James G. Lecce

Tissue distribution of mammalian sulfhydryl oxidase

Sulfhydryl oxidase activity is present in cow, goat, sow, human, and rat milks, and can also be measured in several rat tissues following homogenization in 1% polyoxyethylene-9-lauryl ether. These include lactating mammary tissue, kidney, and pancreas. Bovine kidney homogenates also exhibit sulfhydryl oxidase activity; however, no activity could be detected in rat thymus, brain, heart, liver, spleen, lung, or small intestinal tissue homogenates. Indirect immunofluorescent staining of tissue sections using rabbit antibodies directed against highly purified bovine milk sulfhydryl oxidase preparations revealed that the enzyme is closely associated with the plasma membrane of lactating cow and rat mammary tissues and the basal-lateral membrane of rat kidney cortex. In addition, the oxidase appears to be associated with endothelial cells lining the capillaries of rat kidney, heart, and small intestine, and centroacinar cells in pancreatic tissue slices also stain for sulfhydryl oxidase. In contrast, liver, brain, and thymus tissues do not exhibit fluorescent staining and appear to be devoid of sulfhydryl oxidase activity.

PORCINE POLYSEROSITIS WITH ARTHRITIS: ISOLATION OF A FASTIDIOUS PLEUROPNEUMONIALIKE ORGANISM AND HEMOPHILUS INFLUENZAE SUIS*

This study stemmed from observation of a disease affecting piglets that were used in nutritional experiments. Because of the nature of the experiments (fat studies), it was necessary to use, as experimental animals, piglets free of colostrum. These pigs were caught at birth and placed in individual cages in an isolation unit. Within 12 hours of birth, 10 to 15 ml. of 10 per cent porcine gamma globulin was administered intraperitoneally in order partially to replace immune bodies usually obtained from colostrum. At the end of 6 to 8 weeks, these colostrum-free pigs had outgrown their original cages and, in order to continue the experiment, space limitations made it necessary to move them from isolation to a pig farm. After approximately 4 days at the pig farm, the following symptoms of disease were observed: anorexia, high fever, weakness in hindquarters, unsteady gait, and swollen joints. Within a week after the first appearance of symptoms, approximately 70 per cent of the pigs died. Post-mortem examinations consistently revealed fibrinous peritonitis, pleuritis, pericarditis, and excess and viscous synovial fluid in the joint cavities. In addition to the polyserositis with arthritis, there was observed a hemorrhagic gastroenteritis and cystitis. The isolation and identification of infectious agents capable of reproducing this syndrome are the subjects of this report.

Effects of Nursing on Growth and Development of Small Bowel Mucosa in Newborn Piglets

Trophic factors in natural milk are potential mediators of the rapid growth of intestine in neonates. To determine whether nursing stimulates growth and development of small bowel mucosa, litters of piglets were divided into suckled and artificially reared groups at birth. The latter animals were raised in an automated feeding device (Autosow) with an artificial diet simulating the nutritional composition of sow milk. At 2, 8, and 15 d of age, animals were killed and 10-cm segments of jejunum, mid-bowel, and ileum were removed. Mucosal homogenates were prepared for enzyme assay and measurement of mucosal mass. Mean body weight, total length of bowel, and circumference of bowel segments did not differ between the two feeding groups at any age studied. As anticipated, mean mucosal ornithine decarboxylase activity decreased (p < 0.001) and measurements of mucosal mass increased (p < 0.001) with age; however, mean values for each of these measures were never greater in the nursed animals in comparison to the artificially reared group in any segment at any age. In addition, levels of disaccharidase activity did not correlate with the feeding regimen. To investigate the possibility that unanticipated growth factors in the artificial diet might have accounted for the apparent lack of trophic effect of nursing compared to artificial rearing, we evaluated the effects of this diet and of sow colostrum on 3H-thymidine incorporation in Swiss 3T3 cells in vitro. Colostrum, but not artificial diet, stimulated greater incorporation of 3H-thymidine than culture medium alone (p < 0.005). The mitogenic activity of colostrum was not inhibited by the addition of neutralizing antibody against epidermal growth factor or IGF-I. In conclusion, the effect of nursing on growth and development of the small bowel mucosa in the piglet does not differ from that of rearing with an artificial diet, despite the finding that porcine colostrum has greater growth-promoting activity than artificial diet in vitro. Neither epidermal growth factor nor IGF-I alone in colostrum appears to be required for this mitogenic effect.

THE EFFECT OF DIET ON THE MATURATION OF THE NEONATAL PIGLET'S SERUM PROTEIN PROFILE AND RESISTANCE TO DISEASE*

The work described in this paper grew from difficulties encountered in raising colostrum-free pigs for subsequent use in experiments dealing with either infectious disease’ or mineral metabolism? To secure such pigs, they either were removed from the sow approximately 2 days before term by hysterectomy or were caught in sterile towels a t parturition. Immediately following birth, colostrum-free pigs were carried to another isolation unit and placed individually in sterilized stainless steel cages where they were fed 6 times a day on a diet consisting of cow’s milk supplemented with minerals and vitamins. In spite of what was considered strict, sanitary conditions, it was not unusual to lose 30 to 60 per cent of the litter. Generally, diarrhea was observed a t about the third day of life, most of the pigs dying between the fourth and the eighth day. Escherickia coli, type 08, frequently were isolated from the blood of morbid and the liver of dead pigs, as well as from the feces of healthy, vigorous pigs? Little future trouble was encountered in raising piglets that survived the first 8 days. I t was possible to circumvent the mortality of the first week, either by continuously treating the pigs with intramuscular injections of chloromycetin or by limiting the intake of food, coupled with intraperitoneal injections of porcine gamma globulin? Although these “tricks” minimized death losses, the lack of weight gain and poor appearance of survivors impressed on us the need for questioning the adequacy of cow’s milk as a food for baby pigs. Nursing pigs born devoid of gamma globulin obtain from the sow a colostrum rich in gamma globulin, whereas pigs fed cow’s milk are deprived of this important bulwark against disease. The importance of colostrum might be inferred from the work with calves, for it has been shown that cow’s colostrum provides the calf, also born essentially gamma globulin-free, with immune globulin. It is thought that the immune globulin contains antibodies to E . coli and thereby protects the calf from diarrhea and death.ss6 Without minimizing the role and the importance of antibodies in disease resistance, it was our intent to investigate other possibilities whereby the mammary secretions of the sow might contribute to the disease resistance of the piglet. Two observations stimulated this approach: (1) even though it was possible to keep piglets alive during the critical iirst week, the colostrum-free piglets had gained little or no weight, while piglets nursing the sow had doubled their weight in this interval; and (2) coincident with approximately a week of age colostrum-free piglets started growing and little future trouble was encountered in raising them. Nevertheless, they were still antibody-free pigs and

Effect of feeding on the cessation of transport of macromolecules by enterocytes of neonatal piglet intestine.

To elucidate further the role of digesta in the cessation of transport of macromolecules to the blood by enterocytes of neonatal piglet small intestine, a portion of the ileum was surgically isolated and vented to the exterior. The ability of this isolated intestine to transport macromolecules was compared with that of the intestine remaining in the digestive pathway in the same piglet. The isolated intestine ceased transporting macromolecules at the same time as the intestine in the digestive pathway, thus implicating a humoral signal that is responsible for the cessation of transport.

Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.

Oral Transforming Growth Factor-α Enhances Jejunal Mucosal Recovery and Electrical Resistance in Piglet Rotavirus Enteritis

ABSTRACT: A randomized, investigator-masked trial determined the effects of oral recombinant human transforming growth factor-α (TGFα) on jejunal mucosal recovery in 75 piglets with rotavirus diarrhea. Rotavirus inoculation of artificially reared piglets induced subtotal (∼50%) villus atrophy and watery diarrhea. Dietary TGFα was associated with significant restoration of villus surface area by 4 d postinoculation (p.i.) and complete restoration by 8 d p.i., whereas saline-treated animals required 12 d for recovery. Jejuna segments from clinically recovered TGFα-treated piglets showed an increase in electrical resistance across the epithelial barrier in vitro which was proportional to villus height. TGFα treatment for 12 d also produced a 30–50% increase in jejunal mucosal mass (protein content and wet weight), compared with the corresponding values in salinetreated piglets and in uninfected controls. However, oral TGFα did not hasten the resolution of diarrhea, enhance the specific activities of jejunal mucosal digestive enzymes, or increase jejunal glucose-stimulated Na+ absorption in vitro. We conclude that dietary TGFα stimulates jejunal mucosal hypertrophy, improves barrier function, and enhances regrowth of villi in rotavirus enteritis; however, it does not facilitate the restoration of functional activity or mucosal digestive enzymes. Oral TGFα can facilitate intestinal epithelial recovery in diseases associated with mucosal damage.

Relationship of incorporation of radioprecursors into protein and phospholipids of the plasmalemma of Guinea pig (neonate) intestinal epithelium and the cessation of uptake of macromolecules (closure).

The incorporation of radioprecursors into intestinal epithelial cell proteins and phospholipids was studied to assess the role of protein and phospholipid synthesis and/or turnover in the cessation of the absorption of macromolecules (closure). Radiophosphorus incorporation into cellular phospholipids was enhanced when pinocytosis was stimulated. The specific activity of cellular and brush border phospholipids and specific phosphatides increased during the period of active endocytosis. No alteration in specific phosphatide percent composition was observed. Radioamino acid incorporation into cellular protein was not influenced by age. These data are consistent with the idea that the period of active macromolecule absorption by the guinea pig intestinal epithelium is defined by the exhaustion of membrane available for endocytosis.