Intestinal permeability was assessed in 12 healthy cats by use of a differential sugar absorption test. A 50-ml isotonic aqueous solution containing a combination of 1.8 g of the disaccharide lactulose and 1.7 g of the monosaccharide mannitol was administered to cats via nasogastric tube. Urine was collected after 6 hours, and all urine samples were analyzed the same day, using a gas-liquid chromatographic technique (GLC) and an enzymatic assay (ENZ). Median urinary recovery of lactulose was 0.27 and 0.54% determined by GLC and ENZ, respectively. Differences between these groups were statistically significant (P = 0.023), and correlation between assays was high (r = 0.94, P < 0.01). Median urinary recovery of mannitol was 1.93 and 2.09% for GLC and ENZ, respectively. There were no statistically significant differences between these groups and the correlation between assays was high (r = 0.85, P < 0.01). The median lactulose-to-mannitol ratio was 0.29, using GLC, and was 0.52, using ENZ. Correlation of these ratios was again high (r = 0.93, P < 0.01).

The antibiotic polymyxin B sulfate is a cationic polypeptide with a unique cyclical configuration and distinct cationic characteristics. In this investigation, polymyxin B was evaluated to determine its ability to prevent synthesis of lactic acid and tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated strain RAW 2647 macrophage-like cell populations. In this context, gradient concentrations of polymyxin B were formulated in the presence of fixed concentrations of lipopolysaccharide fractions from Escherichia coli (B4:0111), E. coli (J5), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and S. typhimurium (Re). Quantitation of TNF-alpha was established by the application of a tissue culture-based biological assay system, using the WEHI 164 clone 13 indicator cell line. Investigations also included evaluation of the ability of gradient concentrations of lipopolysaccharide fractions from E. coli (B4:0111), E. coli (J5), K. pneumoniae, P. aeruginosa, S. minnesota, and S. typhimurium (Re) to form a complex with polymyxin B. This was established through application of high-performance thin-layer chromatography techniques. On the basis of the known molecular characteristics of lipopolysaccharide, its lipid A-core subfractions, and polymyxin B, these results imply that cytoprotective properties of polymyxin B are attributable to direct interaction and subsequent complex formation. More specifically, the mechanism by which polymyxin B exerts affinity for lipopolysaccharide fractions is proposed to occur through attractive ionic interactions established between the cationic diaminobutyric acid residues of polymyxin B and the mono- or diphosphate group(s) of the lipid A-core moiety. It is highly probable that this molecular phenomenon is accompanied by hydrophobic interactions established between the terminal methyloctanoyl or methylheptanoyl groups of polymyxin B and the saturated carbon chains of the lipid A-core subfraction of lipopolysaccharide fractions.

We purified porcine whey lactoferrin by affinity chromatography on a heparin-sepharose column, followed by high-performance liquid chromatography. Molecular mass of purified lactoferrin (PLF) is 78,000 daltons. The iron-binding activity of PLF had a UV/ visible-light absorption spectrum indistinguishable from that of human and bovine lactoferrins (absorbance ratio [465 nm/280 nm] approx 0.046). The growth ratio of WIL-2 cells in PLF-supplemented medium is 70% of that in serum-containing medium. The aforementioned characteristics are similar to those of human and bovine lactoferrins. Immunoblot analysis, using polyclonal antibody raised in rabbits against porcine whey lactoferrin, revealed high specificity for PLF, and low cross-reactivity with commercial human and bovine lactoferrins.

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (PSGAG) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of PSGAG/ml for 48 hours, after which the medium was replaced with medium containing similar doses of PSGAG and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the PSGAG for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation. Polysulfated glycosaminoglycan caused a significant (P < 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to PSGAG were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [K(AV)] = 0.40), and was not influenced by PSGAG treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that PSGAG in vitro, at the dosages evaluated, caused a decrease in proteoglycan synthesis, had little effect on labeled proteoglycan degradation, did not influence the size of large monomer, and caused a modest increase in the concentration of keratan sulfate in proteoglycans synthesized by osteoarthritic equine chondrocytes.

A murine IgM monoclonal antibody causing bacterial agglutination was used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling efficiency were > 99% when evaluated by ELISA, agglutination testing, and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.

We used size-exclusion high-performance liquid chromatography (HPLC) to investigate the properties of the 2 isoforms of vitamin A-containing (holo) retinol-binding protein (RBP) in animals: the form that is bound to transthyretin (holo-TTR-RBP), and the form that does not bind to TTR (holo-free RBP). We also used radial immunodiffusion to measure immunologically active RBP (apo+ holo RBP). We compared the isoforms of RBP in animals with those of human beings to determine which animal is the best model of human RBP. Size-exclusion HPLC detected holo-free and holo-TTR-RBP in every animal species studied. Apparent concentration of holo-TTR-RBP varied among species: that of rabbits and dogs much greater than that of apes, sheep, goats, monkeys, rhinoceroses, felids, rats, human beings, and deer greater than that of pigs, zebra, and bison greater than that of penguins. Dogs have unusual RBP chromatograms; they have high concentration of RBP, but also appear to transport much of their vitamin A on proteins other than RBP, Human RBP antibody preparations could detect apo + holo RBP immunologic activity only in apes, monkeys, and felids. Apes and monkeys appeared to have complete cross-reactivity to human RBP antibodies. Felids may have substantial, but partial, cross-reactivity. Apes and monkeys appear to be the most relevant animal models for study of human RBP transport. However, there is a need for less-expensive models. Further research is needed, but in the interim, rats or sheep may be satisfactory for some purposes.

Labor and delivery stimulate increased release of catecholamines and endogenous opioid peptides in neonates. Catecholamines promote adaptation to the extrauterine environment after birth. Enkephalins are stored together with catecholamines in the adrenal medulla and have an inhibitory effect on catecholamine release. We investigated the influence of labor and neonatal hypoxia on epinephrine, norepinephrine, and met-enkephalin release in calves. Blood samples were taken from the umbilical artery before rupture of the umbilical cord and from the jugular vein repeatedly after birth. Highest plasma norepinephrine concentration was found in calves delivered at the end of gestation (term calves) before umbilical cord rupture. In calves delivered before the physiologic end of gestation (preterm calves), norepinephrine values increased after cord rupture, but remained lower than values in term calves. Epinephrine release followed a similar pattern, but norepinephrine was clearly predominant. In term calves, met-enkephalin values were significantly higher than values in preterm calves. In calves of both groups, met-enkephalin release increased after cord rupture. During birth, the increase in catecholamine release seems to take place earlier than that of enkephalins. Norepinephrine-dominated stimulation during expulsion of the calf might be followed by increasing enkephalinergic inhibition after cord rupture and onset of respiration. Reduced release of catecholamines and enkephalins in preterm calves may be connected with delayed adaptation to the extrauterine environment.