SEPTUM FORMATION IN THE DESMID XANTHIDIUM (CHLOROPHYTA): EFFECTS OF CYTOCHALASIN D AND LATRUNCULIN B SUGGEST THE INVOLVEMENT OF ACTIN MICROFILAMENTS

Septum formation and mitosis were investigated by light and electron microscopical techniques as well as through the completion of inhibitor experiments in the unicellular desmid Xanthidium armatum (Bréb.) Rabenh. In untreated cells, numerous endoplasmic reticulum (ER) cisternae permeated the mitotic apparatus and secretory vesicles, and ER formed a band in front of the linearly growing septum, indicating the predetermined direction of septum growth. Under the influence of cytochalasin D (CD), the vesicle/ER band lost its proper orientation, which led to a malformed septum wall; moreover, abnormal septum branches could potentially have developed. Whereas the septum of an untreated cell only grew at its edge, the CD-induced branches (also with a vesicle/ER band in front) represented additional growing zones. These observations indicated that actin filaments were involved in establishing, maintaining, and orienting the "preforming" vesicle/ER band and, thus, the later septum. Latrunculin B (LB) had more severe effects on septum formation than did CD. Only small accumulations of septum material were found at the septum edge, and no aberrant growth of the septum occurred in LB-treated as in CD-treated cells. This could be explained by the more rapid disturbance of all actin-driven processes after LB treatment, which was assumed, because even low concentrations of the drug rapidly inhibited cytoplasmic streaming.     

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat

Summary The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 M, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2–3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4–5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment.Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cisor trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.     

Catalase positive particles from pig lung

Summary In pig lung tissue catalase positive particles (CPs) are abundant especially in type II pneumocytes and in Clara cells.In both cell types they occur circular, oval or elongated membrane profiles surrounding a moderately electron dense matrix lacking a crystalline core. In Clara cells and in part of type II pneumocytes they are located as individual particles without any evident morphological relation to other cell organelles. In part, of type II pneumocytes 5–8 particles are forming a group and their close relation to agranular endoplasmic reticulum cisterns is evident. The particles can be purified from lung homogenates by fractionated pelleting and subsequent rate sedimentation in a sucrose gradient using a zonal rotor. The catalase rich fraction bands in the middle of the gradient whereas cytochrome oxidase and part of the acid phosphatase sediments at its heavy end. A second part of acid phosphatase stays at the light end of the gradient and — according to morphological control — seems to correspond to lamellar bodies of the type II pneumocytes. The purified catalase positive particles do not contain hydroxyacid and d-aminoacid oxidases thought to be characteristic H₂O₂ producing enzymes of peroxisomal systems. The buoyant density of the particles (d=1.195 g/cm³) is lower than that of liver peroxisomes.Cytochemical controls of the peroxisomal pellets exhibit the particles partly uniformly filled with reaction product, partly irregularly stained.     

Disturbance of the secretory pathway inMicrasterias denticulata by tunicamycin and cyclopiazonic acid

Summary Both tunicamycin, an inhibitor of N-linked glycosylation of proteins, and cyclopiazonic acid, which inhibits the Ca²⁺-dependent ATPase in the ER, influence the secretory pathway at the ER level and lead to a cessation of cell growth inMicrasterias. Electron microscopical investigations reveal that the mode of action of the two inhibitors differs. While tunicamycin treatment results in a disintegration of the Golgi bodies into small vesicles, cyclopiazonic acid prevents products being supplied from the ER, resulting in the dilatation of ER cisternae and a reduction in the number of Golgi cisternae, combined with a loss of dictyosomal activity. The disturbed cell wall formation under tunicamycin indicates that N-linked glycosylation of proteins is required for normal cell growth inMicrasterias. Moreover, our studies reveal that changes in cytoplasmic free calcium concentration, as a consequence of ATPase inhibition in the ER by cyclopiazonic acid, may inhibit wall material secretion by interrupting the normal ER-dictyosome association.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. Xanthi and Ky 17 which were cultured in the presence of 100 or 200 g/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.     

Characterization of S-layers from mesophilic bacillaceae and studies on their protective role towards muramidases

High resolution ¹³C NMR combined with chemical analysis were used to study the formation of metabolites from [1-¹³C]-labelled glucose by the salt-tolerant yeast Debaryomyces hansenii after transfer to media containing 8% NaCl. Time course spectroscopy of an aerobic cell suspension showed [1,3-¹³C]glycerol as the predominant end product. Perchloric acid extracts revealed additional less prominent incorporation of label into arabinitol, trehalose, glutamic acid, and alanine. The incorporation into trehalose and arabinitol showed a transient increase after shift to the high salinity medium. It is concluded that glycerol and arabinitol are the major organic solutes in D. hansenii, the production of glycerol being strongly induced by high salinity. Analysis of labelled extracts of D. hansenii after transfer to 8% NaCl media containing [1-¹³C]- or [6-¹³C]glucose, demonstrated that glucose is dissimilated via a combination of the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway, with the former playing a major role in glycerol formation and the latter in arabinitol production. The almost exclusive labelling of C5 of arabinitol from [6-¹³C]glucose indicates that the pathway to arabinitol proceeds via reduction of ribulose-5-phosphate.Abbreviations used NMR nuclear magnetic resonance - EMP Emden-Meyerhof-Parnas - PP pentose phosphate - GAP glyceraldehyde phosphate - DHAP dihydroxyacetone phosphate - ppm parts per million     

The occurrence of various non-ΔF508 CFTR gene mutations among Hungarian cystic fibrosis patients

Summary Cystic fibrosis (CF) is an autosomal recessive disease caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the major mutation (F508) in the Hungarian population is 64%. To identify other common mutations in CF families from Hungary, 30 nonF508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621+1GT), intron 10 (1717–1GA), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. In 6 of the 30 non-F508 CF chromosomes the following mutations were detected: R553X, G542X, 1717–1GA, W1282X, and N1303K. After analysis of the above eight mutations, 30% of CF chromosomes are as yet undefined and further analysis is planned.     

PBN spin trapping of free radicals in the reperfusion-injured heart. Limitations for pharmacological investigations

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trap technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (a_N = 1.39 mT, a_H = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7 ± 0.5 U/ml, p < 0.001) or 5 M vitamin E (11.7 ± 0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7 ± 4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 ± 20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5 ± 15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10 ± 0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6 ± 11.8%; VF 19.42 ± 3.64 min/30 min). The Ca antagonist verapamil (0.1 M), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4 ± 6.8% (p < 0.05), 39.6 ± 12.7% (p < 0.05) and 52.4 ± 2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3 ± 6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.     

Speciation and Phylogenetic Distances within Paxillus s. str. (Basidiomycetes, Boletales)

Abstract: This study aimed to elucidate the phylogenetic relationships within Paxillus 5. str. Collections of the Paxillus involu-tus complex from different habitats have been compared. DNA sequence data were generated from the nuclear encoded 28S gene and the ITS region. Phylogenetic analyses of the two data sets showed three clearly differentiated groups within Paxillus s. str. Collections from gardens and park areas under Betula, Cory-lus, Populus, Quercus and Tub showed to have a very close relationship to the North American species Paxillus vernalis , but are separated from P. involutus collections found in forests as well as from P. filamentosus . Possible taxonomic conclusions are discussed.