the nursery period.

Preparation of Micrometer Size Budesonide Particles by Precipitation.

Particles of different shape, size and crystallinity of budesonide, a hydrocortisone streroid, were prepared by precipitation. The method is based on taking advantage of the different solubility of this material in certain organic solvents (ethanol, acetone) and water or alcanes. In principle, to solutions of budesonide, water was added until the solids precipitate or the same solute in mixed solvents was carefully separated by evaporation of the more volatile component at room temperature. The morphology, size, and stability of the resulting particles were sensitive to the evaporation technique, stirring, and presence of a stabilizing agent. Copyright 2000 Academic Press.

Age-related difference in tamoxifen disposition.

PURPOSE: To investigate the pharmacokinetic aspects of tamoxifen, such as the pharmacokinetic-pharmacodynamic (toxicity and clinical response) relationship and the influence of hepatic dysfunction, age, treatment duration, and associated chemotherapy on tamoxifen pharmacokinetics. PATIENTS AND METHODS: Three hundred sixteen patients with breast cancer (247 postmenopausal women) were investigated. Mean age was 58 years (age range, 29 to 85 years). One hundred seventeen patients received tamoxifen as single therapy (adjuvant, 60; neoadjuvant, 17; metastatic, 40); 292 of 316 received 30 mg daily. We obtained 794 blood samples at steady state. Tamoxifen and metabolites, N-desmethyltamoxifen, N-desdimethyltamoxifen, primary alcohol, and 4-hydroxytamoxifen were measured by HPLC. RESULTS: Serum concentrations of tamoxifen and metabolites showed a wide asymmetrical distribution. Median and extremes were 347 nmol/L (not detectable [ND] to 1677) for tamoxifen, 572 nmol/L (ND to 3132) for N-desmethyltamoxifen, 109 nmol/L (ND to 795) for N-desdimethyltamoxifen, and 59 nmol/L (ND to 390) for primary alcohol. 4-Hydroxytamoxifen was detectable in 9.5% of the samples (ND to 162 nmol/L). Neither the absolute nor the relative concentrations of each compound showed significant variations during treatment. Chemotherapy concomitant with tamoxifen slightly increased the tamoxifen blood concentration. Hepatic dysfunction had no obvious effect on drug concentrations, an exception being a slight reduction in the relative proportion of tamoxifen. The influence of age revealed that concentrations of tamoxifen and metabolites increased significantly with age: women younger than 40 years had a tamoxifen plus metabolite median concentration of 802 nmol/L compared with 2428 nmol/L for women older than 80 years. In the 28 patients in whom tamoxifen-related side effects developed, the proportion of demethylated metabolites was higher than that in patients in whom toxicity did not develop. There was no difference in drug concentrations between responding and nonresponding patients. CONCLUSION: Despite the tremendous interpatient variability in drug concentrations, the present data show that tamoxifen and metabolite concentrations significantly increase with age.

Production and characterization of monoclonal antibodies against the H-Y antigen.

Monoclonal antibodies against the H-Y antigen were produced using spleen cells from female C57BL/6 mice hyperimmunized with cells from syngeneic males. Anti-H-Y positive clones were detected by enzyme immunoassays. Supernatant fluids from Daudi cell cultures and testicular cell preparations taken from mice, rabbits or calves served as presumptive sources of H-Y antigen. In addition, testis supernatant from genetically sterile mice was used. Male specificity was ascertained by the fact that the antibodies could be absorbed with spleen cells from male but not from female mice. Binding of the antibodies to H-Y antigen on the surface of male and female cells, obtained from a number of tissues and species, was confirmed by an indirect immunofluorescence assay. Several monoclonal antibodies appeared to be positive in all assays tested, suggesting that the molecule conferring the H-Y antigenicity lacks species-specificity and appears to be identical for soluble and membrane-bound H-Y antigen.

The DNA content of cerebral cortex neurons. Determinations by cytophotometry and high performance liquid chromatography.

Previous work from our laboratories has indicated that the DNA content of rat cerebral cortex neurons increases postnatally to a level of slightly above 3c, where 2c denotes the diploid DNA complement. We have re-evaluated this concept by using various cytophotometric assays and a novel high performance liquid chromatography (HPLC) technique. The latter consists of digesting the DNA in isolated neuronal nuclei by a mixture of DNA-degrading enzymes followed by analysis of the resulting deoxynucleosides by HPLC. We find that the various methods fall into two groups. The first gives evidence of a postnatal DNA (or histone) increase, while the second does not. The first group (DNA increase) comprises cytofluorometry for DNA following Schiff-type staining with fluorochromes 2,5-bis-(4-aminophenyl)-1,3,4-oxadiazole (BAO) and pararosaniline, ultraviolet absorption scanning for DNA and cytofluorometry for histones following staining with sulfaflavine at pH 8. The second group (no DNA increase) consists of cytofluorometry for DNA following staining with the DNA-complexing agents mithramycin, chromomycin A3, 4',6-diamidino-2-phenylindole (DAPI) and bisbenzimide (Hoechst 33258), as well as the newly developed HPLC technique. Since the HPLC technique measures DNA by a direct chemical approach without interference from other nuclear constituents or from higher order packaging in the chromatin, and detects at least 94-95% of the total DNA contained in neuronal nuclei independent of the developmental stage, we infer that the HPLC technique and, by consequence, the cytochemical assays of the second group reflect true DNA values. Therefore, we propose that cerebral cortex neurons retain a diploid DNA level throughout development.

Cytofluorometric DNA base determination for the investigation of heterochromatin and heterochromatin amplification.

The measurement of chromomycin A3/DAPI fluorescence ratios is shown to allow base content determinations in eu- and heterochromatic regions of interphase cell nuclei. The base content values obtained in chromocenters and euchromatin of Scilla sibirica agree with those measured earlier [12] on the band and non-band areas of the chromosomes of this species. In Sinapis alba three different heterochromatin types, with regard to base content, can be discerned. The heterochromatin amplification observed in the polyploid nuclei of Sinapis roots and hypocotyl involves either all three heterochromatin types, with little resulting change of the total nuclear base content, or only one or the other of them, with a measurable shift of the base content.

Monoclonal antibody to streptococcal group B carbohydrate: applications in latex agglutination and immunoprecipitin assays.

Two monoclonal mouse antibodies with specificities for group B streptococcal capsular antigens were evaluated in assays for the identification of group B streptococci (GBS). One of these antibodies (A9) was shown to precipitate group B carbohydrate antigen in reactions with both purified group B antigen and antigen present in autoclave or enzyme extracts of GBS. A9 antibody was also specific for group B antigen in gel diffusion reactions with extracts of Lancefield group A, B, C, D, F, and G streptococci and was a highly sensitive reagent in detecting soluble group B antigen by counterimmunoelectrophoresis. Antigen extracted from all five serotypes of GBS was shown to be precipitated by A9 antibody. A second monoclonal antibody (C8) was reactive with intact GBS but did not precipitate soluble antigen in bacterial extracts. In contrast with what has been shown for polyclonal rabbit anti-group B antiserum, neither antibody was significantly inhibited in binding or precipitation assays by high concentrations of free rhamnose or other monosaccharides of carbohydrates found in group B antigen. Rhamnose, the most abundant carbohydrate of the group B antigen, does not appear therefore to be an immunodominant determinant in the binding of A9 or C8 antibody. The epitopes of both monoclonal antibodies are exposed on the surface of live as well as heat-fixed GBS cells. A9 antibody-coated latex particles were compared with a commercially available polyclonal latex agglutination reagent and shown to be equally sensitive and specific in the detection of soluble group B antigen in urine and cerebrospinal fluid from patients with GBS infections. Because of its uniformity and defined antigen specificity, which includes reactivity with all five serotypes of GBS, A9 antibody offers the potential of an improved immunodiagnostic reagent for the identification of GBS.

Use of soluble and Staphylococcus aureus-immobilized second antibody compared in a radioimmunoassay for human alpha-fetoprotein.

Staphylococcus aureus cells coated with rabbit anti-goat immunoglobulin G were compared with soluble second antibody in a radioimmunoassay for human serum alphafetoprotein. Recovery of primary immune complexes with use of the bacterial immunoadsorbant (a) allowed second-antibody incubation time to be shortened from 3 h to 20 min at 37 degrees C, (b) decreased by 80% the amount of second antibody required per assay, thereby significantly reducing assay costs, and (c) provided a convenient and reproducible method for separation of free and bound antigen. The ease of preparation and the uniformity and stability of the S. aureus second-antibody reagent are additional advantages that may be useful in other radioimmunoassay procedures.

In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins.

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.

Purification and properties of a nicotinamide adenine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism.

Glycerol:NAD+2-OXIDOREDUCTASE (EC 1.1.1.6) was purified to homogeneity from a mutant of Escherichia coli K12 that uses this enzyme, instead of ATP:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit of 39,000 daltons. During electrophoresis under nondenaturing conditions, the protein migrates as two bands. These two forms, both of which are enzymatically active, appear to be dimers and octomers of the same subunit. The optimal pH for the oxidation of glycerol is about 10, and that for the reduction of dihydroxyacetone is about 6. Glycerol dehydrogenation is highly activated by NH4+, K+, or Rb+, but strongly inhibited by N-ethylmalemide, 8-hydroxyquinoline, 1,10-phenanthroline, Cu2+, and Ca2+. The enzyme exhibits a broad substrate specificity. In addition to glycerol, it act on 1,2-propanediol and several of its analogs.

The fluorescence and bright field microscopic demonstration of cathepsin B in human fibroblasts.

Cathepsin B was demonstrated cytochemically in human fibroblasts with Z-Ala-Arg-Arg-2-(4-methoxy)naphtylamide as substrate. The enzyme was visualized in the bright field microscope with the diazonium salt Fast Blue B as coupling reagent and in the fluorescence microscope with 5-nitrosalicylaldehyde. With both methods cathepsin B was found in small granules distributed throughout the cytoplasm.

Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes.

Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary ("reverse") banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.

Cytophotometric study of nuclear differentiation during pollen development in Tradescantia paludosa.

During pollen development the dry weight, total protein, histone, DNA, arginine, and lysine content were analysed by cytophotometric methods in partially isolated nuclei. The amount of analysed substances increased from the end of the meiosis to the mitosis of the microspores to the double of the initial values. After mitosis the ratio histone/DNA remained almost unchanged in both vegetative and generative nuclei. On the other hand a large difference in the ratio non-histone protein/DNA could be observed, the vegetative nucleus containing more non-histone protein than the generative nucleus. The rate of RNA synthesis being higher in the vegetative nuclei, these non-histone proteins may have some function in nuclear activation. The DNA of the generative nucleus is duplicated before anthesis, whilst in the vegetative nucleus the DNA content remains constant.

Isolation of nontoxinogenic mutants of Vibrio cholerae in a colorimetric assay for cholera toxin using the S49 mouse lymphosarcoma cell line.

A mouse lymphosarcoma (S49) cell line that is growth-inhibited by agents that elevate intracellular concentrations of adenosine 3':5'-cyclic phosphate was used in a sensitive and convenient colorimetric assay for cholera toxin. S49 cells suspended in Dulbecco's modified Eagle's minimal essential medium containing 10(-5)--10(-6) M RO 20-1724, an analogue of 4-(3,4-demethoxybenzyl)-2-imidazolidinone and a phosphodiesterase inhibitor, were growth-inhibited by subnanogram concentrations of cholera toxin. Effects of toxin were detected by the absence of a yellow pH change (phenol red indicator) which normally accompanies the production of acid metabolites by lymphoma cells. An assay using S49 cells grown in microtiter plates, which is capable of detecting 10 pg of cholera toxin or 0.01 units of cholera antitoxin, was used in screening for nontoxinogenic mutants of Vibrio cholerae strain 569B. The properties of two mutants of the Tox--phenotype, which lacked biologically and immunologically detectable toxin products, are described.