The cyclosporins.

This review presents the progress and some aspects achieved during recent years with cyclosporin sources, chemistry, biological activities, side effects, biosynthesis and metabolism. Although incomplete the results indicate future research trends and some white spots to be studied in the near future to afford unique insights into cell biology and to improve the search for similar and even more specific agents based on rational drug design.

[Various aspects of regulating the synthesis of ergot alkaloids]. / Nekotorye aspekty reguliatsii sinteza érgoalkaloidov.

The regulation of ergot alkaloid (EA) biosynthesis both at the genetic level and at the level of physiological realization is discussed. The genes involved in EA synthesis are shown to be under rigorous metabolic control. Circumstantial evidence links the initiation of the EA metabolism to changes in some parameters viz morphological feature, concentrations of enzymes and their substrates, contents of nutrients, and external stress.

Physiological controls and regulation of ergot alkaloid formation.

Innovation and technical development of ergot alkaloids (EA) has moved closer to scientific research. Circumstantial evidence presently links the initiation of EA metabolism to changes in a range of parameters--morphology, concentrations of enzymes and their substrates, nutrients and external stress. The biosynthesis of EA begins at the level of the genetic information apparatus and continues at the level of physiological expression. EA and their formation play a role in the physiology of the production organism. Insufficient insight into Claviceps physiology hampers the deployment of computers in the control and regulation of the EA process. Knowledge of physiological controls and genetic manipulation are the principal tools of modern EA production. In principle it is now possible to improve EA yields by a concerted breeding of the ergot fungus by sexual and parasexual genetic engineering.

Formation of cyclosporins by Tolypocladium inflatum.

Submerged cultures of the low-production strain of Tolypocladium inflatum DSM 63544 formed a mixture of cyclosporins (CS) consisting of CS-A, CS-B, "CS-3" and "CS-4". Glucose, sucrose and maltose were highly favored for biomass production but provided a different physiological state necessary for CS biosynthesis. Not only the magnitude of CS production but also the proportion of individual components of the CS mixture were affected by the C source. Intensive CS synthesis was in correlation with the formation of CS-3. Lower yields of CS were accompanied by an increased proportion of CS-A in the CS complex. The best specific production of CS was achieved on the glucose medium, the highest yield of CS-A on the maltose medium. There was no remarkable relationship between the biomass formation and the intensity of CS synthesis.

Formation of conidia in a saprophytic strain Claviceps paspali producing simple lysergic acid derivatives.

A non-mutant saprophytic strain C. paspali which forms conidia both on a solid medium and during submerged fermentation is described. Conidiation proceeded in parallel with culture growth and production of alkaloids. The effect of composition of culture media on the intensity of conidiation is described.

New trends in microbial technology.

Microbial technology includes not only the production of materials in bioreactors, or the production of new catalysts by genetic engineering but extends to aspects of both human and animal health care, waste and pollution management, enhanced oil recovery, mineral leaching, advanced plant breeding, diagnostics and analytical equipment, biosensors, bioelectronics and renewable energy system based on biomass feedstocks. National strategies of industrialized countries are being developed which identify microbial technology as a substantial factor in the attainment of industrial and economic goals. Although extremely promising microbial technology is not a quick fix and its application will only arise as a result of systematic programme of research and development. Such programme requires a broad base of disciplinary underpinning in molecular biology, genetics and bioengineering. The development of expertise of this kind in the tertiary educational institutions is the essential starting point. It should be developed by appropriate programmes and networking systems.

Substrate regulation of elymoclavine formation by some saccharides.

Regulation of the production of clavine alkaloids, especially elymoclavine, by sucrose, maltose, and mixtures of these saccharides was studied in submerged cultures of strains Claviceps purpurea 129/35 and Claviceps sp. SD-58. The data were statistically processed on an EC 1040 computer. Fermentation medium containing sucrose (80 g/l) in combination with glucose (20 g/l) was the best for elymoclavine formation. Retarded release of glucose from maltose increased the formation of elymoclavine and suppressed the synthesis of undesirable extracellular glucans. Carbon source can affect both the total amount of produced alkaloids and the relative proportion of individual clavines in the alkaloid mixture.

Regulation of the immune response by ergot alkaloids.

We tested the effect of 33 ergot alkaloids (natural and semisynthetic) on the induction of antibody formation in isolated spleen cells by antigen in tissue cultures. The results are quantitatively expressed as the number of antibody-forming cells. The group of natural ergot alkaloids (ergopeptines) exerted an effective inhibitory action on the antibody response. Compounds derived from lysergic acid are completely inactive in this respect. A new, very active semisynthetic drug (Table 1, compounds 2) derived from inactive lysergol was described. The compounds interacting with alpha-adrenergic-receptors on lymphocytes (methoxamine, phentolamine, prazosin act synergistically with the inhibitory effect of ergopeptine alkaloids.

Saprophytic production of clavine alkaloids and activity of hydroxymethylglutaryl-CoA reductase.

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids.

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.

Quantitative changes of the alkaloid complex in a submerged culture of Claviceps paspali.

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 +/- 1 degrees C resulted in an increased production of total alkaloids from the original value of 100-200 micrograms/mL to more than 2000 micrograms/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites, 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. alpha-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80% in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids, i.e. lysergamide and the corresponding minor isomers, increased.

A psychrophilic strain Actinoplanes sp. 220.

The strain designated Actinoplanes sp. 220 differed in its characteristics from other strains of the genus Actinoplanes listed in Bergey's Manual (1974). The strain belongs to psychrophilic culture growing within the range of 0-30 degrees C. The optimal temperature for growth on yeast--malt agar is 10-23 degrees C. Cultures transferred at 23 and 28 degrees C differed in morphological and physiological properties, enzyme activity and pigmentation in standard media. Submerged culture transferred at 28 degrees C inhibited growth of Bacillus subtilis ATCC 6633 and ATCC 9945. LL-2,6-Diaminopimelic acid was chromatographically detected in the submerged mycelium of this culture. This compound was not found in the mycelium of the original culture transferred at 23 degrees C. The cultures did not substantially differ in the composition of other amino acids contained in larger quantities in the mycelium.

Fine structural localization of alkaloid synthesis in endoplasmic reticulum of submerged Claviceps purpurea.

Acetyl coenzyme A (CoA) carboxylase (EC 6.4.1.2), an enzyme catalyzing the synthesis of malonyl-CoA, was cytochemically localized in endoplasmic reticulum (ER) of sclerotia-like cells of submerged Claviceps purpurea Tul. producing clavine alkaloids. The enzymic activity was structurally bound in unit membranes of ER strands which, later on, evolved into vacuoles containing lipoprotein material. The reaction product was absent from ER in nonvacuolized filamentous hyphae and ovoid asexual spores containing numerous lipid globules; it was also absent from ER in the mycelium of submerged C. purpurea strain producing no alkaloids. In view of our previous morphogenetic observations and the available biochemical evidence, the observed localization of acetyl-CoA carboxylase was assumed not to coincide with fatty acid biosynthesis but to represent sites of alkaloid synthesis.

A simple method for preparing specimens of Claviceps purpurea for scanning electron microscopy.

The method is a modification of freeze-drying and includes also a simultaneous coating during rotation. It utilizes a common vacuum coating apparatus with a modified miniature electric motor. The cells of C. purpurea in the resulting specimen retain their original shape.

Microcycle sporulation in the Claviceps purpurea 244.

The mutant strain Claviceps purpurea 244 forming hyphae composed mainly from sclerotium-like cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 x 6.5 micrometer). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 microgram/ml clavins, with elymoclavin predominating (82%). The parent strain Claviceps pururea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85%) at a concentration of 4 mg/ml.

Variability of requirements to fermentation media in Claviceps purpurea strains.

The alkaloid-synthesizing activity of the saprophytic Claviceps purpurea strains was variable. This fact was partly due to different requirements of the cultures to nutritional conditions. Not only single strains, but also formation of intra- and extracellular alkaloids differed in this respect. In case of extracellular alkaloids, the influence of medium was more expressive. A positive correlation between accumulation of cell polysaccharides and the level of intracellular alkaloids was evident. Constant or decreasing cell proteins were favourable for alkaloid formation.

The relationship between conidiation and alkaloid production in saprophytic strains of Claviceps purpurea.

A direct relationship between alkaloid synthesis and conidia formation was observed in a series of mutants of the saprophytic fungus Claviceps purpurea 129 which produces high quantities of clavine alkaloids (4500 microgram-ml-1). Sclerotia-like mutants forming mostly arthrospores and only a low number of conidiospores were exceptional in that they were characterized by an inverse relationship between alkaloid synthesis and conidial formation. Conidiospores of the parent strain 129 differed both in their size and ultrastructure. They include oval microconidia (2 X 3.5 micrometer) and prolonged macroconidia (2 X 10.5 micrometer). Both conidial types originated on short lateral sporophores terminated by phialids. Most conidia were characterized by electron-dense cytoplasm with a number of lipidic inclusions; a minority of conidia contained granular cytoplasm and vacuoles with membrane invaginations. Bicellular conidia were only rarely observed.