[Pathways of the synthesis of glutamic acid by cephalosporin C-producing Acremonium chrysogenum]. / Puti obrazovaniia glutaminovoi kisloty u Acremonium chrysogenum, produtsenta tsefalosporina C.

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.

[Ultrastructure of liposomes (interliposomal bonds)]. / Ul'trastruktura liposom (mezhliposomal'nye sviazi).

Interliposomal bonds (ILBS) analogous to intercellular bonds (ICBs) in microbial cultures were detected by electron microscopy in the liposomal materials obtained after encapsulation of substances of various chemical structure. Possible nonspecific formation of the bonds between biological membrane-limited objects (ILBs and ICBs) was suggested and formation of such bonds in liposome encapsulated drugs was believed to be of importance.

[Incorporation of an antineoplastic drug aclarubicin into liposomes in relation to the conditions of its encapsulation]. / Vkliuchenie v liposomy protivoopukholevogo antibiotika aklarubitsina v zavisimosti ot uslovii inkapsulirovaniia.

The results of liposome drug encapsulation of aclarubicin (aclacinomycin A), an antitumor antibiotic, are presented. The method of flow detergent dialysis was applied. Conditions providing maximum encapsulation of aclarubicin (the ratio of lipid components and the lipid/detergent ratio), as well as conditions providing stability of liposomal emulsion (the presence of antioxidants, stearic acid and cholesterol) were defined.

[Obtaining liposomal preparations of various biologically active substances by the method of detergent flow analysis]. / Poluchenie liposomal'nykh preparatov razlichnykh biologicheski aktivnykh veshchestv metodom detergentnogo protochnogo analiza.

It was shown that detergent dialysis could be successfully used for liposomal encapsulation of substances belonging to different chemical groups with diverse therapeutic activity such as rifampicin, aclarubicin, amphotericin B, pefloxacin and insulin. Liposome encapsulation of substances poorly soluble or insoluble in aqueous media was likely the most promising. The optimal incorporation depended on both the composition of the lipids forming the liposomes and the properties of the compounds being encapsulated.

[Inactivation of free and liposome-encapsulated aminoglycosides by enzymes of resistant strains of bacteria]. / Izuchenie inaktivatsii svobodnykh i vkliuchennykh v liposomy aminoglikozidov fermentami rezistentnykh shtammov bakterii.

Liposome-encapsulated gentamicin, sisomicin, paromomycin, amikacin, dibekacin and some other aminoglycoside antibiotics were prepared by the method of detergent dialysis. The entrapment level was higher with respect to paromomycin, gentamicin and sisomicin. After a 1-hour incubation of the liposome-encapsulated paromomycin in the presence of a cell-free preparation of 3'-1-phosphotransferase from Escherichia coli there was observed no inactivation of the antibiotic. Free paromomycin incubated under the same conditions in the presence of the enzyme was inactivated by 50 per cent. After a 3-hour incubation of paromomycin in the presence of 3"-1-phosphotransferase the antibiotic activity in the incubation mixture was not detected. The liposome-encapsulated paromomycin preserved about 50 per cent of the activity after its 18-hour incubation in the presence of the enzyme. Inactivation of gentamicin by a cell-free preparation of 2"-adenylyltransferase (Salmonella oranienburg 1376) was also markedly lowered when it was used encapsulated in liposomes.

[Isolation and characteristics of mitochondrial DNA from Acremonium chrysogenum]. / Vydelenie i kharakteristika mitokhondrial'noí DNK iz Acremonium chrysogenum.

Circular mDNAs 26.85 and 26.94 kb in length were isolated from two isogenic strains of A. chrysogenum producing cephalosporin C. The strains differed in antibiotic production capacity. Restriction analysis of the mDNAs was performed with using 6 endonucleases. Comparison of the restriction data revealed identity of mDNAs. A restriction map of the mDNAs was constructed. It is useful as a basis for further studies with molecular cloning.

[Cloning in Escherichia coli of a mitochondrial DNA fragment from Acremonium chrysogenum containing a region responsible for DNA replication]. / Klonirovanie v Escherichia coli fragmenta mitokhondrial'noi DNK Acremonium chrysogenum, soderzhashchego uchastok, otvetstvennyi za replikatsiiu.

A bireplicone plasmid pSU901,4.6 kb in length, was constructed on the basis of plasmid pUC19 and the pstIB fragment, 1.9 kb in length, from mitochondrial DNA of A. chrysogenum. Based on the hybrid plasmid pSU901 and kanamycin resistance determinant, an autonomically replicating vector for A. chrysogenum, a culture producing cephalosporin C, is being constructed.

[Biosynthesis of 14C- and 35S-lincomycin using different sources for the label]. / Biosintez 14C- i 35S-linkomitsina pri ispol'zovanii razlichnykh istochnikov metki

The sources of radioactive labels were chosen for biosynthesis of labeled lincomycin. The levels of the label incorporation into lincomycin were high with all the sources used when the lincomycin-producing organism was cultivated on the synthetic medium as compared to the complex medium. Incorporation of the label into lincomycin was most effective when I14C- or 214C-thyrosine was used as the precursors. These precursors were effective in both the complex and the synthetic media. In production of significant amounts of the labeled lincomycin I14C- or 214C-sodium propionate, I14C- or 142C-sodium acetate and 14C-protein hydrolysate may be used as the sources of the radioactive carbon. In production of 35S-lincomycin K235SO4 may be used as the source of the label. The optimal conditions for biosynthesis of 14C- and 35S-lincomycin were developed (concentration of some components of the medium, time of the label addition and others).

[Biochemical characteristics of the determinants of drug resistance to gentamycin from clinical strains of gram-negative microorganisms]. / Biokhimicheskaia kharakteristika determinantov lekarstvennoi ustoichivosti k gentamitsinu iz klinichekikh shtammov gramotritsatel'nykh mikroorganizmov.

Three to four aminoglycoside inactivating enzymes were detected in cell-free extracts of clinical strains of gram-negative bacteria including Pseudomonas, Escherichia, Serratia, Enterobacter and Klebsiella and in cell-free extracts of their transconjugants. The clinical strains were selected by the feature of gentamicin resistance. All the strains contained AAC(3), APH(3') and APH(3"). In addition to these enzymes 12 out of 25 investigated strains contained AAD(2"). The biochemical characteristics of the gentamicin resistance determinant cloned from the clinical strains on the plasmid vector pUC 19 were studied. By the size of the insertion element pAA4 was the smallest among the constructed hybrid plasmids determining gentamicin resistance. It was shown that just this plasmid determined formation of the two gentamicin inactivating enzymes: AAC(3) and AAD(2") in the transformants carrying it.

[Inhibitor of aminoglycoside phosphotransferases from Micromonospora sp.--a producer of sisomicin]. / Ingibitor aminoglikozid-fosfotransferaz iz Micromonospora sp.--produtsenta sizomitsina.

A substance inhibiting the activity of aminoglycoside-3-'-phosphotransferases of both the actinomycetous and the bacterial origin was detected in the filtrates of the culture fluid and mycelium of Micromonospora sp. producing sisomicin. The activity of the substance against the aminoglycoside inactivating enzymes of different types was studied. It was shown that the quantity of the inhibitor in the culture fluid of Micromonospora sp. correlated with intensity of sisomocin production. Under conditions not providing production of the antibiotic the inhibitory activity was lacking. The inhibitor was purified by ion exchange chromatography on Amberlite CG-50 and KM-cellulose (NH+4 form), gel filtration through Sefadex G-50 and preparative paper chromatography. Stability of the inhibitor at different pH values and different temperatures, its sensitivity to certain enzymes and behaviour in high voltage electrophoresis were studied. The results of ultrafiltration showed that the molecular weight of the inhibitor was relatively low: less than 1000 D.

[Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. Its isolation, purification and properties]. / Aminoglikozid-3'-fosfotransferaza iz Actinomyces fradiae. Vydelenie, ochistka i svoistva.

Aminoglycoside phosphotransferase was isolated from the mycelium of Act. fradiae, the neomycin-producing organism, with paromomycin, neomycin and to a less extent ribostamycin being substrates of aminoglycoside-phosphotransferase. It was purified to homogenous state. The maximum activity of the enzyme preparations was observed at pH 7.7--7.8;KM for neomycin and paromomycin was about 20 micron and KM for ATP was 150 micron. Mg2+ ions were necessary for the enzyme activity. None of the divalent cations tested could replace the magnesium ions in the reaction of phosphorylation catalyzed by the enzyme. High sensitivity to the ionic strength of the buffer was characteristic of the enzyme. It lost about 80 per cent of the initial activity at a concentration of KC1 equal to 1.0 M. The molecular mass of the enzyme from the mycelium of Act. fradiae was determined by the method of gel-filtration through sefadex G-100. It was about 22,000. High stability was characteristic of the enzyme. The fingings indicate that aminoglycoside phosphotransferase from Act. fradiae differs from the described aminoglycoside-3'-phosphotransferases isolated from antibiotic resistant bacteria.

[Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. The identification of its inactivation product]. / Aminoglikozid-3'-fosfotransferaza iz Actinomyces fradiae. Identifikatsiia produkta inaktivatsii.

Neomycin phosphate was obtained as a result of neomycin phosphorylation with aminoglycoside-phosphotransferase from Act. fradiae. It was isolated from the reaction mixture and purified. Successive ion exchange chromatography on columns with Amberlite IRC-50 (NH+4 form), Dowex 1 X 10 (OH- form) and Amberlite CG-50 (NH+4 form) was used for purification of the inactivation product. The findings of the elementary analysis of neomycin phosphate showed the presence of 1 mole of phosphorus per 1 mole of the antibiotic. From the results of the chemical analysis, IR- and NMR-spectrometry neomycin phosphate and neamine phosphate obtained from it by methanolysis were identified as neomycin-3'-phosphate and neamine-3'-phosphate, respectively. The data indicate that the enzyme isolated from Act. fradiae is aminoglycoside-3'-phosphotransferase.

[Streptomycin-3"-phosphotransferases from streptomycin-resistant cells of Escherichia coli strains]. / Streptomitsin-3"-fosfotransferazy iz kletokh ustoichnvykh k streptomitsinu shtammov Escherichia coli.

Streptomycin-3"-phosphotransferases were isolated and purified from E. coli cells containing plasmids 836, pBS52 or R6K, which determine the microorganisms resistance towards streptomycin and dihydrostreptomycin. Phosphorylation of the 3"-hydroxylic group of dihydrostreptomycin was demonstrated by [13C]-NMR spectrometry. It was shown that streptomycin-3"-phosphotransferase, whose synthesis is determined by plasmid 836 (as well as by plasmid R6K), differs from the analogous enzyme, whose synthesis is operated by plasmid pBS52 in some properties, e. g. dependence of the initial reaction rate on concentrations of antibiotics and ATP, pH-optimum, sensitivity to the buffer ionic strength, stability, etc. Besides, the antiserum against streptomycin-3'-phosphotransferase detected by plasmid pBS52 does not produce cross immunological reactions with the other enzyme.

[Production of liposomal preparations of streptomycin and dihydrostreptomycin]. / Poluchenie liposomal'nykh preparatov streptomitsina i digidrostreptomitsina.

The effect of the conditions of the formation of liposomal preparations of streptomycin and dihydrostreptomycin on incorporation of the antibiotics into the liposomes was studied. The liposomes were obtained with the detergent (cholate) method modified by the authors. The modification implies preliminary application of a 20 per cent antibiotic buffer solution on columns for gel filtration of a mixed mycellar antibiotic solution (20 per cent). The volume ratio of the preliminary applied buffer solution and the mixed mycellar solution is higher than 4:1. The new procedure is simple, readily reproduced, providing formation of the liposomal preparations characterized by high levels of the antibiotic incorporation (about 300 micrograms per 1 mg of lecithin). The preparations are stable with regard to the antibiotic release. The liposomes do not aggregate on storage for a long period (more than 6 months). The liposomal preparations thus formed may be useful as intravenous dosage forms of the antibiotics.

[Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]. / Aminoglikozid-3'-fosfotransferaza I iz kletok E. Coli 182 s mnozhestvennoi ustoichivost'iu k aminoglikozidnym antibiotikam.

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.

[Aminoglycoside acetyltransferase from Streptomyces fradiae--a producer of neomycin]. / Aminoglikozid-atsetiltransferaza iz Streptomyces fradiae--produtsenta neomitsina.

An enzyme inactivating aminoglycosides by acetylation was isolated from S. fradiae 918 producing neomycin. By the data on the substrate specificity the enzyme was identified as aminoglycoside-3-acetyltransferase. Localization of aminoglycoside-3-acetyltransferase in the cells of the culture was determined. The enzyme could be detected in the spores and mycelium of S. fradiae already at the early hours of the fermentation process. The enzyme was not detected in the fermentation broth filtrate. The activity of aminoglycoside-acetyltransferase did not depend on the cultivation conditions providing different antibiotic production levels.