Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat.

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.

Nonendoscopic removal of percutaneous endoscopic gastrostomy tubes: morbidity and mortality in children.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) tubes are often removed by cutting the tubing at skin level and allowing the internal components to pass through the gastrointestinal tract. This technique is commonly used in adults, but little information is available concerning its safety in younger patients. METHODS: To assess the safety of this approach in children, the clinical courses of all patients who had undergone PEG tube removal in our pediatric gastroenterology unit over a 3-year period were reviewed. RESULTS: Five of 11 patients in whom the internal components were allowed to pass developed significant complications. Three required subsequent endoscopic removal of the internal component due to persistent vomiting, one died from complications of esophageal perforation caused by the retained internal component, and one developed a gastrocutaneous fistula containing the retained bumper 2 years after PEG tube removal. Significant complications occurred more often in the younger and smaller patients. CONCLUSIONS: Small children are at greater risk than adults for developing serious complications associated with unremoved PEG tube internal components. If passage of the internal components cannot be confirmed after 2 weeks, chest and abdominal radiographs should be obtained.

Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease.

Reovirus type 3 has been implicated in the origin and pathogenesis of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but routine detection of this virus in hepatobiliary tissues from affected infants by culture and histological techniques has been unsuccessful. In this study, oligonucleotide primers specific to the M3 genome segment of reovirus 3 (Dearing) were used in a reverse transcriptase-mediated polymerase chain reaction technique to develop a sensitive and specific assay for the detection of reovirus 3 RNA in formalin-fixed, paraffin-embedded patient samples. Optimal reaction conditions were determined by testing infected murine tissues and preserved human liver tissue supplemented with reovirus 3. Archival specimens from 50 infants, including 14 with extrahepatic biliary atresia, 20 with idiopathic neonatal hepatitis, and 16 age-matched controls, were evaluated. Successful amplification of human albumin complementary DNA from the preserved tissues confirmed the presence of intact RNA in every patient specimen tested. Analysis of the amplification reactions by agarose gel electrophoresis and Southern blot hybridization detected the presence of reoviral RNA only once in a single patient sample. These results do not support a strong role for reovirus 3 in the development of neonatal cholestatic liver disease. The recent association of other RNA viruses of the Reoviridae family with murine liver disease and human extrahepatic biliary atresia indicates that continued investigation into a viral cause for idiopathic neonatal hepatobiliary disease is warranted.

Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase.

A 5417-base pair (bp) region of Pseudomonas aeruginosa PAO chromosomal DNA containing the mmsAB operon and an upstream regulatory gene (mmsR) has been cloned and characterized. The operon contains two structural genes involved in valine metabolism: mmsA, which encodes methylmalonate-semialdehyde dehydrogenase; and mmsB, which encodes 3-hydroxyisobutyrate dehydrogenase. mmsA and mmsB share the same orientation and are separated by a 16-bp noncoding region. The transcriptional start site for the operon has been pinpointed to a cytidine residue located 77 bp from the translational start site of the operon. mmsR is located on the opposite strand and begins 134 bp from the translational start site of mmsA. MmsR has been identified as a member of the XylS/AraC family of transcriptional regulators and appears to act as a positive regulator of the mmsAB operon. Sequence comparison of MmsA to other proteins in the data bases revealed that MmsA belongs to the aldehyde dehydrogenase (NAD+) superfamily. MmsB shares a 44% amino acid identity with 3-hydroxyisobutyrate dehydrogenase from rat liver. Mutants with insertionally inactivated mmsR, mmsA, and mmsB grow slowly on valine/isoleucine medium and exhibit reduced enzyme activity in cell-free extracts compared to P. aeruginosa PAO.

Relapsing pneumococcal bacteremia in immunocompromised patients.

Relapse of Streptococcus pneumoniae bacteremia after appropriate therapy is thought to be rare, even in immunocompromised patients. We describe three immunodeficient patients who experienced repeated episodes of pneumococcal bacteremia within 8 weeks after receiving appropriate therapy. Serotyping and DNA fingerprinting of respective isolates strongly suggested that each patient's bacteremic relapse was caused by the same pneumococcal strain. Relapsing and recurrent infections with an identical pneumococcal strain, especially in immunodeficient individuals, may be more common than is generally appreciated.

A Cryptosporidium parvum genomic region encoding hemolytic activity.

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.