Calcium transport in cells of Streptomyces chrysomallus var. macrotetrolidi-- a producer of macrotetrolide antibiotics]

The dynamics of calcium accumulation by cells in batch cultures and by washed cells was defined with a radiotracer procedure for Streptomyces chrysomallus var. macrotetrolidi producing ionophore macrotetrolide antibiotics. It was shown that macrotetrolides added to the cultivation medium could regulate intracellular contents of calcium by participating in cation transport. Moreover, possible functioning of a Ca-ATPase system for the calcium active transport in the streptomycete cells was demonstrated.     

Early identification of streptothricin group antibiotics]

Methodical approaches to detecting cultures producing streptothricins at the early stages of screening new antibiotics were developed. The approaches are based on chromatographic and electrophoretic mobility of streptothricins and the products of their hydrolysis in the extracts from agar cultures of actinomycetes. Application of the method for screening new antibiotics is illustrated with an example. Nine strains of actinomyces with broad antibacterial spectra isolated from soil samples were studied and 6 of them belonging to 4 species were shown to produce streptothricins in agar cultures. The new streptothricin-producing culture S. roseolilacinus was isolated.     

Use of the method of enriching of soil samples with calcium carbonate for isolation of Actinomyces

Possible application of a modified procedure for treatment of soil samples with calcium carbonate under humid conditions to isolation of actinomycetes was studied. The procedure proved to be rather efficient in regard to increasing the number of the isolates including representatives of rare genera as compared with the routine methods. The number of microorganisms isolated from the soil samples treated with calcium carbonate under humid conditions was 2 orders higher than that from untreated soil samples. 1033 actinomycete cultures were isolated from the soil samples treated in accordance with the above procedure and only 597 cultures were isolated from the untreated soil samples. The antibiotic activity of the isolates was studied and their taxonomic position at the genus level was determined. The preliminary treatment of soil samples equally stimulated development of actinomycetes belonging to different genera. It is advisable that the described procedure for isolation of actinomycetes from soil samples in screening of cultures producing new antibiotics be used in combination with other selective methods of isolation.     

Various methodological approaches in controlled screening of antibiotic producers among the group of Coryneform bacteria]

A methodical system for directed screening of cultures producing broad-spectrum antibiotics among soil saprophytic coryneform bacteria was developed. To isolate such cultures, it was recommended to use the glucose-yeast medium supplemented with malt extract (No. 18/3) and soybean-glucose medium with sodium sulfate and cobalt chloride (No. 20/3). The preliminary alkaline treatment of the soil substrates and the use of acidic soil samples were found to favour isolation of the Mycobacterium type cultures. It was recommended to use gram-negative tests microbes with relatively low antibiotic resistance for screening cultures producing broad spectrum antibiotics. Various agarized and liquid fermentation media were compared in regard to detection of antibiotic activity in the soil coryneform bacteria. The corn medium supplemented with protein-vitamin concentrate, glucose, lactose and starch (No. 116) proved to be the most efficient.     

A system for screening natural immunosuppressors

Criteria for directed screening of antibiotics with immunosuppressive action were defined. The first stage included screening of cultures producing antiaspergillous antibiotics. At the second stage, the antibiotics whose antifungal activity decreases in the presence of insulin (at the background of calcium salts) and erythromycin and increases in the presence of verapamil were selected. The screening of antibiotic-producing cultures among 123 strains of mycelial fungi and 181 strains of actinomycetes resulted in isolation of 3 fungal cultures and 2 actinomycetes which produced antibiotics corresponding to cyclosporine A as evidenced by thin-layer and high performance liquid chromatographies.     

Direct screening for producers of antibiotics among the Micromonospora

A test system was developed for screening organisms producing antibiotics of definite chemical groups or mechanisms of action. The system includes efficient selection of cultures belonging to a definite microbiological taxon (genus Micromonospora), investigation of their biological and taxonomic features, the use of specific selective media with high concentrations of definite antibiotics for isolating antibiotic-producing cultures from natural substrates, the use of specific methods for antibiotic chemical isolation at the initial stages of the screening and chromatographic study of the screened compounds. The system provided efficient screening of valuable antibiotics in a short period.     

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczyński et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108–2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.     

Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

Radio-frequency rectification in electrogenic and nonelectrogenic cells ofChara andNitella

Summary Electrogenic cells ofChara braunii andNitella flexilis were placed in a pulse-modulated radio-frequency electric field of up to 6000 V/m. Their vacuolar resting potentials were found to experience submillivoltdepolarizing offsets (typically 140 V at 250 kHz) which were relatively indepencent of temperature, increased linearly with resting potential from a zero near –210mV, and had a cutoff (putatively due to ion transit times) near 5 MHz. By contrast, nonelectrogenic cells experiencedhyperpolarizing offsets (typically 1100 V at 250 kHz) which increased in magnitude with increasing temperature, were independent of resting potential, and had a transit time cutoff near 10 MHz.The ionic mobilities inferred from these cutoff frequencies are somewhat higher than would be expected for active transport and presumably reflect passive conductance mechanisms which therefore must be presumed different for the electrogenic and nonelectrogenic states.     

Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides.

Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.     

Taxonomic characteristics of soil Mycobacteria and their antibiotic properties]

Morphological, cultural and chemotaxonomic properties of 12 gram-positive soil cultures isolated were studied by using a test system developed for screening the organisms producing broad-spectrum antibiotics among Nocardiaforms (Coryneforms). The cultures were found to belong to Actinomycetales, Nocardioforms, Mycobacteriaceae and Mycobacterium. The saprophytic rapidly growing soil mycobacteria showed antibiotic activity against a large number of gram-positive and gram-negative test microbes including those belonging to Pseudomonas and Proteus resistant to the majority of the antibiotics currently used in medicine.     

Screening of antineoplastic antibiotics using a rapid method of evaluating their cytostatic effect on tumor cells

The study was aimed at development of a rapid method for estimating in vitro cytostatic action of antitumor antibiotics on the basis of intensity of inclusion of labeled precursors into nucleic acids of tumor cells which should be useful in primary screening of antibiotics with potential antitumor activity. The method was applied to substances isolated from 600 cultures of actinomycetes. 35 antibiotics showed antitumor activity; 8 of them were studied in detail. They proved to be novel antibiotics having antitumor activity in animals.     

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.     

Selective transport of Pb2+ and Cd2+ across a phospholipid bilayer by a cyclohexanemonocarboxylic acid-capped 15-crown-5 ether

A cyclohexanemonocarboxylic acid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for Pb2+ and Cd2+, transporting them across a phospholipid bilayer membrane. Transport studies were carried out using 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) vesicles containing the chelating indicator 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2). Data obtained at pH 7.0 using this system, show that the synthetic ionophore transports divalent cations with the selectivity sequence Pb2+ > Cd2+ > Zn2+ > Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. Selectivity factors, based on the ratio of individual initial cation transport rates, are 280 (Pb2+/Ca2+), 62 (Pb2+/Zn2+), 68 (Cd2+/Ca2+), and 16 (Cd2+/Zn2+). Plots of log initial rate versus logM(n+) or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 cation-ionophore complex, but that complexes with other stoichiometries may also be present. The ionophore transports Pb2+ and Cd2+ by a predominantly electrogenic mechanism, based upon an enhanced rate of transport that is produced by agents which dissipate transmembrane potentials. The rate of Pb2+ transport shows a biphasic pH dependence with the maximum occurring at pH approximately 6.5. The high selectivity for Pb2+ and Cd2+ displayed by the cyclohexanecarboxylic acid-capped 15-crown-5 ether suggests potential applications of this ionophore for the treatment of Pb and Cd intoxication, and removal of these heavy metals from wastewater.     

Chloride transport in embryonic cells: effect of ethanol and GABA

Ethanol and GABA (gamma-aminobutyric acid) and their interaction on 36Cl-influx were analyzed in cultured embryonic palate and limb mesenchymal cells in order to determine whether ethanol exerts its teratogenic action through a GABA receptor involved in embryogenesis. Cl- transport in secondary cultures of C57BL/6 palate mesenchymal cells was shown to consist of three systems including the electroneutral Cl-/HCO3- exchange (50%) and Na+/K+/Cl- cotransport (30%) pathways and the voltage-dependent Cl- channel (20%). Treatment with DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or SITS (4-acetamido-4'-isocyano-stilbene-2,2' disulfonic acid) in SWV palate cells inhibited the Cl-/HCO3- exchange pathway, while treatment with DIDS and bumetanide inhibited both the exchange and cation cotransport pathways, the residual Cl- influx inferred to be the electrogenic pathway. Inhibition of Cl- transport by anthracene-9-carboxylic acid confirmed the presence of the electrogenic Cl- pathway. It was observed that the rate of Cl- transport was significantly greater in palate cells of C57BL/6 mice than those of SWV mice. Also the rate of Cl- transport was significantly greater in secondary cultures of palate cells from C57BL/6 mice than from primary cultures of limb cells from the same strain. No evidence could be obtained that ethanol (10 to 100 mM) or GABA (3 X 10(-5) M) or their combination stimulated total Cl- influx in either C57BL/6 or SWV palate mesenchymal cells, putative voltage-dependent Cl- influx in C57BL/6 palate cells, or total Cl- influx in primary cultures of C57BL/6 limb mesenchymal cells.     

Chloride transport pathways and their bioenergetic implications in the obligate acidophile Bacillus coagulans.

The protonophore-mediated collapse of the large delta pH that acidophiles maintain across their cytoplasmic membranes was augmented by the presence of Cl-, and Cl- influx into the cells occurred evidently in response to the protonophore-induced increase in the inside-positive membrane potential (+ delta psi). In respiring cells, the addition of Cl- but not SO4(2-) salts caused a rapid and precipitous decrease in the + delta psi. A Nernstian relationship between the imposed transmembrane K+ gradient and the valinomycin-induced K+ diffusion potentials was observed when everted membrane vesicles were loaded with K2SO4 or KH2PO4 but not when loaded with KCl or KNO3. Thus, electrogenic Cl- transport occurred in Bacillus coagulans. In addition, a nonelectrogenic temperature-sensitive Cl- transport mechanism, with the net Cl- efflux coefficient (PCl-) ranging from 1.5 x 10(-4) to 6.1 x 10(-6) cm/s, accounted for the massive Cl- efflux from Cl(-)-loaded cells. Thus, B. coagulans, despite its dependence on the + delta psi and therefore the need to exclude anions, apparently possesses specific mechanisms for Cl- permeation. Active cells of B. coagulans prevented Cl- accumulation from attaining an electrochemical equilibrium, maintaining a delta micro Cl- of ca. -63 mV. B. coagulans therefore also possesses an energy-dependent mechanism for Cl- exclusion from the cells.     

Activating the NaK pump with monensin increases aminoisobutyric acid uptake by mouse fibroblasts

Monensin rapidly tripled the initial rate and extent of α-aminoisobutyric acid accumulation by Swiss 3T3 cells. This ionophore catalyzes the electroneutral exchange of external Na for cellular protons and stimulates the NaK pump by suppling it with more Na. The stimulation of the NaK pump and α-aminoisobutyric acid uptake exhibited a similar dependence on monensin concentration. Ouabain prevented monensin from increasing α-aminoisobutyric acid transport. Aminoisobutryic acid transport was more than doubled at low doses of monensin that activated the NaK pump by elevating cell Na without significantly changing cell K. The rapid activation of α-aminoisobutyric acid transport is probably due to the hyperpolarizing effect of stimulating the electrogenic NaK pump. The stimulation of the NaK pump is quiescent fibroblasts by serum or growth factors may be sufficient to activate the Na-dependent amino acid transport systems.     

A novel method for the observation of membrane transporter dynamics.

A new method is proposed for measuring the dynamic properties of a membrane transporter by means of steady-state fluxes. Any voltage-sensitive transporter will give a flow of substrate in the presence of a steady-state periodic membrane potential. The periodic steady-state flow, averaged over one period, is a flux that can be measured by traditional steady-state techniques, such as the radioactive tracer method. The average flux, solely due to the periodic field, is described by a set of Lorentzian functions that depend on the applied periodic field amplitude and frequency. The normal mode amplitudes and frequencies of these Lorentzians are model-independent parameters of the transport mechanism. Measurement of the average flux as a function of the applied periodic frequency permits determination of system relaxation times as the reciprocals of the midpoints of the Lorentzian curves, which in turn can be used to estimate individual rate constants of specific models. It was found by simulation of a six-state model of the electrogenic Na+/glucose cotransporter, using published estimates of the model rate constants, that the periodic field effects can be large and rich with measurable details that can be used to study the mechanism thoroughly. The new method serves in this case to complement and expand on the information obtainable by means of the voltage clamp method. It was also found by means of simulations of a nonelectrogenic six-state cotransporter model that experimentally measurable effects are expected and that results can be used to distinguished among alternative kinetic models as well as to estimate individual rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytomorphological and cultural characteristics of tylosin-producing strains with different antibiotic activity]

Cytomorphological and cultural characteristics of highly and low active collection strains of Streptomyces fradiae producing tylosin were studied. The strains were grown on agarized and liquid media. It was shown that unlike the low active strain, the highly active one was less sporogenic, the difference being more pronounced when tylosin was added to the agarized medium. When the strains were grown in the fermentation medium there were detected differences between them in the growth type and microcolony structure at the early stages of the fermentation process. During intensive synthesis of tylosin spheric structures were found to form near the hyphal surfaces. The phenomenon was previously observed in cultures producing other antibiotics. In the low active strain the structures were single while in the highly active strain they formed in large numbers.     

Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.