Werner Müller

Elektronenmikroskopische Untersuchungen zum Formwechsel der Kinetochoren während der Spermatocytenteilungen von Pales ferruginea (Nematocera)

Longitudinal thin sections of preselected spermatocytes were studied with the electron microscope. The kinetochores of autosomes and sex chromosomes show a characteristic change of their form during the meiotic divisions. Just after nuclear membrane breakdown the kinetochore profiles have the form of circles, in early prometaphase they have flame shape, and in metaphase appear as straight zones. As early as prometaphase I two sister kinetochores are discernible in each kinetochore region of a dyad. In prometaphase the sister kinetochores of the sex chromosomes are connected with each other through condensation zones which are continuous with both kinetochores. A double line structure is often seen in kinetochores and condensation zones. The morphological change of kinetochores can be asynchronous, as is especially conspicuous in the sex chromosomes. —The mitotic apparatuses of Pales behave in hexylenglycol medium like mitotic apparatuses of marine eggs. Crystalloids (Fuge, 1970) and microfilament bundles (Bajer and Molè-Bajer, 1969) occur in mitotic apparatuses in early and middle prometaphase.

Complete nucleotide sequence of band 3 related anion transport protein AE2 from human kidney

The cDNA coding for the complete human band 3 related anion exchange protein AE2 has been cloned from human kidney mRNA. The protein is encoded by a mRNA of approx. 3885 nucleotides containing an open reading frame of 3720 nucleotides. The AE2 protein consists of 1240 amino acid residues and has a molecular mass of 136,805 Da.

Schutzversuche gegen S. typhimurium-Infektion an Mäusen. Chemische Zusammensetzung und biologische Wirksamkeit verschiedener Extrakte aus Salmonella-R-Mutanten

Abstract It is not clear as yet how far defined structural components in gram-negative bacteria participate in the induction of immunity to (gram-negative) infections. A number of bacterial components have been considered as playing a role in this respect. These components include the O-antigenic determinants and the more ubiquitous structures of the core portion (Ra-Re) of lipopolysaccharides. Also proteins have been discussed in this connection, especially surface proteins that are exposed under in vivo conditions. In addition, even internal constituents such as nucleic acids have been thought to play a role in induction of immunity to infection. Since it is not yet clear whether all of these components are necessary, we have reinvestigated this question. In the present communication we compare a number of bacterial extracts with respect to their chemical composition and their ability to protect mice against infection to S. typhimurium . Bacteria from a Salmonella typhimurium S form and from 5 R-mutant strains from S. typhimurium and S. minnesota were subjected to extraction with guanidine thiocyanate, urea and veronal buffer. Subsequent analysis revealed significant differences with respect to yield and composition of the antigenic material thereby obtained. With live bacteria, highest yields were obtained by extraction with guanidine thiocyanate (13–23% of the bacterial dry masses) and lowest with veronal buffer (1–3%), while yields obtained by urea were between these two. In the latter case prior killing of the cells with phenol significantly increased the amount of material obtained. One essential difference among the isolation procedures lay in their efficiency for the quantitative extraction of cell wall proteins, which constituted the major part of all extracts. In contrast, with all the procedures the amounts of lipopolysaccharide, phospholipid and nucleic acid obtained were comparable and were relatively low. The highest protein (about 90% of extracted material) and thereby correspondingly lowest LPS- and phospholipid content were exhibited by the guanidin thiocyanate extracts. The lowest protein-(60–80%) and highest LPS and phospholipid content however, were found in the veronal extracts. SDS-polyacrylamide gel electrophoresis of the guanidine thiocyanate and urea extracts from the same bacterial strains showed the presence of similar polypeptides, however in a different quantitative distribution. In all extracts obtained by the two methods 18 common polypeptides were present. The veronal buffer extracts of the mutants on the other hand showed a lower number of polypeptides of which only 5–6 were common. None of the above methods led to the isolation of significant amounts of proteins of porin complex origin. In the individual extracts up to 30 polypeptides were detectable. In active immunization experiments using NMRI mice it could be shown that the extracts afford protection against infection with S. typbimurium . Protective activity was measured as the LD 50 in immunized animals. All extracts from a S. typbimurium Ra mutant (and also those from the S form) obtained by the above mentioned procedures showed similar activity. In contrast, in the case of 3 other R-mutants the urea extracts were superior to the others in their ability to protect against following infection while in the case of another mutant the guanidine thiocyanate extract showed the highest protective capacity. In the case of the most active extracts the degree of protection was comparable to that obtained by immunizing with acetone-killed bacteria. The protective activity in the various extracts did not correlate with the content of lipopolysaccharide, phospholipid or nucleic acid. This poses the question of how far ubiquitous protein antigens play a role in immunity to gram-negative infections.

Proteins from salmonella R-mutants mediating protection against Salmonella typhimurium infection in mice I. Preparation of proteins free from lipopolysaccharide using various chromatographic methods

Different chromatographic methods are described to remove traces of LPS from proteins extracted from the surface of several R-mutants of Salmonella. The protein preparations obtained by these methods were found to contain less than 0.001% of beta-hydroxymyristic acid as determined by gas liquid chromatography. This corresponds to less than 0.003 to 0.006% of R-form-LPS (depending on the chemotype of the mutant) and less than 0.014% of S-form-LPS. From some protein mixtures LPS could be removed by repeated gel filtration on a column of Sepharose CL-6B. Recycling gel filtration proved to be more effective considering the high recovery of protein in much shorter time. Ion exchange chromatography on DEAE Sepharose CL-6B was also useful in some cases. Thus, using one or the other method LPS free protein preparations could be obtained with extracts obtained from 6 out of 7 bacterial strains.

Osmotic shock fluids from salmonella R-mutants. Chemical composition and protective capacity against S. typhimurium infection in mice.

Salmonella R-mutants of different chemotype were subjected to osmotic shock treatment according to the method of Willis et al. (18). Chemical composition (protein, LPS, palmitic acid and nucleic acids) as well as the polypeptide patterns of the shock fluid and hypertonic fluid (in which the cells were suspended before shock treatment) were determined and compared with material extracted by urea treatment carried out to get protein from the cell surface. The results indicate that even components from the cell surface have been released by shock treatment. All the extracts mentioned above showed protective activity in mouse against infection.

Chemische und biologische Eigenschaften von Revertanten aus einer Salmonella typhimurium-Rd1-Mutante

Abstract Two S-form-revertant strains were isolated from a S. typhimurium Rd 1 culture on account of their phage resistance. In microbiological and serological (O-agglutination) characterization — as well as in stability tests (agglutination in auramin and saline and heating at 100 °C) — the behaviour of the two strains was the same as that of the wild type. The two strains were found to be indistinguishable from the wild type strain also with respect to the chemical composition of their lipopolysaccharides. Thus the amount and proportion of fatty acids and sugar residues as well as the number of repeating units in the O-chain were all identical. In contrast, the isolated revertants were similar to the Rd 1 mutant with respect to their auxotrophic markers methionine and tryptophane, to the absence of flagella as well as to the reduced content of cyclopropane fatty acids (C 17 , C 19 ). Protein analysis revealed no significant qualitative or quantitative differences between the wild type strain and the two revertants with respect to the major proteins of their outer membranes. The sensitivity of the revertants to crystal violet, erythromycin and rifamycin SV was intermediate between the wild type and the Rd 1 mutant. Their temperature maximum in nutrient broth was 43 °C, the retardation in growth at this temperature corresponding to that of the Rd 1 mutant. At 37 °C, however, the growth rate of the revertants was identical to that of the wild-type, while that of the Rd 1 mutant was slower. Addition of sodium chloride to the growth medium rendered the temperature dependent behaviour of the mutants and revertants similar to that of the wild type. Studies in NMRI mice revealed that the revertants, also with regard to their virulence, occupy an intermediate position between the mutant and the wild type. Nevertheless their ability to afford protection to Salmonella typhimurium infection following active immunization with acetone killed cells was as high as that of the wild type. The results show that the biologic behaviour of S. typhimurium is determined by the type of lipopolysaccharide it contains but also to a large extent by other cell-wall constituents.