Permeabilization ofNeurospora crassa hyphae with toluene-ethanol and filipin

Young hyphae ofNeurospora crassa were made permeable to UDP-glucose and trypan blue by treatment with toluene-ethanol and filipin. Less than 2% of treated cells survived treatment with 8% and 16% toluene-ethanol, while 25% survived treatment with 4% toluene-ethanol. Similarly, 98% of treated cells were killed by treatment with 16 g/ml filipin. Electron microscopy revealed that toluene-ethanol-treated cells lost pieces of plasma membrane and contained a number of vacuole-like structures; filipin-treated cells were less affected. Both filipin- and toluene-ethanol-treated cells were able to incorporate UDP-glucose into insoluble material (likely glycogen and glucan).     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

Interaction of Zn2+ and Cu2+ ions with glyceraldehyde-3-phosphate dehydrogenase from bovine heart and rabbit muscle.

1. Binding of Zn2+ and Cu2+ ions to GAPDHs from bovine heart and rabbit muscle resulted in a partial loss of enzymatic activity of both enzymes, in a time and metal ion concentration dependent manner. Cu2+ ions caused a much larger decrease of the activity than Zn2+ ions. 2. Addition of NAD+ or EDTA to either enzyme resulted in a protective effect on GAPDH activity. A similar protective effect was observed following addition of 2-mercaptoethanol to the enzyme solution. 3. The association constant for GAPDH-Zn2+ complex, calculated from equilibrium dialysis data, was 0.9 x 10(4) M-1 for the bovine heart GAPDH and 1.3 x 10(4) M-1 for the rabbit muscle enzyme. The association constant for GAPDH-Cu2+ complex was the same for both enzymes, 11.3 x 10(4) M-1. 4. Equilibrium dialysis data also revealed that in either enzyme the specific sites, binding the metal ions, are identical or very similar, and independent from each other. They are situated in the most conserved part of the enzyme molecule. 5. Some zinc was found in GAPDH preparations from bovine heart. It is discussed if Zn2+ ions could have a kind of modulation effect on GAPDH activity.     

Preliminary crystallographic studies of urease from jack bean and from Klebsiella aerogenes.

Ureases from both jack bean (Canavalia ensiformis) seeds and Klebsiella aerogenes have been crystallized by the hanging drop method. The plant-derived urease crystals are regular octahedra analogous to those obtained by Sumner. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group F4(1)32, with a = 364 A, and appear to contain one or two subunits in the asymmetric unit. Using a synchrotron source, the crystals diffract to near 3.5 A resolution. Crystals of urease from K. aerogenes belong to the cubic space group I23 or I2(1)3, with a = 170.8 A and appear to contain a single catalytic unit per asymmetric unit. The crystals diffract to better than 2.0 A resolution and are well suited for structural analysis.     

Sex-based differences in vigor and site preferences of Juniperus communis subsp. nana

Dioecious alpine juniper has been influenced by human impacts, management and climate changes; hence, its present populations are remnant fragments of its former distribution in central Europe. Complex environmental shifts have had fatal consequences for growth, reproduction and health of juniper and hence for its population structure. We asked the questions: 1) what was the population size structure, the sex ratio and the health status of individuals? 2) How were the population parameters linked with the environmental conditions and surrounding vegetation? The study area was close and above the upper forest limit in central Europe, the Hrubý Jeseník Mts. The parameters of each juniper individual and its environmental conditions were obtained, Ellenberg's indicator values and habitat categories were also determined. Proportions of sex categories, shrub size and environmental conditions were investigated to assess relationships between the population categories and environmental factors. We revealed linkage among the shrub size, health vigor, vegetation cover, a habitat and environmental factors. While there was equal ratio of females and males (1:1), small individuals of undetermined sex predominated that reducing the effective population size. Juniper health vigor was associated with surrounding vegetation cover. Individuals of undetermined sex were smaller than those of remaining sex categories and prefer specific habitats at higher elevation, underlining the effect of abiotic conditions on sex performance and ratio. The observed pattern, which was attributed to shift in land-use practices in the middle of 20th century and climate changes, could be improved by management. We recommended experimental local grazing and mowing accompanying by profound and continuing assessment of interaction among environmental factors and juniper performance.     

Effect of angiotensin II on protein phosphorylation in PC12 cell line

Two subtypes of angiotensin II receptors have been characterised so far: AT1 and AT2. In PC12W pheochromocytoma cells, only AT2 receptors have been found (acting probably through G1 proteins or via G protein-independent mechanism). Here, dynamic changes in phosphorylation pattern in PC12W cells upon induction of angiotensin II and under influence of redox agents were investigated. PC12W pheochromocytoma cell line was preincubated with angiotensin II, then incubated with redox agents. After lysis the cells were subjected to Western-Blotting technique with antiphosphotyrosine and anti-ERK2 antibodies, as well as phosphotyrosine phosphatases and kinases activity was measured. Angiotensin II through its AT2 receptor induced dephosphorylation of tyrosines of the proteins in the range of 60 to 150 kD in PC12W cells. The obtained phosphorylation pattern suggests that AT2 receptors may act comparably to leukocyte CD45 receptor pathway. Treatment of PC12W cells with H2O2 resulted in significant decrease in phosphotyrosine phosphatases activity. It could be assumed that signal transduction based on protein phosphorylation might be controlled by cellular redox mechanisms.     

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.