Search

We isolated and characterized Xenopus laevis c-myc cDNAs from an oocyte-specific library. These cDNA clones encompass 2.35 kilobases of the X. laevis c-myc RNA and contain the entire coding domain of 1,257 nucleotides of the 419-amino acid-long X. laevis c-myc protein. The 2.7-kilobase X. laevis c-myc mRNA is expressed in the oocyte, maintained in the egg, and is present throughout the early cleavage stages of embryogenesis. At the time of transcriptional activation in the embryo the c-myc RNA levels show a significant decline and then reaccumulate continuously throughout the remainder of premorphogenic development. At the early neurula stage of embryogenesis the pattern of c-myc RNA expression is elevated in the mesoderm with respect to the endoderm and ectoderm. In the adult X. laevis the c-myc mRNA is expressed in some (e.g., skin, muscle) but not all differentiated tissues. The X. laevis c-myc protein migrates as a doublet of 61,000- and 64,000-dalton species. Both species are phosphorylated in oocytes and somatic cells, exhibit extremely short half-lives of less than 30 min, and are localized to the nuclear fraction of somatic cells. By contrast, the oocyte protein shows both cytoplasmic and germinal vesicle distribution and appears to be stable.

The feasibility of early weaning systems depends in part on the control of rotaviral infection in newly weaned pigs. Diets containing antibodies to rotavirus were tested to determine whether deaths (rotaviral infections) could be controlled in pigs that were farrowed “conventionally” at a commercial farm and reared artificially at our research facility. Three sources of antibody were tested: (1) fresh milk from a cow immunized against swine rotavirus, (2) fresh pooled cows' milk and (3) pooled cows' colostrum. Pigs were from two farms where the level of rotavirus in the farrowing environment was thought to be typical of that in North Carolina and beyond. In trial 1, on farm I, pigs that had nursed for about 1 day were brought in groups to the isolated, fumigated research facility. This trial was designated “all in, all out.” Under these conditions, the presence of antibody in the diet, prepared either from milk from the immunized cow or from pooled cows' milk, was effective in reducing mortality: (mortality rate among pigs fed these diets was 4.5% by 2 weeks of age vs27% for the controls; P<.05). In trial 2, on farm I, the level of rotavirus was built up by the continuous introduction of newly weaned 1-day-old pigs into our rearing environment. This trial was called “continuous.” Under these conditions, the diets used in trial 1 were ineffective in controlling mortality (65% or more deaths; P<.05). In trial 3, on farm II, pigs whose survival was thought to be unlikely were weaned at 1 to 5 days of age and brought in groups to the fumigated rearing facility. This trial was called “all in, all out; disadvantaged.” Under these conditions, the diets containing the low level of antibody, either from the immunized cow's milk or from pooled cows' milk, were partially effective (mortality rates of 23%, P<.05, and 34%, P<.1, respectively, vs53% for the controls). The most effective diet contained cows' colostrum diluted at 1:10 (3% deaths, P<.05), but a diet containing the cows' colostrum diluted at 1:20 was ineffective (55% deaths).

The importance of identifying and quantifying the level of contaminants on the surface of biomaterials has long been recognized as a significant step towards predicting the biocompatibility and rate of healing of implantable devices. Yet suitable techniques for characterizing the surface chemistry of soft and flexible biomaterials with an uneven surface contour such as those used in vascular prostheses have only recently been made available. As a first step towards determining the effect of surface contaminants on the healing behaviour of vascular prostheses, this study used X-ray photoelectron spectroscopy (XPS) to characterize the surface chemistry of 14 virgin polyester arterial prostheses from a wide variety of sources. Generally speaking, all surfaces appeared relatively clean. However, the results indicate that certain prostheses were not manufactured with Dacrorf-like fibres, and that some exhibit significantly carbon-rich surfaces. This suggests that the surface of these devices has been modified either by a hydrocarbon lubricant or by vapour phase hydrocarbons during fabrication.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.

Large numbers of a reovirus-like agent were visualized with electron microscopy in bacteria-free gut homogenates obtained from piglets with a fatal diarrhea resembling transmissible gastroenteritis. The syndrome, of vomiting, diarrhea, dehydration, and death, was reproduced in piglets artificially infected with these bacteria-free gut homogenates. Reovirus-like particles persisted in serial piglet passage and none was seen in uninfected, asymptomatic controls. Hyperimmune sera (made in recovered piglets) aggregated the reovirus-like particles, as judged by immunoelectron microscopy, and neutralized the infectious agent. The cytoplasm in enterocytes on infected intestinal epithelium fluoresced when this hyperimmune sera was used in an indirect fluorescent antibody test. Feeding cow colostrum or diets containing porcine gamma globulin protected infected piglets. No cytopathogenic effect was noted in infected tissue cultures, nor did this agent affect neonatal guinea pigs, hamsters, mice, and rats. The agent did not agglutinate human O or A erythrocytes.

Since the turn of the century, Escherichia coli has been implicated in the etiology of weanling diarrhea (colibacillosis). However, rotavirus--a virus that destroys enterocytes--has been shown recently to be causally associated with weanling diarrhea of pigs. The role of both rotavirus and hemolytic enteropathogenic E. coli in weanling diarrhea was assessed in this study. Pigs from a closed herd were farrowed and weaned by two markedly different systems: an "intensive care sanitary" system and a "conventional unsanitary" system. Pigs weaned at 3 weeks of age in the sanitary system usually experienced a rotaviral diarrhea about 16 days postweaning. No hemolytic E. coli were detected in feces from these pigs. Peers weaned at the same time by the unsanitary system commenced diarrhea 3 days postweaning. Rotavirus and nonhemolytic E. coli were detected in the feces at the onset of diarrhea and for a few days thereafter. Then, the aerobic fecal flora shifted to nearly pure hemolytic enteropathogenic E. coli. About 10 days later, the diarrhea waned, and the fecal flora shifted back to nonhemolytic E. coli. This hemolytic E. coli shedding pattern could not be duplicated in artificially inoculated sanitary pigs unless they were inoculated with the hemolytic E. coli during a rotaviral-associated diarrhea. Otherwise, the shedding of hemolytic E. coli was fleeting, and the diarrhea, if present, was mild. Pigs developed humoral antibodies to the rotavirus but not to the hemolytic E. coli. We conclude that rotavirus damages the epithelium of the small intestines, which changes the luminal environment to one that favors colonization by enteropathogenic E. coli.

Piglets weaned abruptly and precociously at 3 weeks of age and placed in a crowded nursery commenced diarrhea 3 to 5 days later. Death losses were low (approximately 6%), but weight gain ceased for 2 weeks. Large numbers of rotavirus (reo-like) particles were seen by electron microscopy in diarrhetic fluids. Sections of intestines showed a loss of adsorptive surface in that villi were shortened and fused with adjacent villi. Immunofluorescence revealed rotaviral antigens within damaged enterocytes. Rotavirus-containing gut fluid was harvested from sick, weaned piglets. This fluid, filtered free of bacteria, was used to inoculate per os colostrum-deprived piglets. These infected piglets developed diarrhea and dehydration, and large numbers of rotaviral particles were seen in their diarrhetic fluid. Also, rotaviral antigens were present in aberrant enterocytes, and the intestinal villi were shortened. Since the weaned piglets (3 weeks old) came from sows that were providing their piglet's intestine with passive antibody protection via milk, we concluded that the abrupt removal of the piglet from the gut-bathing antibody combined with the stress of weaning produced a neonate vulnerable to the ubiquitous rotavirus. Similar circumstances may prevail and operate in exacerbating rotaviral diarrhea in neonates of other species of mammals.