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Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 235 records · Page 13Linked to original sources
PubMed1985

Detection of rotaviruses by nucleic acid hybridization with cloned DNA of simian rotavirus SA11 genes.

We developed a dot-blot hybridization assay to detect rotaviral RNA sequences in tissue culture or in clinical samples. 32P-labeled cloned cDNA probes of the simian rotavirus SA11 specifically detected rotaviral RNA sequences and were more sensitive for detecting SA11 than was the commercial enzyme-linked immunosorbent assay Rotazyme test. A full-length probe of SA11 gene 6 detected 2.5 X 10(5) SA11 particles or approximately 0.27 ng of purified SA11 dsRNA. Combined probes from genes 6 and 9 detected 0.135 ng of purified SA11 dsRNA. The assay detected group A rotaviruses from different subgroups and serotypes, but the sensitivity of RNA detection varied from 0.5 to 31 ng when RNA from heterologous strains of virus was analyzed. An analysis of coded stool samples correctly identified 31 (91%) of 34 samples positive for rotavirus by electron microscopy and 100% of 26 samples negative for rotavirus by electron microscopy. Preliminary experiments also showed the assay has potential to directly characterize (subgroup and serotype) rotaviral isolates.

Cloning, Molecular↗
PubMed1985

Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3.

We prepared monoclonal antibodies against two serotypically distinct rotavirus strains: Wa, a serotype 1 virus of human origin, and rhesus rotavirus, a simian serotype 3 virus. Monoclonal antibodies which react specifically with VP7 of each serotype were identified by hemagglutination inhibition tests, plaque reduction neutralization studies, and solid-phase immunoassays which used wild-type and reassortant strains of rotavirus. An enzyme-linked immunoassay was designed which utilizes two of these antibodies to correctly identify serotype 1 and serotype 3 viruses.

Animals↗
PubMed1984

Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein.

The genes of the RF strain of bovine rotavirus have been cloned into pBR 322 following the synthesis and hybridization of cDNA transcribed from both strands of in vitro polyadenylated genomic RNA. Cloned rotavirus DNAs were assigned to most of the 11 genomic RNA segments by Northern blot hybridization. The complete sequence of gene 6 that codes for the major inner capsid protein has been determined. The gene is 1356 nucleotides long and possesses an unique long open reading frame that could encode a protein (397 amino acids) of similar size to the known gene 6 product. Comparison of the RF bovine rotavirus gene 6 sequence with the sequence of the simian rotavirus gene 6, showed these genes to be very similar in nucleotide sequence (87% homology). Most of the base changes are silent and the predicted amino acid sequences are almost identical (97% homology).

Amino Acid Sequence↗
PubMed1984

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.

Base Sequence↗
PubMed1984

Pathogenesis of rotavirus-induced diarrhea. Preliminary studies in miniature swine piglet.

The pathogenesis of diarrhea caused by rotavirus infection was studied in miniature swine piglets. The animals were inoculated orally with 2 X 10(7) plaque-forming units of porcine rotavirus (OSU strain). During the height of diarrhea, intestinal function was investigated by in vivo perfusion of a 30-cm segment of proximal jejunum and a 30-cm segment of distal ileum. Absorption of Na+ and water decreased and 3-O-methylglucose transport was markedly reduced, P less than 0.01 compared to control animals. Mucosal lactase and sucrase levels were depressed in both the jejunum and ileum, P less than 0.001. Na+,K+-ATPase activity was significantly depressed only in the ileum, P less than 0.001. These changes were associated with a marked reduction in villous height, suggesting that the diarrhea could be an osmotic diarrhea due to nutrient (carbohydrate) malabsorption. Fresh stool samples were obtained and analyzed immediately for NA+,K+, osmolarity, glucose, and lactose; the osmotic gap was also determined. Stool osmolarity continually increased from 248 +/- 20 mosm/liter prior to inoculation to 348 +/- 20 mosm/liter at 75 +/- 1 hr postinoculation (P less than 0.005); the majority of the fecal osmotic gap could be accounted for by the amount of lactose present in the stools. Stool sodium increased from 34 +/- 6 mM prior to inoculation to a maximum of 65 +/- 4 mM at 53 +/- 1 hr postinoculation, P less than 0.001. There was no significant change in potassium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Ultrastructural localization of rotavirus antigens using colloidal gold.

Colloidal gold was used to localize six of the ten known proteins of the simian rotavirus SA11 within infected cells by ultrastructural immunocytochemistry. Monospecific or monoclonal antibodies to selected structural and nonstructural proteins were the primary antisera. The major outer capsid glycoprotein, VP7, was associated with nonenveloped particles, with particles de-enveloped by Triton X-100 and with both nuclear and cytoplasmic inclusions. The protease-sensitive outer capsid protein, VP3, was also found on nonenveloped and de-enveloped particles. The major inner capsid protein, VP6, was accessible to antibodies on some of the nonenveloped particles (presumably single-shelled particles) and on the de-enveloped particles. A monospecific antibody to the gene 11 product, believed to be a precursor to a minor structural protein, VP9, reacted strongly with viroplasmic inclusions. Virus particles were weakly labeled by this antibody. NS35, a nonstructural SA11 protein, was found only in the viroplasms. NS29, a nonstructural glycoprotein, was localized to the cytoplasmic side of the endoplasmic reticulum membrane and to the inside of enveloped virus particles. These data support the hypothesis that NS29 facilitates budding of the virus particles and acquisition of the outer capsid layer.

Animals↗
PubMed1984

Search for evidence of a viral aetiology for inflammatory bowel disease.

The aetiology of the inflammatory bowel diseases. Crohn's disease, and ulcerative colitis, is still obscure. A viral aetiology has been proposed, based in part on reports that filtrates prepared from tissues of patients with inflammatory bowel disease induce cytopathic effects in tissue culture cells. Our attempts to culture viruses in many cell lines from filtrates prepared from the tissue of 95 patients have been negative, except for one case in which cytomegalovirus was isolated from the tissue of a Crohn's disease patient. Our studies confirm previous reports that intestinal tissue filtrates induce cytopathic effects in inoculated cell cultures, but the effect we observed is non-specific; cytopathic effects were induced in most cell lines tested and with similar frequency irrespective of whether the intestinal filtrates were prepared from Crohn's disease patients, ulcerative colitis patients, or non-inflammatory bowel disease controls. Electron microscopy studies of tissue culture cells exhibiting cytopathic effects have not revealed virus particles. Characterisation of the cytopathic effect inducing factor showed that it was incapable of serial passage in tissue culture, too small to be a conventional virus, resistant to inactivation by ultraviolet light, and heat stable. Our results suggest that the observed cytopathic effect was caused by a non-replicating cytotoxic factor, or factors, released from intestinal tissues of both inflammatory bowel disease and non-inflammatory bowel disease patients.

Animals↗
PubMed1984

RNA electropherotypes of human rotaviruses from North and South America.

Between April 1979 and December 1982, viral agents were found in 231 of 695 children admitted to the Texas Children's Hospital with gastroenteritis. Electron microscopic analysis showed that rotaviruses were the most common viral agents, and a seasonal pattern of rotavirus disease was observed. The migration patterns of the RNA segments of these rotaviruses on electrophoresis in polyacrylamide gels were compared with those of rotaviruses collected from other areas of the United States of America and from Argentina, Colombia and Mexico. A number of different RNA electropherotypes were found, including some patterns not previously reported.

Child↗
PubMed1983

Rotaviruses code for two types of glycoprotein precursors.

Rotaviruses are nonenveloped viruses that code for two glycoproteins: a structural glycoprotein (VP7) and a nonstructural glycoprotein (NS29). The precursor to VP7 (37K) was shown to contain a 1.5K cleavable signal sequence. The 37K precursor was authentically processed (signal sequence cleaved and the polypeptide "core" glycosylated) when synthesized in a cell-free system supplemented with dog pancreatic microsomes. Similar experiments were performed with the nonstructural glycoprotein precursor (20K); however, the 20K precursor contained an integral (noncleavable) signal sequence. Both precursors were inserted into membranes cotranslationally and both glycosylated products underwent posttranslational oligosaccharide processing. The results suggest a morphogenetic scheme for the simian rotavirus SA11.

Glycoproteins↗
PubMed1983

Two types of glycoprotein precursors are produced by the simian rotavirus SA11.

The rotavirus genome codes for two glycoproteins: an outer capsid structural glycoprotein (VP7, apparent molecular weight 38,000 (38K)) and a nonstructural glycoprotein (NS28K). The synthesis of these glycoproteins was analyzed in infected cells and in a cell-free system derived from rabbit reticulocyte lysates supplemented with dog pancreatic microsomes. The data showed a 37K product synthesized in the cell-free system is the precursor to the 38K glycoprotein and that the 37K polypeptide contains a cleavable signal sequence (apparent molecular weight 1.5K). The 37K polypeptide was glycosylated in vitro in the presence of microsomal membranes to a polypeptide of 38K that was immunoprecipitated by monospecific antiserum to VP7. Endo H digestion of the 38K polypeptides from either infected cells or the cell-free system produced polypeptides of identical molecular weight, 35.5K (the glycoprotein precursor lacking the signal sequence). These results were confirmed by comparative studies with a variant of SA11 that is defective in glycosylation of VP7. Similar experiments with the 20K precursor to the 29K nonstructural glycoprotein showed the 20K polypeptide contains a noncleavable signal sequence. Both glycoproteins were inserted into microsomal membranes and were processed via oligosaccharide trimming.

Amino Acid Sequence↗
PubMed1983

Detection of antigenically distinct rotaviruses from infants.

Antigenically distinct rotaviruses, i.e., viruses morphologically identical to conventional rotaviruses by electron microscopy, yet lacking the common group antigen(s) detected by an enzyme-linked immunosorbent assay, were found in 2 of 51 fecal samples from Bulgarian infants with rotavirus gastroenteritis. These antigenically distinct viruses contained 11 segments of double-stranded RNA, but they demonstrated a unique RNA migration profile after electrophoresis of the genome RNA in polyacrylamide gels. This report confirms the presence of a new group of rotaviruses in humans. The significance of these viruses is currently unknown, and specific diagnostic tests must be developed for epidemiological studies to determine their role as human and veterinary pathogens and to evaluate their impact on proposed vaccine development programs.

Antigens, Viral↗
PubMed1983

Effects of tunicamycin on rotavirus morphogenesis and infectivity.

The functions of the two rotavirus glycoproteins were investigated by using tunicamycin and a variant of SA11 rotavirus having nonglycosylated VP7. Results showed that glycosylation of VP7 is not required for normal viral morphogenesis and infectivity and suggested that the nonstructural glycoprotein is involved in assembly of the outer capsid.

Glucosamine↗
PubMed1983

Biochemical mapping of the simian rotavirus SA11 genome.

Biochemical mapping experiments of the simian rotavirus SA11 genome were performed to determine which double-stranded RNA segment coded for each of the viral polypeptides. Viral RNA transcripts were synthesized in vitro by using the endogenous viral RNA polymerase and fractionated by electrophoresis in acid-urea agarose gels. The fractionated transcripts were translated in two cell-free systems: micrococcal nuclease-treated reticulocyte lysates and wheat germ extracts. The polypeptide products were identified by polyacrylamide gel electrophoresis and partial peptide analysis and compared with polypeptides synthesized in infected cells or found in purified virus. The RNA segment that coded for each transcript was determined by hybridization of the fractionated transcripts to the double-stranded RNA genome and analysis of the hybrids by electrophoresis in polyacrylamide gels. Primary gene products were assigned for 10 of the rotavirus transcripts and 10 of the double-stranded RNA segments. The coding assignments are as follows: the inner-capsid polypeptides, VP1, VP2, and VP6, were assigned to segments 1, 2, and 6, respectively; the major outer-capsid polypeptides, VP3 and VP7, were assigned to segments 4 and 9, respectively; segments 5, 7, and 8 coded for nonstructural polypeptides with molecular weights of 53,000, 34,000, and 35,000, respectively; segment 10 coded for the 20,000-molecular-weight precursor to the 29,000-molecular-weight glycosylated nonstructural polypeptide; and segment 11 coded for a 26,000-molecular-weight polypeptide that may be the precursor to the minor outer-capsid polypeptide VP9. Several methods were used to determine the product of gene segment 3, and the problems associated with the identification of this gene product are discussed.

Animals↗
PubMed1983

Double-blind comparison of bismuth subsalicylate and placebo in the prevention and treatment of enterotoxigenic Escherichia coli-induced diarrhea in volunteers.

Enterotoxigenic Escherichia coli cause most traveler's diarrhea in Third World countries. We tested bismuth subsalicylate as prophylactic therapy and as treatment for enterotoxigenic E. coli-induced diarrhea. Thirty-two healthy hospitalized volunteers were challenged orally with enterotoxigenic E. coli, strain H10407 (serotype 078:K80:H11). Administration of 600-mg doses of bismuth subsalicylate or placebo was begun 8 h before bacterial challenge. Doses were taken at 8 h and 2 h before, and at 2 h and 4 h after, the E. coli challenge and were continued four times a day for 3 additional days. The maximum prophylactic bismuth subsalicylate dose was 9.6 g. Those experiencing diarrhea were rerandomized to receive bismuth subsalicylate or placebo, given as 300 mg every 30 min for a total of 2.4 g of bismuth subsalicylate, in eight doses. Diarrhea occurred in 9 of the 16 (56%) subjects receiving placebo and in 2 of the 15 (13%) subjects receiving bismuth subsalicylate, p less than 0.03. This study confirms the effectiveness of bismuth subsalicylate in preventing traveler's (enterotoxigenic E. coli) diarrhea, and shows that bismuth subsalicylate in other than liquid form is effective. Enterotoxigenic E. coli were recovered less frequently from those receiving bismuth subsalicylate than from those receiving placebo, suggesting that bismuth subsalicylate prevents diarrhea by reducing the number or multiplication of enterotoxigenic E. coli. In vitro studies revealed that bismuth subsalicylate and its components each were bactericidal at concentrations possibly attained during the clinical trial.

Antidiarrheals↗
PubMed1983

DNA hybridization studies of the association of Pseudomonas maltophilia with inflammatory bowel diseases.

An infectious etiology has been suggested for the inflammatory bowel diseases, Crohn's disease and ulcerative colitis, and an association of cell wall-defective variants of Pseudomonas maltophilia and Pseudomonas-like group Va bacteria with Crohn's disease has been reported by Parent and Mitchell. Seven of the Parent-Mitchell isolates were compared by using DNA hybridization and six were identical and similar, but not identical, to a type strain of P. maltophilia. The seventh isolate showed extensive homology with VARC, a reference strain of group Va organism, but not with P. maltophilia. Pseudomonas DNAs were radiolabeled by nick translation and used as probes for homologous DNA in hybridization experiments involving 48 different tissues. The presence of DNA with sequences homologous to those of P. maltophilia was detected in three of 23 Crohn's disease samples, two of 10 ulcerative colitis samples, and none of 15 control samples. There was no hybridization with VARC or Pseudomonas aeruginosa probes. We were unable to culture cell wall-defective organisms from patients' tissues but have detected pleomorphic organisms in hypertonic cultures of 14 of 53 Crohn's disease specimens, none of six ulcerative colitis specimens, and none of 11 control specimens. None reverted to normal bacteria. These results do not support an exclusive association of P. maltophilia with Crohn's disease but rather suggest a possible association of P. maltophilia with IBD. Technical limitations currently preclude definitive conclusions regarding the significance of this association. Although we demonstrated the presence of DNA sequences with homology to P. maltophilia DNA in tissues of some patients with IBD, the role, if any, of these bacteria in the pathogenesis of IBD has yet to be established.

Base Sequence↗