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Biomedical subjects

G W Welling

Publications and source records attributed to G W Welling.

At least 109 records · Page 6Linked to original sources
PubMed1982

Association of germfree mice with intestinal microfloras obtained from "normal" mice.

A cultured microflora obtained from the caecum of a "normal" mouse was given to 4 groups of germfree mice and was supplied 1x, 2x, 3x and 4x respectively at 5-day intervals. Another group received a 10(-7) dilution of the caecal flora while a group associated with an 'SPF' flora served as control. The difference (measured by 8 parameters) between mice supplied with the cultured flora or with a 10(-7) dilution, both given once only, was small. Supplying the flora 3x resulted in more 'normal' mice compared with mice which received the flora once or twice. The caeca of specified-pathogen-free mice contained more bacteria per gram (microscopic bacterial count), less aerobic and anaerobic bacteria per gram (viable counts), while the yield as percentage of the microscopic bacterial count was lower as compared with the group to which a cultured flora was supplied 4 times.

Animals↗
PubMed1980

Biochemical effects on germ-free mice of association with several strains of anaerobic bacteria.

The effects of the following changes throughout the association of germ-free mice with increasing numbers of anaerobic bacteria were studied: (i) elution patterns obtained by gel-filtration chromatography of caecal diffusates; (ii) concentration of beta-aspartylglycine in caecal and faecal contents; (iii) polypeptide patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of caecal supernatants; (iv) free amino acid content of caecal supernatants; (v) faecal bile acids, analysed by gas-liquid chromatography; (vi) colonization-resistance. The results indicate that monitoring the normalization (association) process can be accomplished in several ways, but the level of colonization-resistance is most easily measured by high-voltage paper electrophoresis of faecal supernatants to determine the concentration of beta-aspartylglycine. During association, the concentration of beta-aspartylglycine decreased and became undetectable after association with 40 to 50 different strains of bacteria. There was a good negative correlation between the level of colonization-resistance and the concentration of beta-aspartylglycine.

Amino Acids↗
PubMed1978

beta-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic-treated mice.

The caecal supernatants from germ-free, antibiotic-treated and control mice were compared with respect to their content of low-molecular-weight substances (less than 3500 mol. wt.). The supernatants contained about the same amount of free amino acids. After acid hydrolysis, the caecal supernatants of germ-free and antibiotic-treated mice showed a 2.9-fold increase in free amino acids, whereas a similar treatment of the supernatant from control mice resulted in a 2.6-fold increase. By gel filtration on Sephadex G-25, and high-voltage paper electrophoresis at pH 3.5 of the fractions eluted after the void volume, it was found that the caecal supernatants of germ-free and antibiotic-treated mice contained a substance more acidic than aspartic acid. Preparative high-voltage electrophoresis, dansylation, amino acid analysis and a specific colour reaction showed the substance to be beta-aspartylglycine. After a minimal 36 h of treatment with neomycin and bacitracin, a high concentration of beta-aspartylglycine was found, and no enterococci and aerobic Gram-negative rods could be cultured from the caecal contents. The possibility that in one mouse the appearance of beta-aspartylglycine was related to a decrease in Gram-negative rods was ruled out by selective elimination of aerobic Gram-negative rods by using polymyxin B. This suggests that other bacteria concomitantly eliminated with the enterococci and aerobic Gram-negative rods, directly or indirectly, could play a role in the accumulation of beta-aspartylglycine.

Amino Acids↗
PubMed1978

Comparison of various immunological methods for distinguishing among mammalian pancreatic ribonucleases of known amino acid sequence.

Fourteen mammalian pancreatic ribonucleases of known amino acid sequence were compared by 1 or more of 3 different immunological methods: standard quantitative micro-complement fixation, spot-plate micro-complement fixation, and inhibition of phage inactivation. It was found that, while the results obtained by the 3 techniques were correlated with one another, the standard micro-complement fixation procedure was most versatile, economical of materials, and easiest to execute. The standard MC'F technique was more sensitive than the spot-plate technique to differences in amino acid sequence. The inhibition of phage inactivation method was more sensitive than the standard method for measuring differences among closely related RNases but proved impractical for amino acid differences over 15%; the MC'F method could be extended to at least 30% sequence differences. The standard method, moreover, readily detected the single amino acid difference between dromedary and camel RNases. A linear relationship was found between immunological distance (y) in the MC'F test and percent sequence difference (x) which fit the equation y = 7x. The strength of the correlation between immunological distance and percent sequence difference is consistent with the proposal that a large fraction of the evolutionary substitutions of amino acids in ribonuclease are immunologically detectable. This could be explained either by a multideterminant hypothesis or by a pauci-determinant hypothesis which says that substitutions occurring outside determinants produce small conformational changes influencing determinant reactivity.

Amino Acid Sequence↗
PubMed1978

Purification and some properties of two extracellular proteolytic enzymes produced by Vibrio SA1.

The purification and characterisation of an extracellular endo and amino-peptidase of the marine Vibrio SA1 is described. The endopeptidase was purified by ammonium sulphate precipitation, gel filtration and affinity chromatography. It had a molecular weight of approximately 31,000, a pH optimum at 7.8 and a temperature optimum at 50 C. The enzyme was rapidly inactivated at 65 C. The aminopeptidase was purified by ammonium sulphate precipitation, gel filtration and preparative polyacrylamide gel electrophoresis. This enzyme had a molecular weight of approximately 21,000, a pH optimum at 8.6 and a temperature optimum at 60 C. Both proteases were inactivated by EDTA while reactivation occurred by Ca2+, Zn2+ and Mg2+ ions. The endopeptidase hydrolysed several peptide bonds in the oxidized B-chain of insulin, particularly those involving amino groups of hydrophobic amino acid residues with bulky side chains. It was unable to hydrolyse synthetic dipeptides, but a number of tripeptides were hydrolysed at a low rate. The aminopeptidase hydrolysed leucinamide and di- and tripeptides containing hydrophobic bulky amino acids as the N-terminal residue. It was concluded that the endopeptidase and the aminopeptidase of Vibrio SA1 possess complementary specificities.

Aminopeptidases↗
PubMed1978

The amino-acid sequence of kangaroo pancreatic ribonuclease.

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.

Amino Acid Sequence↗
PubMed1977

Reinvestigation of the primary structures of red deer and roe deer pancreatic ribonuclease and proline sites in mammalian ribonucleases.

The sequences of amino acid residues 15-23 of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) pancreatic ribonuclease and the identity of residue 99 in roe deer ribonuclease are corrected. Earlier results are explained by the cleavage of an Asp-Pro bond in both enzymes during the treatment with CNBr in 70% formic acid and by wrong interpretations of amino acid analyses. Proline residues, which occur at a number of positions in several mammalian ribonucleases, can be accommodated in a model of bovine ribonuclease S without disrupting the conformation of the main chain.

Amino Acid Sequence↗
PubMed1976

Proteolytic enzymes as structural probes for ribonuclease BS-1.

Trypsin, pepsin and subtilisin have been used as conformational probes for the structure of bovine seminal ribonuclease BS-1 by studying, under definite conditions, their effects on the seminal enzyme, a dimeric protein made up to two identical subunits; on bovine pancreatic monomeric ribonuclease A (EC 3.1.4.22) with a polypeptide chain homologous to that of the seminal ribonuclease subunit chain; and on a monomeric, active and stable derivative of seminal ribonuclease. The results show: (1) that the C-terminal regions of the pancreatic and the seminal proteins are very similar as they appear to fit in an identical way to the active site of pepsin; (2) that the resistance of the N-terminal region of ribonuclease BS-1 to subtilisin is not due to the dimeric structure of the protein, but to the conformation of this region, where an essential feature is the presence of a proline residue at position 19; (3) that the monomer of ribonuclease BS-1 is resistant to tryptic action only when bound to the partner monomer in the quaternary structure of the protein. This indicates that dissociation of the seminal ribonuclease makes some potentially susceptible susceptible bond or bonds available to trypsin either through a conformational change of the protein subunit, or by simply exposing the protein area hidden at the intersubunit interfaces.

Animals↗
PubMed1976

The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease.

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.

Amino Acid Sequence↗
PubMed1976

Carbohydrate in pancreatic ribonucleases.

A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62 and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated. Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).

Animals↗
PubMed1976

Allelic polymorphism in arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease.

Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.

Alleles↗