Gerda Groen

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.

EVOLUTION OF MAMMALIAN PANCREATIC RIBONUCLEASES

Pancreatic ribonucleases form a group of homologous proteins found in considerable quantities in the pancreas of a number of mammalian taxa and a few reptiles (Barnard, 1969; Beintema et al.,1973). The ribonuclease activity varies greatly in different species. Large quantities are found in ruminants and species that have a ruminant-like digestion, and in a number of species with cecal digestion (Fig. 1). Barnard (1969) proposed that an elevated level of pancreatic ribonuclease is the response to the necessity of digesting large amounts of ribonucleic acid derived from the microflora of the stomach of ruminants. This explanation agrees with the observation of Dobson and Wilson (1980) that the level of stomach lysozyme is also elevated in several ruminants and species that have a ruminant-like digestion.

ISOLATION OF DETERGENT-EXTRACTED SENDAI VIRUS PROTEINS BY GEL-FILTRATION, ION-EXCHANGE AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND THE EFFECT ON IMMUNOLOGICAL ACTIVITY

Virus envelope proteins were isolated from Triton X-100 extracts of purified Sendai virions by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The fusion protein F, the matrix protein M and the tetrameric and dimeric form of the HN protein were isolated by gel-filtration HPLC with a solvent containing 0.1% sodium dodecyl sulphate. HN and F were also isolated by ion-exchange HPLC with 0.1% Triton X-100 in the eluent. Reversed-phase HPLC was performed on a C1 column with acetonitrile as the organic solvent. Especially the F1 and F2 component of the fusion protein F were obtained in pure form. The immunological activity of the proteins after HPLC was determined with an enzyme-linked immunosorbent assay (ELISA). After gel-filtration and ion-exchange HPLC, proteins still reacted with antiserum to the intact virus while proteins purified by reversed-phase HPLC did not react.

The primary structure of giraffe pancreatic ribonuclease

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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.

The preparation and primary structure of S-peptides from different pancreatic ribonucleases.

In 1955, Richards [l] described the isolation of ‘an active intermediate produced during the digestion of ribonuclease by subtilisin’. The characterisation and separation of the non-covalently linked components was described 4 years later [2] . Ribonuclease S* possesses full enzymatic activity and the same holds for the enzyme reconstituted from S-peptide and S-protein. The involvement of S-peptide residues in the binding of S-peptide to S-protein and in the enzymatic activity of the reconstituted RNase S’ has been studied by using synthetic S-peptide analogs [3,4] the cleavage by subtilisin takes place in an external loop. Klee [5] and Gold [6] did not succeed in cleaving the RNases from rat and snapping turtle with subtilisin. In this study, we present the successful cleavage with subtilisin Carlsberg of the RNase from goat, brindled gnu, giraffe, reindeer, dromedary, and red kangaroo and the isolation of the corresponding S-peptides. Differences in the observed behaviour are compared with predicted differences in conformation.

Isolation of sendai virus F protein by anion-exchange high-performance liquid chromatography in the presence of Triton X-100

Purified Sendai virions were treated with Triton X-100. The detergent extract containing the fusion protein (F) and the haemagglutinin-neuraminidase protein (HN) was subjected to anion-exchange high-performance liquid chromatography on a Mono Q (Pharmacia) column with 0.1% Triton X-100 in phosphate-buffered saline. HN was not retained by the column while elution with a salt gradient resulted in several peaks containing mainly or only F protein.

Immunologic comparison of pancreatic ribonucleases

Abstract Ouchterlony double immunodiffusion and micro-complement fixation were used in cross-reactivity studies with 9 pancreatic ribonucleases differing 3–28% in amino acid sequence and rabbit antisera to cow, gnu, reindeer and whale ribonuclease. Generally a correlation was observed between the extent of cross-reactivity and amino acid sequence resemblance. The antiserum against whale ribonuclease however, reacted to a larger degree with several antigens than expected from the amino acid sequence difference. Dromedary ribonuclease, which differs at 26% of the positions, showed the highest cross-reaction. By comparing the antigenic reactivities and the differences in sequence, an attempt was made to localize antigenically relevant regions.

The amino acid sequence of GNU pancreatic ribonuclease

During the course of a study on the evolutionary history of pancreatic ribonucleases, several species belonging to the family of the Bovidae have been investigated. The amino acid sequences of four pancreatic ribonucleases from two subfamilies: the Bovinae, (cow [l] and American bison (F. J. Muskiet, G. W. Welling and J. J. Beintema, unpublished) and [2]) and the Caprinae (sheep [3,4] and goat [4]) have been determined. Here we present primary structure of pancreatic ribonuclease from brindled gnu or wildebeest (Connochaetes taurinus), member of another subfamily of the Bovidae, the Hippotraginae.

Antigenic reactivity of artiodactyl pancreatic ribonucleases with antiserum to cow ribonuclease A

Abstract The antigenecity of six pancreatic ribonucleases differing up to 11% in amino acid sequence, and of a synthetic cow ribonuclease fragment (residues 1–14) was determined with antiserum to cow ribonuclease A, using the modified phage technique. From the results, conclusions could be drawn about the locations of antigenically relevant regions in cow ribonuclease A.