Biomedical subjects
Publications and source records attributed to E Kurowska.
Heterodimeric composition and amino acid sequence of hLH is reviewed. The hormone isoforms and methods of their assay are summarized. Releasing of the hormone, its control by synthetic antagonists and agonists and their significance for therapy are described. Structure and function of hLH receptor are presented.
Non-competitive enzyme-linked immunosorbent assay (ELISA) for rabbit anti-BPTI antibody is described. The antibody was purified by affinity chromatography on Sepharose-BPTI column and then separated into two antibody fractions on Sepharose-trypsin (BPTI) column. The fraction I antibody, not retarded on the second biosorbent column, efficiently inactivated BPTI, but the fraction II antibody, eluted from the column with glycine buffer, pH 2.2 was firmly bound to active BPTI. This proves that BPTI has at least two epitopes.
Using specific rabbit antibody, one non-competitive (sandwich) and three variants of competitive enzyme-linked immunosorbent assays for basic pancreatic trypsin inhibitor (BPTI) were elaborated. For the assays peroxidase-antibody, two peroxidase-BPTI and beta-galactosidase-BPTI conjugates were applied. The best was competitive assay with peroxidase-BPTI conjugated by two step glutaraldehyde procedure. By this assay we were able to determine 1-10 ng BPTI/ml (0.007-0.07 KIU/ml) and the presence of human blood serum did not affect the assay of the inhibitor.
The Swatek's method was further simplified for the assay of penicillin amidase activity. The absorbance of colour obtained during determination of 6-aminopenicillanic acid was dependent on concentration of 4-dimethylaminobenzaldehyde and on temperature. Antiodies induced in rabbits with one molecular form of penicillin amidase from E. coli PCM 271 (PA-1 or PA-2) did not cross-react with the other amidase form. No differences in substrate specificity on inactivation with SDS and in alkaline medium between the two amidase forms were observed. Concentrated urea inactivated PA-2 irreversibly and PA-1 reversibly. N-Bromosuccinimide inactivated almost completely only PA-1. Two E. coli PCM 271 strain variants were separated by microbial selection. Each of them produced only one amidase form. Also two amidase forms were found in cells of E. coli ATCC 11105, whereas E. coli ATCC 9636 and ATCC 9637 synthesize only PA-1.
By manifold immunizations of rabbits with virgin or modified trypsin inhibitor III from squash seeds and trypsin inhibitor II b from cucumber seeds, specific antibodies were produced. In double immunodiffusion the anti-squash inhibitor antibody also gave weak precipitate arcs with inhibitor I from squash, inhibitor II from summer squash and with inhibitor I from zucchini, but not with inhibitor II b from cucumber seeds. The genus of Cucurbita trypsin inhibitors, preincubated with the antibody, lost their antitrypsin activity. The antibody showed a significantly weaker effect on the activity of the inhibitor from cucumber sees. 1H-NMR and CD spectra also confirm structural differences between trypsin inhibitors from the genus of Cucurbita and the cucumber (genus of Cucumis) inhibitor.
A simple and sensitive method of studies on interactions of antibodies with the light forms of gamma-glutamyltransferases was elaborated using polyacrylamide gel electrophoresis and the fluorescent technique for localization of the enzyme. Rabbit antibodies against bovine and rat kidney enzymes were used. gamma-Glutamyltransferases from various organs of the same animal were immunologically indistinguishable, but distinct immunological differences were observed between the enzymes from various animal species. The new method of studies on immunological reaction between antibody and the enzymes appeared more sensitive than the double immunodiffusion method.
R404 plasmid aggregate is composed of two conjugative and two nonconjugative plasmids. Plasmid aggregate reconstructed from separated plasmids had the same genetic properties as the original R404 plasmid aggregate. It was found that plasmids of R404 factor could be transferred in conjugation in twelve different sets. These twelve genetically distinct classes of transconjugants formed only six groups differing in phenotypic characters.
Transformational separation of plasmids from R404 plasmid aggregate found in Salmonella enteritidis strain was performed. Three classes of transformants differing in their resistance patterns were isolated. Genetic properties of the transformants suggest that their resistance is determined by single plasmids. Plasmid pCK3 (Tra-ApCbCrSuSm) and pCK4 (Tra-ApCbCrCm) are nonconjugative while plasmid pCG1 (TraApCbCrSuSmTcKmNm) is conjugative. Separation of all plasmids of R404 plasmid aggregate allowed to determine their genetic properties and the manner of conjugational transfer of R404 plasmid aggregate R-determinants.
The genetic relationship of four plasmids separated from R404 plasmid aggregate has been characterized by investigating entry exclusion and intracellular incompatibility. It has been found that the four plasmids exist in an autonomous state in cells carrying the aggregate. They are compatible and the two conjugative plasmids possess different traS genes.
Conjugational transfer of R404 factor, found in Salmonella enteritidis strain, was examined. Six derivative forms differing in resistance pattern were isolated. The manner of conjugational separation during transfer of R404 factor from E. coli to E. coli strain was the same as from the original strain of S. enteritidis to E. coli strain. During following conjugation, all isolated forms except one, could give forms differing in a resistance pattern from the parenteral ones. This one form, from which different conjugational forms could not be separated, was assumed to be a single plasmid and was designated pCK2. All other forms, including the original R404 factor were assumed to be plasmid aggregates.
Three r-determinants (Cb Cm Cf) out of eight present in R404 factor were transferred in transformation of Escherichia coli by the plasmid DNA isolated from minicells. The r-determinants acquired in this way formed the replicon tr404-1 and were untransferable by conjugation unless another self-transmissible plasmid was present in recipient. The evidences are presented in support of hypothesis that the transfer events consist in infection of transformant cells with sex factor, recombination between tr404-1 replicon and the sex factor and finally transfer of the new formed structure to the host cells of the sex factor.
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