Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Die submontanen krautreichen Buchenwälder auf Silikatböden der westlichen Tschechoslowakei

Die submontanen krautreichen Buchenwälder auf Silikatböden in der westlichen Tschechoslowakei (Tschechische Sozialistische Republik) zerfallen in vier Assoziationen: 1.Tilio cordatae-Fagetum Mráz 1960 em.Moravec 1977, 2.Tilio platyphylli-Fagetum Klika 1939, 3.Melico-Fagetum Seibert 1954 und 4.Carici pilosae-Fagetum Oberdorfer 1957. DasTilio cordatae-Fagetum ist an niedrigere Bergländer Mittel-, Süd- und Westböhmens gebunden. DasTilio platyphylli-Fagetum ist eine lokal bedingte Assoziation des Westteils des Gebirges ?eské st?edoho?í. DasMelico-Fagetum kommt in Nord- bis Ostböhmen und Nord- und Mittelmähren vor. DasCarici pilosae-Fagetum ist auf die Karpaten beschränkt (ausnahmsweise greift es auf den Ostrand des Böhmischen Hochlandes über). Die Buchenwälder des Hügellandes bei Jevany (Mittelböhmen) und des Gebirges ?elezné hory gehören, trotz dem Vorkommen in submontanen Lagen, demDentario enneaphylli-Fagetum Oberdorfer ex W. etA. Matuszkiewicz 1960 an. Die Zusammensetzung der Assoziationen ist durch pflanzensoziologische Tabellen belegt und ihre Verbreitung auf einer Punktkarte erfasst.     

Kinetics of uridine uptake and incorporation into RNA in tobacco pollen culture

The capacity of pollen tubes to utilize exogenous uridine during 8 h of cultivation in shaken suspension in a sugar-mineral medium was examined by continuous and pulse labelling with³H-uridine. The increase of uptake with increasing concentration of the nucleoside indicated a saturable transport system with an approximate K_m of 9.4 × 10⁻⁶ M and 12.5 × 10⁻⁶M as determined in 1-h and 6-h cultures, respectively. Maximal uptake took place at the beginning of germination reaching a rate of about 2 nmol h⁻¹ per 1 mg of dry pollen at 0.1 mM external uridine. The uptake activity decreased with the time of pollen tube growth to less than one third during the 8-h cultivation period. Moreover, the level of radioactivity taken up initially decreased later on during continuous cultivation in the presence of³H-uridine. The uptake took place against a concentration difference and the onset and rate of uridine release depended on its exogenous concentration. The activity of the nucleoside incorporation into RNA increased during the first 4 h of cultivation, decreasing later on. The proportion of RNA radioactivity in continuously labelled pollen tubes grew steadily during 6 h and reached 2.5% with respect to soluble pool at 0.4 μM uridine. The time course of RNA labelling was independent of uridine concentration within the range of 0.4 μM to 40 μM but this concentration rise resulted in an about fiftyfold increase of the total amount of external uridine incorporated.     

Stoicheiometry of dicyclohexylcarbodiimide-ATPase interaction in mitochondria

1. The oligomeric dicyclohexylcarbodiimide (DCCD)-binding protein of mitochondrial ATPase was studied using (a) the relationship between [¹⁴C]DCCD binding and inhibition of ATPase activities and (b) the analysis of the kinetics of inhibition. 2. The [¹⁴C]DCCD binding to bovine heart mitochondria is linearly proportional to the inhibition of ATP hydrolysis up to a 50% decrease of the original activity resulting in 0.6 mol DCCD bound covalently to the specific inhibitory site (Hous?t?k, J., Svoboda, P., Kopecký, J., Kuz?ela, S?. and Drahota, Z. (1981) Biochim. Biophys. Acta 634, 331–339) per mol of the fully inhibited enzyme. 3. Kinetics of the inhibition of both the ATPase activity (heart and liver mitochondria) and ADP-stimulated respiration (liver) reveal that 1 mol DCCD per mol ATPase eliminates both the synthetic and the hydrolytic activities. It is inferred that the activity-binding correlation underestimates the number of DCCD-reactive sites. 4. The second-order rate constant of the DCCD-ATPase interaction (k) is inversely related to the concentration of membranes, indicating that DCCD reaches the inhibitory site by concentrating in the hydrophobic (phospholipid) environment. 5. At a given concentration of liver mitochondria, comparable k values are obtained both for the inhibition of ATP hydrolysis (k=5.35·10²M^?1·min^?1) and ADP-stimulated respiration (k=5.67·10²M^?1·min^?1). 6. It is concluded that both the synthetic and the hydrolytic functions of ATPase are inhibited via a common single DCCD-reactive site. This site is represented by one of the several polypeptide chains forming the oligomer of the DCCD-binding protein. The inhibitor-ATPase interaction does not exhibit cooperativity, indicating that the preferential reactivity towards DCCD is an inherent property of the inhibitory site.     

Transformation ofStreptomyces lincolnensis protoplasts with plasmid vectors

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.     

The effect of mineral nutrition on the growth and maintenance components of respiration during heterotrophic growth of barley seedlings

Spring barley seedling were grown in the dark for 21 d and respiration rates of the whole plant (including the seed), of the shoots, and of the roots were determined. A function describing the growth and maintenance components of respiration was interpolated through the experimental points and its parameters in plants under different mineral nutrition were compared. The plants grown in a complete nutrient solution showed the highest growth rate in the initial phase of development and thus reached the maximum respiration rate earlier than plants in the other variants. The highest proportion of substrate was respired in the shoot. Plants grown under deficiency of phosphorus and magnesium had a slower respiration rate than plants grown in the complete nutrient solution (NP), whereas the amount of respired substrate in plant parts was similar to that recorded in the NP plants. Plants grown in distilled water showed the lowest growth efficiency and respirated the highest proportion of substrate in the root.     

Reversal of IAA-induced inhibition of flowering by aminoethoxyvinylglycine inChenopodium

Aminoethoxyvinylglycine (AVG) applied as a droplet (3 l, 0.1 mM) to the plumule of seedlings of both the short-day plantChenopodium rubrum and the long-day plantChenopodium murale counteracted to a great extent or even canceled the inhibition of flowering due to exogenous indole-3-acetic acid (IAA). This effect was more pronounced with the two substances administered simultaneously than with later application of AVG alone. AVG by itself in some cases promoted the percentage of flowering in bothChenopodium species. Application of IAA to the shoot apex was shown to elevate ethylene production in both species, whereas application of AVG alone was shown to suppress it. Thus, ethylene may be considered an active agent of flowering inhibition brought about by IAA application.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head

Summary Four monoclonal antibodies that discriminate between structural domains of alpha-(TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of -tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon.