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M K Estes

Publications and source records attributed to M K Estes.

At least 253 records · Page 14Linked to original sources
PubMed1982

Localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry.

Virus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, mol. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.

Animals↗
PubMed1982

Identification, synthesis, and modifications of simian rotavirus SA11 polypeptides in infected cells.

The synthesis and processing of simian rotavirus SA11 polypeptides was investigated after infection of MA104 cells. [35S]methionine- or 3H-amino acid-labeled cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was maximal 3 to 5 h postinfection, and 12 major viral polypeptides were detected. Immunoprecipitation and peptide mapping experiments, demonstrated five viral structural proteins (125,000 daltons [125K], 94K, 88K, 41K, and 38K). Three proteins (53K, 35K, and 34K) were identified as nonstructural by comparison of their partial proteolysis maps with those from polypeptides of similar molecular weight synthesized in vitro from viral RNA transcripts. Assignment as to structural or nonstructural status of two other primary gene products (26K and 20K) remains tentative. Pulse-chase experiments and tunicamycin blockage of glycosylation revealed cotranslational or post-translational modifications (or both) and precursor-product relationships of several of the polypeptides. Tunicamycin inhibition of glycosylation identified a 35.5K polypeptide which was proven to be the precursor to the 38K structural glycoprotein by immunoprecipitation and peptide mapping analyses. Tunicamycin treatment of infected cells also resulted in the disappearance of other glycoprotein species (23K to 29K) and in the concomitant build-up of an unglycosylated 20K polypeptide, suggesting a precursor-product relationship between those polypeptides. Labeling with [3H]glucosamine or [3H]mannose suggested that the rotavirus glycoproteins contained high mannose oligosaccharides. The effects of amino acid analogs on rotavirus polypeptide synthesis and processing were also investigated.

Amino Acids↗
PubMed1982

The effect of bran on bowel function in constipation.

Bran is widely used to treat a variety of gastrointestinal disorders. With few exceptions, investigative work has focused on the effect of bran in normal subjects. Despite its widespread use, there have been no systematic studies of bran in constipation. Wheat bran is the "gold standard" because no other natural fiber has yet been shown to be as effective in increasing fecal bulk. We evaluated the effect of two brans, wheat and corn (20 g/day), on fecal weight, fecal moisture content, bowel movement frequency, intestinal transit time, and symptoms in 10 constipated (less than or equal to 3 movements/wk) but otherwise healthy women. The administration of bran was associated with a significant increase in fecal weight (157%), bowel movement frequency (55%), and decrease in intestinal transmit time (50%). Percentage fecal moisture increased only with wheat bran (67.4-72.1%), whereas corn bran was significantly better than wheat bran in relieving symptoms of constipation.

Adult↗
PubMed1981

Proteolytic enhancement of rotavirus infectivity: molecular mechanisms.

The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specifically, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those of known structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).

Animals↗
PubMed1981

Identification of rotavirus particle types.

Negative-contrast electron microscopy of purified rotavirus particles reveals two particle types: single-shelled and double-shelled particles. The relationship of these particle types, seen by negative staining, to the enveloped and various types of nonenveloped particles seen in thin sections of virus-infected cells was determined. Thin-section and negative-contrast electron microscopic analyses were performed on cell lysates from simian rotavirus. SA11-infected cells and on highly purified double- and single-shelled particles. In thin sections, double-shelled particles appeared as smooth-edged ovals containing dense nucleoids, whereas single-shelled particles had ragged edges and threads of material extending from their centers. The majority of nonenveloped particles seen in thin sections of infected cells were identified as double-shelled particles. Enveloped particles showed typical membrane structure and were observed rarely in crude rotavirus stocks, although they constitute about 10% of the particles within infected cells. It is hypothesized that the enveloped form is a transient one and the envelope is lost in the endoplasmic reticulum of the host cells. Finally, the 50-55 nm type IV particles seen within lysosome-like bodies in infected cells were identified as subviral particles formed from input virions.

Animals↗
PubMed1980

Ultrastructural study of rotavirus replication in cultured cells.

A systematic ultrastructural analysis of the replication cycle of the simian rotavirus SA11 in permissive MA104 cells was performed under reproducible conditions. At 8 h p.i., small areas of viroplasm were seen adjacent to swollen vesicles of the rough endoplasmic reticulum (rer) containing a few 80 to 90 nm virus particles. At later times, the size and number of these inclusions increased and the rer contained large numbers of the 80 to 90 nm particles as well as 52 to 65 nm particles. Infected cells eventually lysed, releasing progeny virus. Other cytological alterations included virus particles sequestered in lysosome-like bodies, 15 to 20 nm tubular structures in the nucleus and/or cytoplasm, convoluted membranes within the rer, filament bundles associated with virus particles, and mitochondria containing 1 to 5 virus particles. In addition, SA11 replication was studied in several less permissive cell lines. The results were similar to those with MA104 cells except that a smaller percentage of the cells were productively infected.

Animals↗
PubMed1980

In vitro transcription and translation of simian rotavirus SA11 gene products.

Rotavirus gene products were examined, with the simian rotavirus SA11 as a model. The endogenous viral RNA-dependent RNA polymerase associated with single-shelled virus particles or with activated double-shelled particles was used to synthesize viral RNA transcripts. Sedimentation velocity sucrose gradient analysis of the RNA transcripts revealed four peaks at 9S, 12S, 14S, and 18S, whereas agarose gel electrophoresis under partially denaturing conditions revealed eight groups of RNA species ranging in molecular weight from 2 x 10(5) to 1.2 x 10(6). The transcripts synthesized in vitro were active in an mRNA-dependent cell-free translation system derived from rabbit reticulocytes. The transcripts directed the synthesis of 11 polypeptides that had molecular weights ranging from 125,000 to 20,000 when analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The products of in vitro translation were compared with polypeptides from purified virus and those synthesized in infected cells. Several of the polypeptides synthesized in vitro were designated as structural polypeptides by comparing the molecular weights determined by polyacrylamide gel electrophoresis analysis or by precipitation with hyperimmune serum prepared against purified virus. Three of the viral structural polypeptides (VP4, -5, and -5a) were not synthesized in vitro as primary gene products, demonstrating that processing must occur for the production of some structural polypeptides. Other in vitro-synthesized polypeptides were tentatively identified as either precursors to the viral glycoproteins or nonstructural polypeptides.

Centrifugation, Density Gradient↗
PubMed1980

Identification of rotaviruses of different origins by the plaque-reduction test.

The plaque assay for the simian rotavirus SA11 was shown to be applicable to the economically important calf and porcine rotaviruses. In addition, neutralization of viral infectivity with specific antiserum before assay (plaque-reduction test) was found to be a useful test to identify the species of origin of rotaviruses.

Animals↗
PubMed1979

Epidemic viral gastroenteritis.

Epidemic viral gastroenteritis is a significant world wide problem. In developed countries, gastroenteritis accounts for significant morbidity and loss of time from work; in the Third World it is the leading cause of mortality among infants and children. Recent technologic advances have been associated with an explosion of research activity. Two virus groups, the Norwalk-like agents and the rotaviruses, are currently accepted as causative agents of viral gastroenteritis in man. The problem of viral gastroenteritis is reviewed both from a current and a historic perspective.

Adult↗
PubMed1979

Rotavirus stability and inactivation.

The stability of the infectivity of Simian rotavirus, SA11, has been analysed and compared to the stability of reovirus type 1. SA11 infectivity was stable to freeze-thawing, sonication, incubation at 25 degrees C overnight or at 37 degrees C for 1 h and to treatment with acid, ether, chloroform and Genetron. In contrast to reovirus, the infectivity of SA11 was more rapidly inactivated by heating at 50 degrees C. SA11 infectivity was inactivated above pH 10.0 and by heating at 50 degrees C in 2 M-MgCl2, but was stabilized by heating in 2 m-MgSO4; reovirus 1 infectivity was enhanced by heating in MgCl2. Both SA11 and reovirus 1 were inactivated by freezing in MgCl2. These results show that rotaviruses and reoviruses can be distinguished by their patterns of inactivation by physical and chemical agents.

Animals↗
PubMed1979

A plaque assay for the simian rotavirus SAII.

A sensitive, quantitative and reproducible plaque assay for the measurement of the simian rotavirus SAII is described. Plaque formation required the presence of the facilitators pancreatin or trypsin and diethylaminoethyl-dextran in the agar overlay. SAII produced plaques in three continuous primate cell lines: MA-104, CV-1 and LLC-MK2. MA-104 cells were the most sensitive.

Animals↗
PubMed1979

Comparison of methods for immunocytochemical detection of rotavirus infections.

Rotavirus infections in intestinal tissues of animals or in tissue culture cells were detected by the immunocytochemical unlabeled soluble enzyme peroxidase antiperoxidase method. Comparison of the immunofluorescence and peroxidase antiperoxidase immunological staining techniques revealed that the two methods are equally sensitive for detection of rotavirus-infected cells. The peroxidase antiperoxidase technique offers the advantages of negligible nonspecific staining reactions, the use of a standard light microscope, the production of permanent slides, and the conservation of immunological reagents. The ability to detect antigens in paraffin-embedded tissues enhances the usefulness of the peroxidase antiperoxidase test for both prospective and retrospective studies.

Animals↗
PubMed1979

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.

Animals↗
PubMed1978

Characterization of DNA-protein complexes from simian adenovirus SA7.

DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.

Adenoviridae↗
PubMed1978

Genetic evidence for a temperature-sensitive lesion in the adenovirus 7 region of the PARA genome.

It has previously been shown that the helper adenovirus (Ad) present in the defective Ad 7-SV40 hybrid population is temperature-sensitive (ts) for replication in human cells. This study has shown that the replication of the defective hybrid virus (PARA) in green monkey kidney cells is also restricted at 40.5 percent. Complementation tests between the parental or transcapsidant PARA populations and SV40 or various Ad serotypes revealed that the ts lesion is located in the Ad region of the hybrid genome. Wild-type (wt) Ad type 7 and Ad type 21 were able to complement the replication of PARA while Ad type 31 was unable to do so. Complementation was more efficient after the PARA genome was transcapsidated to the wt isolates of helper Ad than during multiple infections with the parental hybrid population. The SV40 function which complements the replication of human adenoviruses in simian cells is not expressed by PARA at the nonpermissive temperature. However, the ts lesion does not affect the expression of SV40 functions coded for by PARA under other conditions, i.e., complementation of the replication of human adenoviruses in transformed monkey cells, and synthesis of T antigen in lytically infected or transformed cells. Although the exact nature of the mutated Ad 7 gene product is unknown, heat inactivation data suggest that it may be a structural protein.

Adenoviridae↗
PubMed1977

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.

Adenoviruses, Human↗