Carbohydrate and pyruvic acid degradation pathways in Fusidium coccineum strains with varying levels of antibiotic synthesis]

A number of enzymes and reactions of glycolysis, pentose-phosphate cycle and degradation of pyruvic acid in strains of F. coccineum with various levels of antibiotic production was studied comparatively. The experiments showed that highly productive strains were characterized by higher activity of the NADP-deficient enzymes of the pentoze-phosphate cycle as compared to the low active strains. The activity levels of glycolytic enzymes, such as fructose-diphosphate-aldolase and 3-phosphoglycerolaldehydehydrogenase did not practically differ. Significant differences were found in the reactions of puryvic acid degradation: the activity of cytoplasmic pyruvatedecarboxylase in the mutant with high antibiotic production level was lower than that in the low productive strain, while oxidation of the pyruvate of the mitochondrial fraction was on the contrary more intensive than in the highly productive strain. Therefore, metabilism in the strains studied was characterized by ever-increasing biochemical changes with an increase in their antibiotic productivity. Lowering of the growth rate of the mutants as their capacity for antibiotic supersynthesis increased and subsequently the anabolic processes became more intensive was accompanied by increasing derepression of the key enzymes of carbohydrate metabolism and in particular NADR-deficient dehydrogenase of the pentose cycle and pyruvatedehydrogenase, significant for fusidin biosynthesis and providing production of the antibiotic of steroid nature by cofactor NADP-H and acetyl-KoA, the primary precursor.     

Characteristic features of protoplast formation and regeneration in Fusidium coccineum

The methods for preparation and regeneration of protoplasts were tested with respect to the strains of F. coccineum markedly differing in their capacity for antibiotic production, sporulation and the growth rate. It was found that the substrate used for the culture growth had a significant effect on the cell wall and sensitivity of the mycelium to lytic enzymes. An enzyme from Hellix pomatia and its combination with lysozyme were used for lysing the culture. The cytological investigation of the time course of the culture lysis revealed a stage-by-stage pattern of protoplast formation by means of fragmentation of the hyphal contents till a ball was formed. Two to 4 protoplasts differing in their size and structure were formed within a cell. The pH value and osmotically stabilizing component had some effect on the rate of protoplast formation. Highly productive strains were characterized by formation of protoplasts heterogenous in their size and by decreased frequency of regenerations. The enzyme-free protoplasts preserved their viability and capacity for germination in osmotically stabilizing media for 72-96 hours of storage at 4 degrees C. On solid media the regeneration frequency reached 38 per cent. The regenerated cells formed colonies morphologically similar to those of the intact culture.     

Relationship between intracellular pH and antibiotic biosynthesis in Fusidium coccineum

A correlation between the value of the intracellular pH and the biosynthesis of fusidic acid was studied by ³¹P-NMR spectroscopy in two strains of the fungus Fusidium coccineum. One of the strains was highly active and the other strain had low activity with respect to the antibiotic production. The position of the orthophosphate resonance in the ³¹P-NMR spectra was considered as a measure of the intracellular pH. In the cells of the highly active strain pH was in the range 7.0–7.5 in the cytoplasm and 6.1–6.25 in the vacuoles. In the cells of the strain with low activity was in the range 7.3–7.9 in the cytoplasm and 6.0–6.2 in the vacuoles. During high antibiotic productivity, the intracellular pH in the highly active strain full sharply, while in the less active strain it effectively did not change. This suggested that the change in the intracellular pH was responsible for the action of the enzymes in the cells and could be a factor defining the function of the cyanide-resistant respiration pathway and consequently the synthesis of fusidic acid in F. coccineum. *** DIRECT SUPPORT *** AG903062 00009     

Morphofunctional characteristics of the development of strains of Fusidium coccineum differing in antibiotic activity in stab cultivation

The object of the work was to study the morphological and functional characteristics of Fusidium coccineum strains producing fusidic acid and differing in the antibiotic activity. The high metabolic activity of the culture is accompanied by the following morphological characteristics: the cells are rich in ribosomes and mitochondria, they have early vacuolisation, are filled with lipid granules, and then the mycelium undergoes autolysis. As strains with a high activity grow, the structure of the cells changes, the number of ribosomes and mitochondria falls down, and the latter are destroyed. For a long time, the cells contain electron-dense granular structures limited with the membrane and capable of transformation into lipid granules and membranous structures. As was shown by cytochemical studies, the structures have not merely proteins and lipids, but also phosphorus compounds. Their functional role in the fungal metabolism is discussed. As soon as super-synthesis of fusidic acid commences, the cells of the highly active strains are filled with lipid granules associated possibly with the steroid antibiotic. These formations are released from the cell during local lysis of the cell wall and the cytoplasmic membrane.     

Sporogenesis, antibiotic biosynthesis and the systematic position of Fusidium coccineum

Cytomorphological study of the wild and isogenic improved strains of F. coccineum revealed that with increasing of the antibiotic potency the character of the strain sporulation changed towards predominant formation of the spore "heads" which is characteristic of the sporogenesis of the genus Acremonium. These data allow one to revise the systematic position of the strains of F. coccineum and to classify them according to Gamsas belonging to Acremonium designated as Acremonium fusidioides. The criteria of the highly potent strains of this organisms are the following: formation of the spore "heads", changes in the event sequence during sporulation, decreasing of the spore index L/B with an increase in the strain potency. These criteria may be used in further improvement on the organism.     

Characteristics of fucidin biosynthesis by a Fusidium coccineum 257 A strain

The physiological features of Fusidium coccineum, strain 257 A, an organism producing fusidin were studied. It was found that increased concentrations of the carbon sources in the medium stimulated production of fusidin, while an increase in the content of various forms of nitrogen differently affected the level of the antibiotic viosynthesis: high concentrations of the amino acid-peptide form of nitrogen of corn-steep liquor decreased, while the protein form of nitrogen was associated with consumption of the significant part of carbon in the medium for formation of the fungus mycelium. Therefore, the concentration of the easily mobilizing forms of nitrogen may be considered as a regulator of the growth process of F. coccineum 257 A and production of fusidin by it.     

Respiratory system of Fusidium coccineum and regulation of biosynthesis of fusidic acid and sporogenesis

Summary The respiratory system, sporulation, and dynamics of alkaline protease formation were studied in three strains of the fungus Fusidium coccineum, differing in their ability to make antibiotics.Oxidative phosphorylation provided most of the energy in high and low activity strains and their respiratory activity was exclusively related to mitochondria functioning.In inactive and low activity strains, the terminal oxidation of reduced equivalents proceeds mainly by the respiratory chain with cytochrome oxidase as the terminal component. In the high activity strain there is a cyanide-resistant alternative pathway which is parallel to the classical cytochrome chain. The complete transition to the use of this pathway coincides with the stage of maximum antibiotic biosynthesis. The induction of the alternative pathway in the high activity strain was not concerned with the inhibition of the cytochrome site of the respiratory chain by fusidic acid. It was shown that the quantity of the antibiotic synthesized and the character of cellular differentiation can be altered by changing the oxidation pathwats used with inhibitors such as chloramphenicol and salicyl hydroxamate.We suggest that there must be common regulation of antibiotic formation, sporulation and induction of the alternative oxidation pathway.     

Characteristics of the oxidative metabolism in strains with varying levels of fucidin biosynthesis]

Oxidative capacity of the fusidin-producing strains with various biosynthetic activity was studied comparatively. The studies showed that by their capacity to oxidize pyruvate and some metabolites of the tricarboxylic acid cycle (acetate, succinate, malate) the strains were arranged in the order reverse to their antibiotic activity. Such regularity was observed during the whole fermentation process and was most pronounced by the 3rd and 4th days (beginning of the idiophase). The rate of glucose oxidation was higher in more active strains. The same regularity was noted in the 2nd phase of the strain development associated with beginning of fusidin biosynthesis. In the 1st phase (the 1st and 2nd days) the strains almost did not differ by their capacity to oxidize glucose. By oxidation of phosphorylated ethers of carbohydrates (glucose-6-phosphate and fructoso-6-phosphate) the strains did not differ. Various fusidin-producing strains oxidized NAD-N and NADP-N approximately with the same rate. It is supposed that mutations leading to increased antibiotic production are associated with changes in acetate metabolism in the direction of more intensive biosynthesis of isoprenoid compounds, potential precursors of the fusidin molecule.     

Energy metabolism by splanchnic tissues of mature sheep fed varying levels of lucerne hay cubes

The objective of this study was to determine the pattern of energy metabolites net flux across the portal-drained viscera (PDV) and total splanchnic tissues (TSP) in mature sheep fed varying levels of lucerne hay cubes. Four Suffolk mature sheep (61.4 ± 3.6 kg BW) surgically fitted with multi-catheters were fed four levels of dry matter intake (DMI) of lucerne hay cubes ranging from 0.4- to 1.6-fold the metabolizable energy (ME) requirements for maintenance. Six sets of blood samples were simultaneously collected from arterial and venous catheters at 30-min intervals. With increasing DMI, apparent total tract digestibility increased linearly and quadratically for dry matter (P < 0.05), quadratically (P < 0.05) with a linear tendency (P < 0.1) for organic matter and tended to increase quadratically (P < 0.1) for NDF. PDV release of volatile fatty acids (VFA) and β-hydroxybutyric acid was relatively low at 0.4 M and then linearly increased (P < 0.05) with increasing DMI. Net PDV flux of non-esterified fatty acids showed curvilinear decrease from 0.4 to 1.2 M and then increased at 1.6 M. The respective proportions of each VFA appearing in the portal blood differed (P < 0.05) with DMI and this difference was more obvious from 0.4 to 0.8 M than from 0.8 to 1.6 M. Heat production, as a percentage of ME intake (MEI), decreased linearly (P < 0.05) with increasing DMI accounting for 37%, 21%, 16% and 13% for PDV and 62%, 49%, 33% and 27% for TSP at 0.4, 0.8, 1.2 and 1.6 M, respectively. As a proportion of MEI, total energy recovery including heat production, decreased linearly with increasing DMI (P < 0.05) accounting for 113%, 83%, 62% and 57% for PDV and 140%, 129%, 86% and 83% for TSP at 0.4, 0.8, 1.2 and 1.6 M, respectively. Regression analysis revealed a linear response between MEI (MJ/day per kg BW) and total energy release (MJ/day per kg BW) across the PDV and TSP, respectively. However, respective contributions of energy metabolites to net energy release across the PDV and TSP were highly variable among treatments and did not follow the same pattern of changes in DMI.     

Biological properties of clinical staphylococcal strains with a varying number of antibiotic resistance markers]

The study of a number of biological properties of 1881 clinical strains of Staphylococcus showed that in the group of the antibiotic resistant staphylococci there was a tendency for different manifestation of some biological properties depending on the number of the resistance determinants. The staphylococcal strains resistant to 5--7 antibiotics differed from those resistant to a less number of the drugs by greater manifestation of the pathogenicity properties: lecithinase, hyaluronidase and hemolytic activity.     

Energy metabolism in osteoclast formation and activity

Osteoclastogenesis and osteolysis are energy-consuming processes supported by high metabolic activities. In human osteoclasts derived from the fusion of monocytic precursors, we found a substantial increase in the number of mitochondria with differentiation. In mature osteoclasts, mitochondria were also increased in size, rich of cristae and arranged in a complex tubular network. When compared with immature cells, fully differentiated osteoclasts showed higher levels of enzymes of the electron transport chain, a higher mitochondrial oxygen consumption rate and a lower glycolytic efficiency, as evaluated by extracellular flux analysis and by the quantification of metabolites in the culture supernatant. Thus, oxidative phosphorylation appeared the main bioenergetic source for osteoclast formation. Conversely, we found that bone resorption mainly relied on glycolysis. In fact, osteoclast fuelling with galactose, forcing cells to depend on Oxidative Phosphorylation by reducing the rate of glycolysis, significantly impaired Type I collagen degradation, whereas non-cytotoxic doses of rotenone, an inhibitor of the mitochondrial complex I, enhanced osteoclast activity. Furthermore, we found that the enzymes associated to the glycolytic pathway are localised close to the actin ring of polarised osteoclasts, where energy-demanding activities associated with bone degradation take place. In conclusion, we demonstrate that the energy required for osteoclast differentiation mainly derives from mitochondrial oxidative metabolism, whereas the peripheral cellular activities associated with bone matrix degradation are supported by glycolysis. A better understanding of human osteoclast energy metabolism holds the potential for future therapeutic interventions aimed to target osteoclast activity in different pathological conditions of bone.     

Cortical pelvic strains with varying size hemiarthroplasty in vitro

Four human pelves were dissected of soft tissues and instrumented with rosette strain gages. The pelves were left intact (not separated at the pubic symphysis or sacroiliac joints) although each of the eight hemipelves was tested separately. The pelves were loaded to simulate single leg stance with use of a wire mesh cemented to the wing of the ilium, representing the abductor muscles. Loading was carried out with the intact hip joint and 1 mm undersized, correct sized, and 1 mm oversized hemiarthroplasty. The correct sized component produced a strain pattern closet to normal. The oversized component was associated with an increase in strain at the medial acetabular dome.     

Energy metabolism in wheat root cells under modification of plasma membrane permeability by antibiotic nystatin

This paper reports changes in ion transport and energy metabolism of plant cells during short- and long-term expositions, resp., to antibiotic nystatin, which is known to specifically bind with plasma membrane sterols to form channels. The excised roots of 5 days old wheat seedlings were used as a model system in this research. It has been shown that treatment of excised roots with nystatin leads to activation of energy metabolism expressed as an increase of respiration and heat production by root cells. Furthermore, in the presence of nystatin increased pH of incubation medium, plasma membrane depolarization and a significant loss of potassium ions were observed. Nystatin-induced stimulation of respiration was prevented by malonate, an inhibitor of succinate dehydrogenase, electron acceptor dichlorophenolindophenol, and AgNO3, an inhibitor of H(+)-ATPase. Based on the data obtained it can be suggested that nystatin-induced stimulation of respiration is related to electron transport activation via mitochondrial respiratory chain, and is connected with activation of plasmalemma proton pump. Moreover, nystatin-induced increase of oxygen consumption was prevented by cerulenin, an inhibitor of fatty acid and sterol synthesis. This indicates that additional sterols and phospholipids may be synthesized in root cells to "heal" nystatin-caused damage of plasma membrane. A supposed chain of events of cell response to nystatin action may by as following: formation of nystatin channels-influx of protons--depolarization of plasmalemma-efflux of potassium ions-disturbance of ion homeostasis--activation of H(+)-ATPase work-increase in energy "requests" for H(+)-ATPase function--increase in the rate of oxygen consumption and heat production. The increased energy production under the action of nystatin, may provide the work of proton pump and synthesis of sterols and phospholipids, which are necessary for membrane regeneration.     

Energy charge and emergence of the coleoptile and radicle at varying oxygen levels in Echinochloa crus-galli

The role of protein synthesis in senescence and in the inhibition of senescence by light and kinetin was studied in barley ( Hordeum vulgare L. cv. Hassan) leaves with different inhibitors of protein synthesis. A comparison of the actions of D- and L-chloramphenicol was made to compensate for the effects of D-chloramphenicol not mediated by inhibition of protein synthesis. The involvement of phytochrome was also studied. The results suggest that: 1) cytoplasmic protein synthesis is required for senescence in the light and in the dark; 2) chloroplasts, in the dark, synthesize protein which accelerates senescence; 3) kinetin inhibits the synthesis by chloroplasts of senescence-accelerating protein; 4) light changes the type of protein synthesized by chloroplasts from those accelerating to those retarding senescence; and 5) lightretar-dation of senescence is mediated by phytochrome and, probably, by photophos-phorylation.     

Energy metabolism of isolated hepatocytes at various levels of oxidative phosphorylation uncoupling]

The energy metabolism changes in isolated hepatocytes at different levels of proton conductivity of cellular membranes were studied. The low doses of the uncoupler which increased hepatocyte respiration rate but did not markedly affect the mitochondrial potential caused: the reduction in total adenine nucleotide contents (ATP + ADP + AMP), the oxidation of mitochondrial NADH, the increase in the rates of glycogenolysis and net flux via phosphofructokinase without any changes in the rates of glucose, lactate and pyruvate accumulation. High doses of the uncoupler which eliminated completely oxidative phosphorylation decreased Atkinson's energy charge down to 0.5, reduced cytoplasmic NADH, induced a further increase in the glycogenolysis rate, increased the rates of glucose and lactate accumulation, heightened glucose-6-phosphate content and lowered contents of 3-phosphoglycerate and 2-oxoglutarate.