Modern directions in studying biologically active substances from basidial fungi

Nonconventional uses of basidial fungi as producers of valuable biologically active compounds are reviewed. Basidiomycetes are now most extensively used in the medical industry to obtain antibiotics, enzymes, lipids, and various drugs acting on the immune system (hypocholesterolemic, antiviral, hypotensive, hypoglycemic, etc.). Recent data on toxic and psychotropic substances of Basidiomycetes and of their aromatic compounds responsible for the so-called fungal flavor are also considered. Finally, the increasing significance of Basidiomycetes for human practice is underlined.     

State of biologically active substances in the mother-placenta-fetus system in pregnancy and labor]

Individual development in the course of a generation has an unidirectional, irreversible, "closed", predominantly structly-determined, predominantly autoprogrammed, autoreproducible character. The characteristics of individual development differ in terms of its processes, stages, types and orders. Ontogenesis and ageing of the organism represent the principal processes of individual development. Individual development has three main stages (progressive stage, "plateau" stage and regressive stage). The types of individual development may be grouped into two categories: elementary and complex. The complex types result from the combining in different ways of the elementary types. Similarly, orders of the generations of individual development and hierarchical orders of individual development may be differentiated. Between the processes, stages, types and orders of individual development certain "combining" relationships may appear. The processes, stages and types of individual development as well as their mutual relationships serve both development and stability.     

Chemistry of biologically active benzoxazinoids

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and its desmethoxy derivative (DIBOA) are major allelochemicals produced by corn, wheat, rye and related monocotyledons. These benzoxazinone derivatives have a wide variety of biological actions, including antifungal and mutagenic activities. Structure-activity relationships of these compounds and their derivatives (benzoxazinoids), the reactivity of benzoxazinoids with nucleophiles, and the substituent effects of the 7-methoxy and 2-hydroxy groups are discussed in relation to the molecular mechanisms of the biological activities.     

Eosinophil chemotaxis induced by several biologically active substances and the effects of apafant on it in vitro

Chemotaxis of guinea pig eosinophils induced by various stimuli in use of a modified Boyden chamber technique in vitro and the effect of a platelet-activating factor (PAF) antagonist, apafant (CAS 105219-56-5, WEB 2086 BS), on it were examined. The eosinophils were obtained by bronchoalveolar lavage from the animals treated by i.v. injection with Sephadex G-200 and purified by Percoll density gradient centrifugation. PAF significantly and potently induced the chemotaxis at a broad range of 10(-17) to 10(-7) mol/l, where no concentration-dependency was observed. Leukotriene B4 also induced the chemotaxis in a concentration-dependent manner at 10(-14) to 10(-12) mol/l and the enhanced migration was not declined until 10(-7) mol/l. Interleukin-5 (IL-5), IL-8 and regulated on activation normal T expressed and secreted (RANTES) only modestly enhanced the chemotaxis in some concentrations at 10(-13) to 10(-7) mol/l with or without significance and with no concentration-dependency while formyl-methionyl-leucyl-phenylalanine (FMLP), a known chemoattractant, increased the migration at 10(-7) to 10(-5) mol/l. Apafant at 10(-8) to 10(-6) mol/l strongly and concentration-dependently inhibited 10(-8) mol/l PAF-induced chemotaxis. However, the drug showed nominal or no influences on their chemotaxis stimulated by the other agonists, at the concentrations of which the enhanced migration was observed. From these results, it is concluded that IL-5, IL-8 and RANTES, different from PAF and LTB4, are not potent stimuli for the eosinophil chemotaxis and that apafant is a selective antagonist of PAF, which is expected to be therapeutically effective for PAF-associated diseases including bronchial asthma.     

Specificity of Pseudomonas aeruginosa serralysin revisited, using biologically active peptides as substrates

The characterization of the specificity of alkaline protease from Pseudomonas aeruginosa has not yet been clearly defined. Some previous results suggested that its specificity was influenced more by amino acids far from the hydrolyzed peptide bond, than by amino acids in P1 or P'1 position. From other data, it was a C-(COOH)-type endoprotease where the preferential amino acid in P1 position was an arginine residue. We have studied the hydrolysis of several biologically active peptides. Many various sites of cleavage have been characterized but no arginine in P1 position was found, despite the presence of arginine in the peptide sequence. In fact P1 and P'1 position could be occupied by various amino acids. It seems unlikely that Pseudomonas alkaline protease may only be considered as a protease specific to arginine in P1 position. On the other hand, we have observed that increase of the peptide chain length led to an important increase of the hydrolysis rate, suggesting an extended number of subsites.     

Automated segmentation of muscle fiber images using active contour models

Absorbed power (PAbs) during exposure to vertical whole-body vibration in a sitting posture was measured on 15 male and 15 female subjects. Different experimental conditions were applied, such as vibration level (0.5-1.4 m s(-2)) and frequency (2-100 Hz), body weight (54-93 kg) and, relaxed and erected upper body positions. Results show that PAbs was strongly related to the frequency of the vibration, peaking within the range of 4-6 Hz. The peak was predominantly located in the lower end of this range for females and for the relaxed sitting position. PAbs increased with acceleration level and body weight. Almost a ten-fold increase in PAbs was observed at the critical frequency when the vibration exposure was raised from 0.5 to 1.4 m s(-2). If risk assessment is based on the assumption that the amount of PAbs, independent of the frequency of the vibration, indicates a hazard, then the ISO-standard 2631 under- and overestimates the risk at frequencies below and above about 6 Hz, respectively. The results also indicate a need for differentiated guidelines for females and males. Many types of vehicles produce whole-body vibration with frequencies which coincide with the range where the highest PAbs was observed. PAbs is a 'new' concept for measurement of whole-body vibration exposure. Although not yet thoroughly evaluated, this measure may be a better quantity for risk assessment than those specified in ISO 2631 since it also takes the dynamic force applied to the human body into account.     

Chemoenzymatic synthesis of chiral biologically active heterocycles

During 1995, among 1105 HIV patients explored in our department, 64 presented a deep fungic infection (5.8%). The yeast was searched for in cerebrospinal fluid, blood, urine, and bronchoalveolar aspiration. Isolated germs were Cryptococcus neoformans (95%), Candida tropicalis (1 case), Saccharomyces cerevisiae (1 case) et Aspergillus fumigatus (1 case). Results of treatment with amphotericin B were: recovery (9%), clinical success (11%), out of sight (14%), letality (66%), relapse (23%) and side effects (19%). We emphasized diagnostical and therapeutical difficulties, and bad prognostic of mycoses in patients with AIDS.     

Development of a membrane oxygenator for ECMO using a novel fine silicone hollow fiber

One of the limitations of conventional silicone hollow fiber oxygenators compared with microporous membrane oxygenators is poor gas permeability. However, the silicone hollow fiber is free from plasma leakage, which is the major life limiting factor of the microporous membrane oxygenator. It has been difficult to fabricate a fine, thin hollow fiber for reduction of resistance to gas permeability because of the poor mechanical strength of conventional silicone materials. The authors developed a novel silicone material with sufficient mechanical strength, and a fine silicone hollow fiber with a diameter of 30 microns and wall thickness of 50 microns, which is approximately half that of a conventional silicone hollow fiber. Using this newly developed silicone hollow fiber, the authors developed a compact extracapillary flow membrane oxygenator. The oxygenator consists of fine silicone hollow fibers inserted in a housing made of polycarbonate. The housing is a cylindrical case, 20 cm long and 55 mm in inside diameter. The hollow fibers are cross-wound. The surface area of the membrane is 2.0 m2, and priming volume is 230 ml. Gas transfer performance of the newly developed oxygenator was evaluated by in vitro experiments. Oxygen and carbon dioxide transfer rates were 195 ml/min and 165 ml/min, at a blood flow rate 3 L/min. The novel silicone membrane oxygenator developed in this study can be used for extended duration in such applications as extracorporeal membrane oxygenation.     

Effect of treatment with cardiac glycosides and biologically active substances on the state of the lesser circulation according to the electrokymographic data

Electrokymography was used to assess by pulmonary densograms and pulmonary artery pulse curves treatment-induced changes in cardiac contractility and lesser circulation in 455 patients with rheumatic heart disease and stage IIA, IIB, and III circulatory insufficiency. Treatment with cardiac glycosides in combination with metabolic preparations (ATP methionine, vitamin B12, inosine-F, and extract of the heart muscle) leads to a more conspicuous reduction in the symptoms of congestion in the lesser circulation than that encountered in treatment with cardiac glycosides alone. The reduction in these symptoms following treatment with ATP and heart-muscle extract may be explained not only by improved myocardial contractility but also by the direct vasodilating effect of adenyl compounds.     

Conversion of thyrotropin heterodimer to a biologically active single-chain

TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a hormone specific beta-subunit. Assembly of alpha- and beta-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site-directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common alpha-subunit to the carboxyl terminal end of hTSHbeta in the presence or absence of hCGbeta carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.     

Purification of biologically active SPARC expressed in Saccharomyces cerevisiae

SPARC (secreted protein, acidic and rich in cysteine) is a secreted, Ca+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC have been mammalian bone, platelets, a basement membrane tumor, and cultured cells; most if not all preparations, however, contain platelet-derived growth factor and one or more serum proteins that bind specifically to purified SPARC. To avoid these contaminants, as well as the toxic lipid moiety associated with endotoxin, we expressed recombinant wild-type and a mutated murine SPARC in two strains of Saccharomyces cerevisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recombinant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimized for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution of Asn98 with Asp98 in the wild-type sequence. Purification of SPARC was achieved by copper-affinity and hydrophobic-interaction chromatography. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endothelial cells: inhibition of cell spreading/disruption of actin microfilaments and competition for the binding of nonrecombinant 125I-labeled SPARC to the cell surface. The availability of biologically active, recombinant SPARC will facilitate investigation of the structural and functional properties of this protein, which is expressed at high levels in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue.     

Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.     

Improved method for the purification of biologically active transcobalamin II

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.     

Application of Kohonen Neural Networks in classification of biologically active compounds

Automated data classification is an indispensable tool in Drug Design. It allows to select homogeneous training sets or to distinguish compounds with required biological properties. The Kohonen Neural Networks (KNN) suggest new means for classification of biologically interesting compounds. In this paper, first, capabilities of KNN in data dimensionality reduction are presented as compared with the capabilities of Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). The advantages of KNN become evident with increasing data dimensionality and size of the training set. Then, new methods are suggested to evaluate the quality of KNN models. Finally, a case study on chemical and biological data is presented. The database studied includes more than 2000 organophosphorous potent pesticides. The Kohonen maps were obtained which allow to distinguish compounds with different biological behavior.     

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

Identification of biologically active sites in laminin an extracellular matrix protein

Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin alpha 1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.