Biological activity of (lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1.

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1ß and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

[Gynecological consultations for young females for treatment of sexual organ disorders]. / Konsultacje ginekologiczne u dziewczat w profilaktyce chorób narzadu plciowego.

The authors presented their consultations with young females at the out-patient clinic from 1983-1995 and compared them with their consultations during an earlier period. According to the authors, the frequency and character of the issues consulted changed as a result of a greater need for this type of help in general. The authors stressed the particular role of establishing thorough medical case histories on the gynaecology of the pubescent period. They concluded that effective child and pubescent female gynaecology could, in nearly every case, protect the patient against disorders and complications during in maturity and over the reproductive period.

A new Mesorhizobium loti HAMBI 1129 phage isolated from Polish soil.

Phage A1 isolated from the rhizosphere of Lotus corniculatus was studied. It had a very narrow host range, as it was active only against Mesorhizobium loti HAMBI 1129. Phage A1 was classified as belonging to C Bradley's group bacteriophages. The latent period of A1 was 120-130 min and a burst size 13-17 particles per cell. The nature of the phage receptor was examined. Lipopolysaccharide from the phage-sensitive strain inactivated phage A1 in contrast to LPS from the phage-resistant bacteria. Purified LPS obtained from M. loti HAMBI 1129 had a high receptor activity with PhI(50) value of 0. 025 microgram/ml.

[Partial dependence of luliberin and its analogs on the immune system]. / Wspólzaleznosci luliberyny i jej analogów z ukladem odpornosciowym.

Luliberin--luteinizing hormone-releasing hormone (LHRH) is the first link in hypothalamus-pituitary-gonads axis which participates in reproduction. This hormone and lutropin are released from immunological cells, however antagonists of LHRH inhibit this action. Some cytokines (IL-1 beta, GM-CSF) inhibit release of LHRH and others (IL-6, IFN-gamma) stimulate this release.

[Luliberin and lutropin--their significance]. / Luliberyna i lutropina--ich znaczenie.

Human lutropin (hLH) is a heterodimeric, glycoprotein hormone released from pituitary by decapeptide luliberin (LH-RH). The both hormones plays an important role in axis hypothalamus-pituitary-gonads responsible for procreation. Many isoforms of hLH have been found and the occurrence of several genetic variants of the hormone in infertile female patients were described. Also genetic mutants of human lutropin choriogonadotropin receptor (LH/CG-R) in male young patients with precocious puberty were proved. Until now several thousands of LH-RH analogs were synthesized and some of them were applied in therapy of several gynecological, and oncological diseases or in vitro fertilization.

Isolation of human lutropin from woman urine and comparison of its properties with pituitary hormone.

Lutropin was isolated from woman preovulatory urine by immunoaffinity chromatography using a column with monoclonal antibody (mAb) anti-beta hLH(37) coupled to Sepharose CL 4B. As it was demonstrated the isolated hormone, as well as pituitary lutropin, were separated in SDS-PAGE into several well visible fractions with 30-94 kDa molecular mass and scarcely visible fractions with 14-20 kDa. All fractions reacted only with mAb anti-alpha hCG(99)-HRP but not with mAb anti-beta hLH-HRP. Pretreatment of pituitary lutropin with PN-Gase F did not affect its electrophoretic pattern. After boiling the hormone with SDS and beta ME no fractions in SDS-PAGE were observed. No substantial differences in affinity chromatography on Con A-Sepharose between pituitary and urinary lutropin were noted. Some differences between these two hormone preparations were observed in assays performed with several ELISA variants. Using two pairs of mAbs anti-beta hLH for ELISA technique no hormone was assayed in urine samples from women, collected between 12 and 16 days of menstrual cycles.

Influence of some factors on immunoassays of human myoglobin.

Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done using commercial Seratec kit for immunoassay of human myoglobin. Addition to the muscle extracts not only concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.

Some parameters influencing immunoassay of human and horse myoglobins.

It was noted that human and horse sera as well as human heart and skeletal muscle homogenates or extracts distinctly decrease immunoassays of purified myoglobins. The assays of homogenate and extract myoglobins could be many times increased by precipitation certain proteins with concentrated ammonium sulfate or sodium chloride. Also in homogenates and extracts incubated for several days increased assays of myoglobins were noted. The obtained results indicate that both myoglobins occur in complex with other tissue component(s).

Investigations on human lutropin from pituitary gland and urine.

Two newly isolated monoclonal anti-beta hLH antibodies did not recognize both human chorionic gonadotropin (hCG) and three synthetic peptide fragments corresponding to beta hLH sequence. These antibodies were applied for ELISA of human luteinizing hormone (hLH), however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of hLH from pituitary and urine with 6 different pairs of monoclonal antibodies (4 anti-beta and 1 anti-alpha-subunit) were noticed. The highest hLH concentration in urine samples collected during 53 menstrual cycles of 6 women was demonstrated at different times of the day. In 14 out of 52 "preovulatory urine" portions, preserved for several weeks, over 80% increase of assayed hLH was shown. The highest assays of hLH from pituitary and urine were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and was eluted with alpha-methyl-D-mannoside as a single peak. However, a small part of hLH from urine was eluted from the column as non retarded peak. When HPLC analysis with DEAE column was performed, pituitary hLH was separated in two fractions while lutropin from urine was eluted as a single peak.

[Isoforms of luteinizing hormone]. / Izoformy hormonu luteinizujacego.

Luteinizing hormone (LH) is a heterodimeric glycoprotein containing varied amount of sialic acid. This is a reason of numerous LH isoforms called also isohormones. The hormone isoforms were separated usually by gel electrophoresis, isoelectrofocusing or chromatofocusing. They differ in biological and immunological activity. Human and some animals LH isoforms were reviewed. Also some genetic mutants of LH are described. Problems of the human isoforms for pathology and diagnostics are presented.

Antigenicity of human and horse myoglobins.

Rabbit antisera against human myoglobin and horse myoglobin cross-reacted with both myoglobins but only one of them recognized human hemoglobin. Two mouse monoclonal antibodies anti-human myoglobin were obtained, but only one of them (No. 49) cross-reacted with horse myoglobin. Antibody No. 49 and rabbit antibodies reacted also with apo-, FITC- and treated with hydrochloric acid or TPCK-trypsin horse myoglobin, but their binding to myoglobin pretreated with NaOH was reduced. Thirteen peptides overlapping sequence of human myoglobin were synthesized on polyethylene pins. Rabbit and mouse polyclonal antibodies reacted with some of these peptides but no reaction was noted with mouse monoclonal antibodies. Two monoclonal antibodies were applied for specific immunoassay of human myoglobin.

[Human myoglobin (hMb) and its diagnostic value]. / Ludzka mioglobina (hMb) i jej wartosc diagnostyczna.

Composition, properties and occurrence of human myoglobin (hMb) were described. Methods for immunological assay of hMb were presented. Localization of antigenic determinants in myoglobin molecule was shown. Value of assay of hMb in serum and urine for early diagnosis of myocardial infarct and some other muscle diseases was discussed.

[Immuno-contraceptive vaccines]. / Szczepionki immunoantykoncepcyjne.

Results of studies on birth control vaccines accomplished by many research groups within the WHO Special Programe of Research, Development and Research Training in Human Reproduction has been reviewed. The mostly investigated contraceptive vaccines contain human chorionic gonadotropin, and therefore structure, biological role and immunogenicity of the hormone were presented. Also, vaccines obtained on the basis of subunit beta of the human chorionic gonadotropin, its heterodimer with subunit beta of ovine lutropin or C-terminal peptide of beta subunit were described. Results of two phases of clinical trials with immuno-contraceptive vaccines were presented. The use of contraceptive vaccines for treatment of some trophoblastic tumors and the idea of using contraceptive recombinant vaccines were also mentioned.

Human serum immunoglobulins binding to synthetic fragments of human chorionic gonadotropin.

Human sera obtained from healthy individuals, patients with cancer diseases, infertile and pregnant women were investigated for the presence of immunoglobulins (Ig) binding to synthetic fragments corresponding to sequence of human chorionic gonadotropin (hCG). Four synthetic peptides corresponding to some regions of alpha hCG and beta hCG subunits, coupled to polystyrene beads and 16 peptides overlapping sequences of alpha- and beta hCG subunits, synthesized on polyethylene pins, were used. IgG and IgM bound to hCG-synthetic peptides were assayed using goat or mouse monoclonal antibodies anti-human IgG labeled with horseradish peroxidase. Most of the investigated sera contained IgG which bound to examined synthetic peptides, but obtained results were dependent on serum donors. Some relationships between results obtained with peptides synthesized on modified pins according to Geysen et al. method and corresponding peptides synthesized by Merrifield method and coupled to beads were noted.

Stability of human lutropin studied with immunoenzymatic technique.

Characteristics of two monoclonal antibodies against human lutropin and their use in enzyme immunoassays of the hormone are presented. Diluted solution of pituitary lutropin was unstable in buffered saline and could be stabilized with some proteins. Concentration of the lutropin in woman urine, collected during "lutropin-peak", was markedly increased during long time storage at 5 degrees C or at room temperature. Using HPLC it was demonstrated that pituitary lutropin, normal urine lutropin and "increased" urinary lutropin were eluted from ion exchange column almost with the same retention time.

[Human luteinizing hormone (hLH)]. / Ludzki hormon luteinizujacy (hLH).

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.

Effect of pretreatment of wells in polystyrene plates on adsorption of some human serum proteins.

Coating of Immulon polystyrene plates with 0.2% casein in phosphate buffered saline (PBS) completely abolishes adsorption of human serum proteins to these plates. However, pretreatment of such plates with PBS or 0.15 M NaCl in deionized water (10 microS) before coating makes possible adsorption of human immunoglobulins G (HIgG) and M but not serum albumin (HSA) and transferrin (HTrf). Effect of pretreatment with PBS on adsorption of HIgG is long-term and rather stable. Results of experiments with radioiodinated HIgG, mouse IgG, HSA and human chorionic gonadotropin confirm the peculiar effect of pretreatment with PBS on casein coating. Pretreatment with deionized water, instead of PBS, markedly diminish adsorption but tap or commercial spring waters (> 1000 microS), even without 0.15 M NaCl, affect adsorption similarly to PBS in deionized water. Super pure water (0.05 microS) even with NaCl used for pretreatment does not influence adsorption of HIgG to plates coated with casein. Distinct differences in adsorption of HIgG and HTrf was demonstrated using solutions for ELISA prepared from super pure, deionized and tap waters.

Search for antibodies for immunoenzymatic assay of human lutropin.

Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of beta hLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of alpha hLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-alpha hCG antibodies. Using 2 monoclonal anti-alpha hCG and anti-beta hLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.

Semiquantitative assay of human chorionic gonadotropin by a simple and fast immunofiltration technique.

An immunofiltration technique for the semiquantitative assay of human chorionic gonadotropin (hCG) was applied in two versions, using different antibodies. One anti-beta hCG subunit was immobilized on a glass microfibre disc in the form of six radially located bars, and the dry disc was placed on a water-absorbing material in a plastic device. A second antibody labelled with horse radish peroxidase conjugate was used in solution. For the colour reaction a solution with tetramethylbenzidine and hydrogen peroxide was used. The number of blue bars appearing on the test disc depended on concentration range of human chorionic gonadotropin. The technique with the monoclonal antibodies, anti-beta hCG and anti-alpha hCG-horse radish peroxidase conjugate, was specific for intact human chorionic gonadotropin, while the technique with the rabbit antibodies, raised against synthetic fragment 122-145-beta hCG and beta hCG-horse radish peroxidase, was useful for both intact human chorionic gonadotropin and its beta-chain. Cross reactions with human lutropin and thyrotropin were negligible. Haemoglobin, urea and various tested drugs did not affect the assay. In the assay of human chorionic gonadotropin in the urine of pregnant women and in sera of patients with trophoblastic diseases, the results from the immunofiltration technique were in accordance with data obtained by classical ELISA and by two commercial kits.

Rabbit antibodies to some sequences of human chorionic gonadotropin and their use for immunoassay of the hormone.

Three synthetic peptides corresponding to amino acid sequences of two human chorionic gonadotropin (hCG) subunits were conjugated to thyroglobulin and used for immunization of rabbits. The highest antibody concentration was found in antisera from rabbits immunized with the peptide corresponding to 122-145 sequence of the hormone beta-subunit. Specificity of antisera and immunoaffinity purified rabbit antibodies were studied. Antibody anti-122-145-beta hCG with antibody anti-beta hCG conjugated to peroxidase were suitable for sensitive and specific enzyme immunoassay of hCG and beta hCG. Good correlation was noted between assay of the hormone in sera of patients with trophoblastic diseases or in urine of pregnant women by ELISA with the rabbit antibody pair and by commercial kit or by ELISA with two monoclonal antibodies.