Characterization of the high-affinity oligosaccharide-binding site of the 205-kDa porcine large granular lymphocyte lectin, a member of the leukocyte common antigen family.

Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non-major-histocompatibility complex-restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [Pospísil M., Kubrycht J., Bezouska K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83-90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205-kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4-azidophenyl (photoactivatable) and 4-hydroxyphenyl (radio-iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N-glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high-affinity ligands to investigate the oligosaccharide-combining site of the 205-kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205-kDa polypeptide constitutes the major cell-surface calcium-independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand-labeled and eleven other peptides proved that the 205-kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high-affinity binding site is localized in the N-terminal 70-kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate-recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O-glycosidically linked lineage-specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell-surface glycoproteins expressed high-affinity binding sites for the oligosaccharide ligands. Thus, N-linked oligosaccharide moieties of cell-surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide-mediated activation of lymphocytes.

Peripheral membrane molecules of leukocytes and NK cytotoxicity.

Some leukocyte effector cell-surface molecules movement toward the adjoining target cells takes place during the reaction of NK cytotoxicity (NK R). The majority of the moving molecules are usually anchored via a divalent-ion-dependent interaction (PMM-M2+). The released PMM-M2+ can interact also with the secreted tumor necrosis factor alfa (TNF-alpha). In agreement with PMM-M2+ movement, the number of TNF-alpha binding sites on the target cell surface increases during NK R. In addition, antibodies against PMM-M2+, as well as D-mannose- or N-acetyl-D-glucosamine-terminated oligosaccharides of PMM-M2+ inhibit NK R. A more detailed analysis of PMM-M2+ with monoclonal antibodies used flow cytometry and cell-surface biotinylation. Only 3 of 31 tested CD antigens (CD2, LAK-1 and CD45) were passed through this first strongly restricted experimental screening. The EDTA-released LAK-1 antigen, but not CD2 and CD45, interact with TNF-alpha and cell surface via a mannose-inhibitable interaction dependent on the presence of Ca2+ ions. The mechanism of possible participation of PMM-M2+ in cytotoxic events is discussed in relation to Ca2+ influx and subsequent cytolysin secretion.

[Analysis of the polypeptide profile in insulin preparations using HPLC]. / Analýza polypeptidového profilu inzulínových preparátu metodou HPLC.

The purity of insulin preparations which is important during clinical administration of this hormone can be assessed in several ways. The authors suggested and tested a method for analysis of the polypeptide composition of insulin preparations which makes quantitative and qualitative evaluation of the insulin and non-insulin character possible, as well as their separation according to the shape and size of biological macromolecules by gel filtration on HPLC equipment. The described procedure makes possible a more detailed analysis of insulin preparations when the method is used in combination with electromigration and immunological estimations.

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans.

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.

Production of quinomycin A in Streptomyces lasaliensis.

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized by Streptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.

Mutants of Streptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites.

Mutants of Streptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, gamma-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described in Streptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, zeta-rhodomycinone and glycosides of epsilon-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases.

Studies on the production of daunomycinone-derived glycosides and related metabolites in Streptomyces coeruleorubidus and Streptomyces peucetius.

Strains of Streptomyces coeruleorubidus ISP 5145, JA 10092 and 39-146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, epsilon-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher RF (0.4-0.7), isolated from strain 39-146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis. Streptomyces peucetius IMI 101 335 differed from Streptomyces coeruleorubidus in an increased production of epsilon-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots. Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo3; glucose was a more useful carbon source for the remaining strains. The activity of Streptomyces coeruleoribidus was inhibited by 1-propanol, Na-propionate, 5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.

The study of variability and strain selection in Streptomyces atroolivaceus. III. Isolation and preliminary characteristics of mutants impaired in the biosynthesis of mithramycin.

Mutants of Streptomyces atroolivaceus blocked in the biosynthesis of mithramycin were isolated both by natural selection and after treatment with mutagenic factors (UV and gamma rays, nitrous acid). Both physical factors were more effective than nitrous acid. The selection was complicated by a high instability of isolates, out of which 20-80% (depending on their origin) reversed spontaneously to the parent type. The primary screening (selection of morphological variants and determination of their activity using the method of agar blocks) made it possible to detect only potentially non-productive strains; however, the final selection had to be performed always under submerged conditions. Fifty-four stable non-productive mutants were divided, according to results of the chromatographic analysis, in five groups differing in production of six biologically inactive metabolites (compounds A-H). The mutants did not accumulate chromomycinone, chromocyclomycin and chromocyclin. On mixed cultivation none of the pairs of mutants was capable of cosynthesis of mithramycin or new compounds differing from standard metabolites. Possible causes of the above results are discussed.