Search PubMed⌕ Search

Biomedical subjects

H Blobel

Publications and source records attributed to H Blobel.

At least 109 records · Page 6Linked to original sources
PubMed1978

[Effects of serums and plasmas of various species on a protease of Staphylococcus aureus. Human alpha2-macroglobulin as an inhibitor (author's transl)].

Serums and plasmas from various animals and man inhibited 85-100% of the activity of a purified protease from Staphylococcus aureus, strain M 135 (table 1). The inhibitory effects of corresponding serums and plasmas were approximately equal. The inhibition in human serum was caused by alpha2-macroglobulin. On the other hand, alpha1-antitrypsin proved to be ineffective (table 2). The complex alpha2-macroglobulin-protease could be separated by vertical polyacrylamide discelectrophoresis. Immunelectrophoretic analysis revealed no changes by the staphylococcal protease neither on alpha2-macroglobulin, nor on other human serum proteins. Only fibrinogen was split into at least 2 components (fig. 1).

Animals↗
PubMed1977

[Studies on the activation of complement by encapsulated and non-encapsulated staphylococci after their extraction with guanidinium chloride (author's transl)].

All demonstrable enzymes and toxins of encapsulated staphylococci (KS) were removed by extraction with guanidinium chloride. The capsules, however, remained apparently intact on the extracted (KS-Gu) staphylococci (fig. 1), as well as clumping factor and protein A. KS and KS-Gu failed to activate complement in the absence of specific antibodies. They showed neither immunadherence (table 1) nor agglutination by an antiserum against C3 (table 2). KS and KS-Gu had no significant chemotactic effects in vitro upon bovine granulocytes (fig. 2).

Agglutination↗
PubMed1977

[Purification of protease from staphylococcus aureus (author's transl)].

380 (80%) of 475 Staphylococcus aureus cultures isolated from humans, cattle and dogs were proteolytically active either on casein or gelatin or both (table 1). Protease-activity could also be demonstrated in experimental body-cavities of rabbits (fig. 1). The enzyme-activity was estimated with azocasein. Protease from S. aureus, M 135 precipitated from the culture supernatant with ammonium sulfate at 65% saturation (table 2). It was purified by 2 filtrations on Ultrogel AcA 44 (fig. 2,3) and subsequent isoelectric focusing between pH 3.5-7.0 (fig. 4). The purified protease yielded only 1 line in the SDS-polyacrylamidegel-electrophoresis, in the gelatin-polyacrylamidegel-electrophoresis and in the double immuno-diffusion test (fig. 5). Its isoelectric point was at pH 4.6, and its highest proteolytic activity between pH 7.5-8.3. The molecular weight was estimated by SDS-polyacrylamidegel-electrophoresis to be near 29.000. The protease-activity was completely inhibited in the presence of EDTA, partially inhibited by Cu2+ and Zn2+ and increased by Mn2+ (table 3).

Animals↗
PubMed1977

[Effects of capsules of Staphylococcus aureus on the rosette formation with B-lymphocytes and macrophages (author's transl)].

After extraction with guanidium chloride nonencapsulated Staphylococcus aureus (S-Gu) and yeast cells (Hefe-Gu) reduced the hemolytic complement activity of normal human and guinea pig sera much more (fig 1) than encapsulated S. aureus (KS-Gu). Following incubation in the fresh sera S-Gu, Hefe-Gu and guanidinium chloride-extracted S. epidermidis, contrary to KS-Gu, had activated C3 on their surface. Therefore, they formed rosettes with human B-lymphocytes (fig. 2,3,4) and peritoneal macrophages from guinea pigs. The adherence-reactions were less frequent. if the pre-incubation was conducted in the heat-inactivated (30 min, 56 degrees C) sera (table 1,2). After pre-incubation in the absence of serum only the protein A-positive S-Gu formed rosettes with B-lymphocytes.

Animals↗
PubMed1977

[Characterization of staphylococci in microtiter-plates (author's transl)].

The conventional methods for the demonstration of coagulase, clumping-factor, fermentation of mannitol, protein A, alpha-, beta-, and delta-hemolysins and acid phosphatase were adapted to a microtitersystem (MTS). This led to a considerable saving in working-time and reagents. The biochemical reactions could be distinctly evaluated in the MTS (fig. 1). In all qualitative determinations there was complete agreement between the results obtained by MTS and the conventional procedures. In the quantitative endpoints there were only minor deviations (fig. 2,3,4). Thus, the MTS appeared to be a suitable procedure for the characterization of pathogenic staphylococci at a large scale.

Agglutinins↗
PubMed1977

[A contribution to purification of staphylokinase (author's transl)].

Optimal production of staphylokinase (SAK) was achieved in a casein hydrolysate yeast extract medium (LO3) containing 0.1 M Na-pyruvate. Addition of the pyruvate reduced significantly the production of protease and facilitated purification of SAK. After concentration of SAK from the culture supernatant by precipitation with ZnCl2 highly purified SAK was prepared successively using DEAE-Sephadex A 25 in a batch-procedure, by chromatography on CM-Sephadex C25, filtration with Ultrogel ACA 54, isoelectric focusing between pH 3.5 and 10 (fig. 2) and refocusing between pH 5-7 (table 1). For fractionation by focusing a device was constructed to record pH and optical density together with the fraction number in one diagramm (fig. 1). The purified SAK-preparation had a specific activity of 16.700 units per mg protein. It proved to be free of hemolysins, coagulase, leukocidin, lipase, nuclease, phosphatase, and protease. It was homogeneous upon analyses by diselectrophoresis, double immunodiffusion and immunelectrophoresis.

Chromatography, Ion Exchange↗
PubMed1977

[Evaluation of phagocytosis of Staphylococcus aureus with the aid of lysostaphin (author's transl)].

The Staphylococcus aureus strains HV 1 and K 807 were lyzed by lysostaphin. S. epidermis E 1 and staphylococci extracted with guanidinium chloride were resistant to lysostaphin-induced lysis. In the phagocytosis of S. aureus lysostaphin proved to be most useful for the differentiation between engulfed and extracellular staphylococci, particularly those attached to the surface of the polymorphonuclear granulocytes. It enabled a better recognition of the phagocytized staphylococci and therefore a more precise analysis of the phagocytosis experiments. A further improvement in the evaluation of phagocytosis was possible by the use of radioisotope labelling of staphylococci. This technique in combination with lysostaphin, might be useful for large-scale phagocytosis studies.

Animals↗
PubMed1977

[Demonstration and partial purification of staphylokinase (author's transl)].

In these studies the fibrinogen plate medium proved to be not sufficiently specific for the demonstration of staphylokinase (SAK). For this purpose we modified the azocasein-test by the addition of plasminogen. Protease, on the other hand, we measured without plasminogen. This enabled us to differentiate between the 2 enzymes. After precipitation with ZnCl2 from the culture supernatant of Staphylococcus aureus strain, V8, SAK could be prepurified by filtration on ultrogel AcA 44 (tab. 1, fig 1). A more than 100-fold increase in specific SAK-activity (in comparison to that in the culture supernatant) to 34.722 units/mg protein was achieved after 2 X isoelectric focusing between pH 3.5-10.0 and refocusing between pH 5.0-7.0 (fig 2). The partially purified SAK was free of protease, coagulase, beta- and delta-hemolysins. DNase, lipase and phosphatases, but it contained minor amounts of alpha-hemolysin. It revealed only 1 band in the SDS-polyacrylamide-gel-electro-phoresis and 1 precipitin-line in the double immunodiffusion test with an antiserum against the SAK preparation after ultrogelfiltration.

Isoelectric Focusing↗
PubMed1977

["Clumping factor"-reactions using staphylococci after their extraction with guanidiniumchloride (author's transl)].

Staphylococci of strain K 807 (ATCC: 31243) have much "clumping factor" (CF) on their surface. Extraction of the staphylococci with 6 M guanidinium chloride, removed all soluble substances, including coagulase, without reduction in CF-activity. The extracted staphylococci proved to be most suitable for the quantitative determination of fibrinogen and fibrin degradation products in a microtiter procedure (fig. 2). The CF-test with staphylococci of strain K 807 was more sensitive compared with the hitherto used strain Newman D2C (table 1). After staining with "Astrazonrot (BBL)" no loss of CF occurred. With the stained staphylococci the CF-reactions became more distinctly visible and gave sharp endpoints.

Agglutination↗
PubMed1977

[No or little chemotactic activity of staphylococcal substances on macrophages (author's transl)].

Chemotactic effects of staphylococcal substances on macrophages from guinea pigs were studied using modified Boyden-chambers. Filters with an average pore-diameter of 12 micrometer allowed optimal migration of the macrophages. The macrophages were obtained from the peritoneal exsudates 96 h after stimulation with sodium-caseinate (fig. 1) and subsequently concentrated by centrifugation in a ficoll-ronpacon gradient (fig. 2, table 1). Casein had strong chemotactic effects on the macrophages with and without fresh guinea pig serum (fig. 3). Staphylococci, before and after extraction with guanidinium chloride, purified protein A and capsular substances were neither cytotaxic nor cytotaxigenic (in the absence resp. presence of fresh serum). Culture supernatants and staphylococci after incubation with their homologous antiserum proved cytotaxigenic, also lysates of granulocytes from the blood. Lysates of granulocytes from the peritoneal exsudates of guinea pigs were strongly cytotaxigenic for the macrophages.

Animals↗
PubMed1976

[Possible relationship between bacterial surface structures, immune-adherence, chemotaxis and phagocytosis (author's transl)].

In the present study we attempted to determine the relationship between bacterial surface structures, immune adherence, chemotaxis and phagocytosis. As shown in table 1 encapsulated bacteria (Klebsiella ozanae, K. pneumoniae, Pasteurella multocida, Streptococcus pneumoniae), Mycoplasma pneumoniae and M. fermentans had a much lower immune adherence-activity than non-encapsulated bacteria (Bacillus cereus, Brucella abortus, Erysipelothrix insidiosa, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, S. typhi). Consequently, the former bacteria, in the absece of specific antibodies, activated complement via the C-3-bypass to a smaller extent than the latter. In modified Boyden chambers the encapsulated bacteria and the mycoplasmas were much less cytotaxigenic than the non-encapsulated bacteria (table 3). Corresponding to this was the chemotactic response in vivo. Finally the rates of phagocytosis were considerably lower with the encapsulated bacteria than with the non-encapsulated ones (fig. 1).

Animals↗
PubMed1976

[Electron microscopic investigations on encapsulated Staphylococci (author's transl)].

Attempts were made to study the surface of encapsulated staphylococci by electron microscopy. After fixation with osmium tetroxide the encapsulated staphylococci showed a "hazy" cell-surface, possibly because of capsular components (Fig. 1). On the other hand, the nonencapsulated staphylococci exposed a "smooth" surface of the cell wall (Fig 1). Similar findings were obtained after the immuneferritintechnique (Fig 2). The staphylococcal capsules could be clearly demonstrated by the "negative contrasting" method with ferritin (Fig 3). The capsules did not significantly enlarge after reaction of the encapsulated staphylococci with their homologous antiserum.

Ferritins↗
PubMed1975

[Leukotactic studies with artificial body cavities in rabbits (author's transl)].

Artificial body cavities (KK) produced by implanting polyethylene balls in the subcutaneous tissues of rabbits proved to be suitable for the in vivo studies of leukotaxis. Na-caseinate induced a cell content per mm-3 KK-fluid of 7000-8500, "clumping-factor" of Staphylococcus aureus that of 9000 and capsular substances of S. aureus a delayed increase to 5300 cells per mm-3 KK-fluid. Paraffinum liquidum caused a somewhat delayed increase in granulocytes and not in macrophages. In all cases the percentage of granulocytes increased with the cell content and reached values up to 96%.

Animals↗
PubMed1975

[Effects of staphylococcal leucocidin in mice (author's transl)].

After intraperitoneal injections of mice with paraffin-oil and sodium caseinate the numbers of macrophages and granulocytes increased respectively as well in their peritoneal exsudate as in their blood. On the other hand, cyclophosphamide markedly decreased the numbers of leucocytes. All mice were more susceptibel to infections with leucocidin (PVL-R)-positive staphylococci than to those with leucodidin-negative staphylococci of comparable hemolytic properties. Generally, mice with increased numbers of leucocytes were relatively more resistant to the staphylococcal infections. Concentrated PVL-R-leucocidin injected intraperitoneally into mice caused a temporary decrease in the number of leucocytes, especially granulocytes and their phagocytic activity.

Animals↗
PubMed1975

[Chemotactic effects of some staphylococcal substances (author's transl)].

Bovine leukocytes, besides rabbit leukocytes, were found to be suitable for chemotactic studies on staphylococci with the modified Boyden-method. The bovine leukocytes could be obtained more easily in larger quantities. Casein, however, had no significant chemotactic effects on bovine leukocytes. Culture supernatants of Staphylococcus aureus and S. epidermidis and the heat-killed staphylococci had cytotaxigenic and to a small degree also cytotaxic effects on the rabbit and bovine leukocytes. The extracted "straphylococcal skeletons", "clumping factor" (CF), protein A and capsular substances were cytotaxigenic. In addition, CF was moderately cytotaxic only for rabbit leukocytes. The chomotactic migration of leukocytes was markedly reduced after their treatment with leukocidin (PVL-R) in subleukocidic concentrations.

Animals↗