The effect of undecanones and their derivatives on tumor angiogenesis and VEGF content.

The in vivo effects of some derivatives of aliphatic ketones (2-undecanone, 3-undecanone, 4-undecanone and their derivatives) on L-1 sarcoma tumor angiogenesis and VEGF content were studied in Balb/c mice. Mice that inhaled 10% solution of 3-undecanone(3-on) or 1% solution of 2-undecanone propylene acetal (Acpr2) for 3 days after tumor cells implantation, presented lower neovascular response measured by tumor-induced cutaneous angiogenesis test (TIA) and lower tumor VEGF content in 5-days tumors, than non-inhaled controls. Other substances presented various effects on tumor VEGF concentration and angiogenesis. Histological examination of lesions collected from mice inhaled Acpr2, or non-inhaled controls, revealed small diffused areas of necrosis in the former group. In both groups, slight to moderate inflammatory infiltrations were seen at the tumor's margin. In Acpr2 group, there were less small blood vessels at tumor's margin than in the control group.

Directed biosynthesis of new enniatins.

New cyclohexadepsipeptides of the enniatin type with potential anthelmintic properties were produced by two different strategies: 1. In vitro synthesis by use of the multienzyme enniatin synthetase, and 2. in vivo precursor feeding of enniatin producing strains Fusarium scirpi and Fusarium sambucinum. The compounds were analyzed by HPLC, various NMR measurements and mass spectrometry. The three N-methyl L-amino acid positions in the enniatin B molecule could be gradually replaced by other (N-methyl) L-amino acids, e.g. alanine, cysteine, threonine and serine. The latter two amino acids yield new enniatins with functional groups in the hydrophobic side chains. Similarly the three D-2-hydroxyisovalerate residues, present in all naturally occuring enniatins, could be substituted by D-2-hydroxybutyric acid and D-lactic acid. Despite its lower yield the in vitro synthesis has the advantage of a broader variety of products formed.

[Reduction of dustiness at non-stationary work stations]. / Ograniczanie zapylenia na niestacjonarnych stanowiskach pracy.

Many industrial processes are responsible for chemical and dust pollution. As the quantities of the substances emitted remain unknown in most cases, the data on the effect of technological parameters on the emission rates are not available. Various technical and organisational projects aimed at reducing dust hazards in rooms and at workposts are listed. Methods of assessing emission rates of pollutants from machines to the workplace air, and the efficiency of local exhaust ventilation are discussed. Results of the study in this area for a manual mechanised tool grinder and a tool grinder machine are presented.

Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase.

N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases and S-adenosyl-l-methionine (AdoMet)-dependent N-methyltransferases. The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes. Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site. The role of four conserved motifs (I, (2085)VLEIGTGSGMIL; II/Y, (2105)SYVGLDPS; IV, (2152)DLVVFNSVVQYFTPPEYL; and V, (2194)ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied. Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding. Further shortening close to motif I resulted in loss of binding activity. Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity. Point mutations converting the conserved Tyr(223) (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to approximately 70%, indicating that Tyr(223) is important for AdoMet binding and that the aromatic Tyr(223) may be crucial for AdoMet binding in N-methylpeptide synthetases.

Biosynthesis of PF1022A and related cyclooctadepsipeptides.

PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D-lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.