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.     

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

Production of rhodomycins inStreptomyces griseoruber 4620

The strainStreptomyces griseoruber 4620 produces, besides the anthracycline antibiotics beromycins, some other anthracyclines of the rhodomycin type. Twelve isolates exhibiting a higher antibiotic activity (up to 2.5×), as compared to the parent strain, were obtained after a spontaneous selection. The following species were isolated from the hydrolysate of mycelial extract: β-rhodomycinone, β-isorhodomycinone, α₂-rhodomycinone and 10-deoxy-β-rhodomycinone, which has not yet been described.     

The effect of ultrasound and its combination with radiation on the genetic material ofSaccharomyces cerevisiae

Ultrasonication at 20 kHz, intensity 35 W/cm² and amplitude 15–25 μm of a diploid strain ofSaccharomyces cerevisiae was found to act as a weak mutagen with maximum efficiency at the 20 % survival of the cells. Under these conditions, the frequency of reversion of the suppressible allele ilv1-92 increased ten times, the frequency of mitotic gene conversion four times. Doses leading to survivals lower than 20 % led to a slight increase in the frequency of cytoplasmic respiration-deficient mutants. Submutagenic doses applied immediately after γradiation or UV light did not substantially increase the effect of these physical agents on the genetic material of the yeast strain investigated. Application of ultrasound prior to UV radiation did not considerably influence the effect of the radiation either.     

The cytotoxic and cytocidal effect of colicin E3 on mammalian tissue cells

The plasma membrane of mammalian cells can mediate the cytotoxic and cytocidal effects of colicin E3. As little as 10² lethal units of purified colicin E3 per cell exert a pronounced cytocidal effect on human epithelial HeLa cells and as little as 10⁴ lethal units per cell also on line L mouse fibroblasts in tissue culture. Cells in complete monolayers are rapidly killed, become spherical and shrink, they are detached from the support and finally autolyzed. The percentage of killed cells in both lines is directly proportional to the multiplicity of colicin used. Theld ₅₀ for HeLa cells is about 30 times lower than for L cells. At the multiplicity of 10⁵ l.u., usually 100 % HeLa cells and 90 % L cells are killed in 2–3 days. Purified colicins E2 and D have no demonstrable cytological effect on HeLa cells, although DNA synthesis in L cells appears to be partly inhibited by colicin E2. The profound effect of colicin E3 on mammalian cells could be interpreted in a similar way as in bacteria,viz. as a specific cleavage of rRNA.     

Long-term change of the littoral Cladocera in the Tatra Mountain lakes through a major acidification event

In our study, we focused on littoral Cladocera living and feeding in shallow shore parts of 46 mountain lakes in the Tatra Mountains (Slovakia and Poland). The studied lakes underwent a major acidification event in the 1980s and are now in the process of recovery. Lakes were divided into three categories based on their sensitivity to acidification: 5 extremely sensitive (ES), 11 acid sensitive (AS), and 30 non-sensitive (NS) lakes. In our study, we included historical data from the literature, and data from sediment core and littoral samples, which together represent the evolution of the littoral communities from a pre-industrial period up to the present. In total, 11 littoral species were found belonging to three cladoceran families. Most of the species were members of the family Chydoridae: Alona affinis, A. quadrangularis, A. rectangula, A. guttata, Acroperus harpae, Alonella excisa, A. nana, Chydorus sphaericus, and Eurycercus lamellatus. One species belonged to each family Daphniidae (Ceriodaphnia quadrangula) and Polyphemidae (Polyphemus pediculus). The most numerous littoral taxa were Alona affinis, Acroperus harpae, and Chydorus sphaericus. All species reacted to decreased pH levels during peak acidification in the 1980s by disappearing from most of the lakes of all categories; the only persisting species was Chydorus sphaericus. Most species returned to the lakes when pH started to increase in the 1990s, although their return was noticeably slower in AS lakes. Alona quadrangularis decreased its distribution range over the studied period; Polyphemus pediculus was mostly detected in the 1910s only. The number of species was highest in all lake categories when dwarf pine was present in the lake catchment. On the whole, the littoral community was richest in NS lakes.     

Element fluxes in watershed-lake ecosystems recovering from acidification: Plešné Lake,the Bohemian Forest, 2001–2005

Fluxes of major ions and nutrients were measured in the watershed-lake ecosystem of a strongly acidified lake, Ple?né jezero (Ple?né Lake), in the Czech Republic in hydrological years from 2001 through 2005. The lake is situated in a Norway spruce forest and has a steep watershed between elevations of 1090 and 1378 m. The average water input and output from the ecosystem was 1372 mm and 1157 mm (37 L km^?2 s^?1), respectively, and the water residence time averaged 306 days. Despite ecosystem recovery from acidification occurring since the late 1980s, the Ple?né watershed was an average net source of 25 mmol SO ₄ ^2? m^?2 yr^?1. Nitrogen saturation of the watershed caused low retention of the deposited inorganic N (< 44% on average) before 2004. Then, the watershed became a net source of 28–32 mmol m^?2 yr^?1 of inorganic N in the form of NO ₃ ^? due to climatic effects (a dry summer in 2003 and a cold winter in 2004) and forest dieback caused by a bark beetle attack in 2004. Nitrogen transformations and SO ₄ ^2? release were the dominant terrestrial sources of H⁺ (72 and 49 mmol m^?2 yr^?1, respectively) and the watershed was a net source of 24 mmol H⁺ m^?2 yr^?1. Ionic composition of surface inlets showed seasonal variations, with the most pronounced changes in NO ₃ ^? , ionic Al (Al_i), and DOC concentrations, while the composition of subsurface inlets was more stable. The in-lake biogeochemical processes reduced on average 59% of the incoming H⁺ (251 mmol H⁺ m^?2 yr^?1 on a lake-area basis). NO ₃ ^? assimilation and denitrification, photochemical and microbial decomposition of allochthonous organic acids, and SO ₄ ^2? reduction in the sediments were the most important aquatic H⁺ consuming processes (358, 121, and 59 mmol H⁺ m^?2 yr^?1, respectively), while hydrolysis of Al_i was the dominant in-lake H⁺ generating process (233 mmol H⁺ m^?2 yr^?1). Photochemical liberation from organic complexes was an additional in-lake source of Al_i. The net in-lake retention or removal of total phosphorus, total nitrogen, and silica were on average 50%, 27%, and 23%, respectively. The lake was a net source of NH ₄ ⁺ due to a cease in nitrification (pH < 5) and from NH ₄ ⁺ production by dissimilation exceeding its removal by assimilation.     

Chlorophyll content of Plešné Lake phytoplankton cells studied with image analysis

Using image analysis, chlorophyll autofluorescence was measured in single cells of green alga Monoraphidium dybowskii and in filaments of cyanobacteria (Pseudanabaena sp. and Limnothrix sp.) in the vertical profile of small acidified mountain lake Ple?né jezero (Ple?né Lake) from May to November of 2003. Cell chlorophyll autofluorescence was converted to cell chlorophyll content using a conversion factor determined by comparing the total autofluorescence of phytoplankton in a microscope field with spectrophotometrically determined total chlorophyll concentration; the conversion factor did not differ between epilimnion (0.5 m depth) and hypolimnion (9 m depth). Vertical patterns of chlorophyll concentration and of cellular chlorophyll content depended on water column mixing: during the period of stable thermal stratification, a metalimnetic peak in total chlorophyll concentration was present and cellular chlorophyll contents in the metalimnion and hypolimnion were notably elevated compared to the surface. Monotonous vertical profiles of both total chlorophyll concentration and cell chlorophyll content were typical for the period of water column overturn. During the stratification period, hypolimnetic Monoraphidium cell chlorophyll content was on average twice as high (maximum difference 2.7-fold) compared to surface values (of 3.2–12.9 fg µm^?3), while in filamentous cyanobacteria (surface cell chlorophyll content of 2.2–13.3 fg µm^?3), the difference was much higher — six-fold on average, with an 11.6-fold maximum value. The values measured with image analysis in 2003 were compared to unpublished values of total phytoplankton biomass-specific chlorophyll concentrations obtained using manual phytoplankton biomass determination and spectrophotometric chlorophyll measurement in 1998 at the same locality. Good agreement was found in seasonal patterns and vertical profiles of chlorophyll between both seasons.