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Biomedical subjects

G W Welling

Publications and source records attributed to G W Welling.

At least 73 records · Page 4Linked to original sources
PubMed1988

Reconstitution of the gastrointestinal microflora of lactobacillus-free mice.

A colony of mice that do not harbor lactobacilli in their digestive tracts but whose intestinal microflora is otherwise functionally similar to that of conventional animals was derived. Methods used to reconstitute the intestinal microflora of the mice included inoculation of the animals with cultures of specific microbes, noncultivable microbes attached to epithelial cells, and cecal contents from conventional mice treated with chloramphenicol. Twenty-six microflora-associated characteristics were monitored by using relatively simple tests to determine the microflora status of the mice.

Animals↗
PubMed1987

Influence of quinolones on throat- and faecal flora of healthy volunteers.

Oral and faecal flora were monitored in ten healthy volunteers who received three different oral dosages of either norfloxacin or ciprofloxacin during five consecutive days. Oral flora was not influenced by either quinolone. At the end of the five day-treatment period aerobic Gram-negative bacilli could no longer be cultured from the faecal samples of any of the volunteers. Enterococci were suppressed in some volunteers or remained present in concentrations as high as before treatment. All ciprofloxacin-treated volunteers became colonized with yeasts in their faecal samples, even during treatment with the lowest dose. As measured by total microscopic bacterial clump counts and the appearance of beta-aspartylglycine in faecal samples neither of the two quinolones appeared to decrease the colonization resistance of the digestive tract.

Adult↗
PubMed1987

Column liquid chromatography of integral membrane proteins.

Biological membranes have as a major function the compartmentation of biological processes in cells and organelles. They consist of a bilayer of phospholipid molecules in which proteins are embedded. These integral membrane proteins, which cross the bilayer once or several times, generally have a higher than average hydrophobicity and tend to aggregate. Detergents are needed to remove integral membrane proteins from the lipid bilayer and they have to be present during further chromatographic purification. Predominantly, four modes of HPLC have been used alone or in combination for the purification of integral membrane proteins. These are based on differences of proteins in size (size-exclusion chromatography, SEC), electrostatic interaction (ion-exchange chromatography, IEC), bioaffinity (bioaffinity chromatography, BAC) and hydrophobic interaction (reversed-phase chromatography, RPC, and hydrophobic-interaction chromatography, HIC). SEC, IEC, BAC and HIC are used under relatively mild conditions, and buffer systems generally contain a non-ionic detergent. RPC generally has a denaturing effect on the protein and should preferably be used for the purification of integral membrane proteins smaller than 50 kD.

Animals↗
PubMed1987

Purification strategies for Sendai virus membrane proteins.

Viral membrane proteins extracted from Sendai virions with the non-ionic detergents decylpolyethyleneglycol-300 and Triton X-100 were used as a model mixture of hydrophobic membrane proteins. The detergent extract contained the fusion protein (F) and the tetrameric and dimeric forms of the hemagglutinin-neuraminidase protein (HN). These proteins were purified by size-exclusion high-performance liquid chromatography (HPLC) in the presence of 0.1% sodium dodecyl sulphate, by ion-exchange and metal chelate affinity HPLC in the presence of 0.1% decylpolyethyleneglycol, and by reversed-phase HPLC without prior removal of the detergent. The tetramer of HN and F could be purified by size-exclusion HPLC after dissociation of a micellar aggregate containing tetrameric HN and multimeric F. The F and HN proteins could be purified by ion-exchange HPLC. Pure F protein could be obtained after metal chelate affinity HPLC. The F protein and the dimer and tetramer of HN could be eluted from a large-pore (100 nm) reversed-phase column, but they were eluted as broad, overlapping peaks. Only after reduction of the virion extract, the relatively small (13-15 kilodaltons) F2 protein could be obtained in pure form.

Chelating Agents↗
PubMed1987

Structural properties and reactivity of N-terminal synthetic peptides of herpes simplex virus type 1 glycoprotein D by using antipeptide antibodies and group VII monoclonal antibodies.

To investigate the contribution of individual amino acids to the antigenicity of the N-terminal region of herpes simplex virus type 1 glycoprotein D, a series of 14 overlapping synthetic peptides within residues 1 to 30 were examined for their reactivity with monoclonal antibody LP14 (a group VII monoclonal antibody; in herpes simplex virus mutants resistant to LP14, arginine 16 is substituted by histidine) and two antipeptide antisera (antipeptide 9-21 and antipeptide 1-23). Maximal binding was achieved with peptides 9-21, 10-30, 9-30, and 8-30 and the chymotryptic fragment 9-17 of peptide 9-21, suggesting that a major antigenic site is located within residues 10 through 17. Lysine 10 was shown to be essential for high reactivity, either by binding directly to the antibody molecule or by stabilizing an ordered structure of the peptide. The importance of ordered structure was demonstrated by a decrease in reactivity after sodium dodecyl sulfate treatment of peptides 9-21 and 8-30.

Antibodies↗
PubMed1986

Determination of beta-aspartylpeptidase activity in human faeces by high-performance liquid chromatography using pre-column derivatization with phenyl isothiocyanate.

Bacterial enzymes are responsible for degradation of beta-aspartyl peptides in the intestinal tract. These peptides, especially the dipeptide beta-aspartylglycine, are useful as indicators of an impaired anaerobic intestinal microflora of antibiotic-treated patients. A method to separate the dipeptides beta-aspartylalanine, beta-aspartylglutamine, beta-aspartylglycine and beta-aspartylserine, using reversed-phase high-performance liquid chromatography and precolumn derivatization with phenyl isothiocyanate, was developed. This method was used to determine beta-aspartylpeptidase activity in faecal supernatants of healthy human volunteers and antibiotic-treated patients with beta-aspartylglycine as substrate. This activity was absent in the antibiotic-treated group, while in individuals with an intact intestinal flora it ranged from 16 to 100% degradation per 18 h. In addition, it was found that faecal enzyme preparations cleaved beta-aspartylglycine at a much lower rate than the other beta-aspartyl peptides.

Amino Acids↗
PubMed1986

Comparison of reversed-phase column materials for high-performance liquid chromatography of proteins.

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems.

Animals↗
PubMed1986

Size-exclusion high-performance liquid chromatography of Sendai virus membrane proteins in different detergents. A comparison of different columns.

Four column packings for size-exclusion high-performance liquid chromatography (HPLC) of proteins with particle sizes from 3 to 13 micron were compared, using 0.1% sodium dodecyl sulphate in the solvent. Their suitability for the purification of hydrophobic membrane proteins was studied with Sendai virus proteins as a model. The calibration curves of the two 13-micron column packings were linear up to high molecular weights. In contrast to this, large proteins (greater than 100-150 kD) were eluted later than expected from the 3- and 6-micron packings. Peak capacities for proteins larger than 20 kD ranged from 4.7 to 5.5. Therefore, purification of complex mixtures of membrane proteins will often require rechromatography by a different mode of HPLC. Non-ionic detergents are suitable for further ion-exchange chromatography. The effect of addition of 0.1% of five non-ionic detergents (three gluco-methylalkanamide detergents, octylglucoside, and decyl-polyethyleneglycol-300) to the solvent was investigated and decyl-polyethyleneglycol-300 was found to be most suitable. Size-exclusion HPLC with this detergent resulted in the separation of micelles of three different sizes, of which the larger two contained exclusively the Sendai virus F protein.

Chromatography, Gel↗
PubMed1986

Predicted membrane topology of the coronavirus protein E1.

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.

Amino Acid Sequence↗
PubMed1986

The influence of antibiotics on gut colonization.

Animal and human studies have suggested the concept of "colonization resistance" of the gastro-intestinal tract, which can be decreased by administration of antibiotics that inhibit the anaerobic portion of the normal flora of the gut. This effect can be prevented by the production, by resistant members of the flora, of bacterial enzymes that inactivate or destroy the antibiotic in question. Possible changes in the prevailing gut flora and the implications for the incidence of different infecting agents and for antibiotic therapy are discussed.

Animals↗
PubMed1986

'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora.

Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal beta-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora.

Animals↗
PubMed1985

Prediction of sequential antigenic regions in proteins.

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.

Amino Acid Sequence↗
PubMed1985

Detection of sendai virus protein by reversed-phase high-performance liquid chromatography combined with immuno-chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to the detection of Sendai virus F2 protein. Immunoaffinity chromatography was used prior to RP-HPLC to increase the specificity. With this combined method, the presence of F2 protein in complex solutions could be demonstrated within 150 min. Using RP-HPLC, F2 protein could be detected in amounts down to 20 ng, which could be reduced to 1 ng by application of a microbore column.

Chromatography, Affinity↗
PubMed1985

Combined size-exclusion and reversed-phase high-performance liquid chromatography of a detergent extract of Sendai virus.

Virus envelope proteins obtained by Triton X-100 extraction of Sendai virions were purified to a high degree by a combination of high-performance liquid chromatography (HPLC) methods. Size-exclusion HPLC on a TSK 4000 PW column with several concentrations of acetonitrile or ethanol-1-butanol in 0.1% hydrochloric acid as eluent was used as the first chromatographic step. Peak fractions were diluted in water and further fractionated on reversed-phase columns (TMS-250 or Vydac 218 TP). Size-exclusion HPLC with 45% acetonitrile in 0.1% hydrochloric acid, combined with reversed-phase HPLC on either column, was most suitable for obtaining highly purified F2 protein. Antibodies obtained after injection of this protein were reactive with the intact virus.

Chromatography, High Pressure Liquid↗
PubMed1985

Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.

Acetonitriles↗
PubMed1985

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein.

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.

Amino Acid Sequence↗
PubMed1985

Isolation by high-performance liquid chromatography and partial characterization of a 57,000-dalton herpes simplex virus type 1 polypeptide.

A Nonidet P-40 extract of HSV-1-purified virions was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The first peak fraction eluted at 25% organic solvent. Polyacrylamide gel electrophoresis showed that it contained a 57,000-dalton polypeptide. The polypeptide was characterized by determination of the amino acid composition and the N-terminal amino acid sequence. Adsorption of the detergent extract before RP-HPLC showed that the polypeptide reacted with monoclonal antibodies LP1 directed against herpes simplex virus polypeptide VP-16.

Amino Acid Sequence↗