M-MuLV-induced leukemogenesis: Integration and structure of recombinant proviruses in tumors

M-MuLV-specific DNA probes were used to establish the state of integration and amplification of recombinant proviral sequences in Moloney virus-induced tumors of Balb/Mo, Balb/c and 129 mice. The somatically acquired viral sequences contain both authentic M-MuLV genomes and recombinants of M-MuLV with endogenous viral sequences. All reintegrated genomes carry long terminal repeat (LTR) sequences at both termini of their genome. In the preleukemic stage a large population of cells exhibiting a random distribution of reintegrated M-MuLV genomes are seen, but during outgrowth of the tumor, selection of cells occurs leaving one or a few clonal descendants in the outgrown tumor. In this latter stage recombinant genomes can be detected. Although these recombinants constitute a heterogeneous group of proviruses, characteristic molecular markers are conserved among many individual proviral recombinants, lending credence to the notion that a certain recombinant structure is a prerequisite for the onset of neoplasia. The structure of these recombinants shows close structural similarities to the previously described mink cell focus-inducing (MCF)-type viruses.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Adhesion of canine and human uropathogenicEscherichia coli andProteus mirabilis strains to canine and human epithelial cells

Escherichia coli strains causing urinary tract infections in dogs produce fimbriae composed of fimbrial subunits closely related to the F12 and F13 fimbriae of human uropathogenic strains [4]. The adhesins carried by the fimbriae of human and canine isolates differ, however, as concluded from a different hemagglutination pattern and from the fact that the dog strains do not agglutinate latex beads coated with P-fimbriae receptor. This possible difference in adhesive specificity was confirmed by experiments in which the adhesion of human and dog isolates to dog kidney epithelial cells (MDCK cells) and human bladder epithelial cells (T24 cells) was compared. Dog uropathogenic strains, in contrast to human uropathogenicE. coli strains, adhere to MDCK cells but hardly to T24 cells. Adhesion to MDCK cells correlates with the presence of F12 or F13 fimbriae on the dog strains. These results suggest that homologous fimbrial subunits can carry different adhesin molecules and that these adhesin molecules can be responsible for species-specific adherence. On the contrary, adhesion of a number of dog uropathogenicProteus mirabilis strains to MDCK and T24 cells was not species specific; it depended on the mere presence of fimbriae.     

Structure,development and function of the branchial sieve of the common bream,Abramis brama,white bream,Blicca bjoerkna and roach,Rutilus rutilus

Synopsis The filter feeding organ of cyprinid fishes is the branchial sieve, which consists of a mesh formed by gill rakers and tiny channels on the gill arches. In order to establish its possible role during growth we measured the following morphological gill raker parameters over a range of sizes in three cyprinid fishes, bream, white bream and roach: inter raker distance, bony raker length, raker width, cushion length and channel width. At any given standard length common bream has the largest inter raker distance, roach the lowest and white bream is intermediate. In the comb model of filter feeding the inter raker distance is considered to be a direct measure of the mesh size and retention ability (= minimal size of prey that can be retained) of a filter. For the three species under study there is a conflict between the comb model and experimental data on particle retention. Lammens et al. (1987) found that common bream has a large retention ability whereas roach and white bream have a much smaller one. A new model, the channel model (Hoogenboezem et al. 1991) has been developed for common bream; in this model the lateral gill rakers can regulate the mesh size of the medial channels on the other side of the gill slit. The present data indicate that this model is not appropriate for white bream and roach. At any given standard length white bream and roach only reach 70% of the raker length of common bream, which means that in this model the gill slits should to be very narrow during filter feeding. The gill rakers consist of a bony raker and a fleshy cushion. The bony rakers have a rather long needle-like part outside the cushion in bream, but not in white bream and roach which have blunt gill rakers. Blunt gill rakers are not suited to reduce the diameter of the medial channels. The comb model seems more appropriate for white bream and roach, but doubts about the validity of this simple model remain. The sum of the areas of the medial channels is an approximation of the area through which water flows in the filter. This channel area therefore gives an impression of the capacity or flow rate of the filter. With this capacity estimation and an estimation of energy consumption we calculated an energy ratio of filter feeding. The energy ratio decreases with increasing standard length with an exponent close to the expected exponent of -0.40. The energy ratio is highest in bream, intermediate in white bream and lowest in roach.     

The rate of uptake and efflux of phosphatidylcholine from human erythrocytes depends on the fatty acyl composition of the exchanging species

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Molecular species composition of membrane phosphatidylcholine influences the rate of cholesterol efflux from human erythrocytes and vesicles of erythrocyte lipid

The efflux of [3H]cholesterol from prelabelled human erythrocytes having modified phosphatidylcholine compositions was measured during 24-h incubations in the presence of unlabelled acceptor liposomes composed of equimolar amounts of egg phosphatidylcholine and cholesterol. The cells were modified by replacement of part of the native phosphatidylcholine with either dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine or dilinoleoylphosphatidylcholine catalyzed by phosphatidylcholine-specific transfer protein from bovine liver. The results indicated that the efflux of [3H]cholesterol was faster from erythrocytes in which the dipalmitoylphosphatidylcholine content was increased from 7 to 25% of the total, than from cells enriched in palmitoyloleoylphosphatidylcholine or dioleoylphosphatidylcholine. Incorporation of dilinoleoylphosphatidylcholine to a level of 13% of the total phosphatidylcholine slowed the rate of efflux of [3H]sterol. The phosphatidylcholine replacements produced no significant differences in cholesterol/phospholipid ratio before or after 24 h of incubation with the acceptor egg phosphatidylcholine-cholesterol vesicles. Using vesicles prepared from erythrocyte lipid, modified to reflect the changes in the phosphatidylcholine composition induced in the whole cells, the same influence of composition on the rate of cholesterol exchange was evident. Enhancement of the dipalmitoylphosphatidylcholine content from 7 to 25% of the total phosphatidylcholine pool increased the rate of [3H]cholesterol efflux, while the addition of the same amount of dilinoleoylphosphatidylcholine slowed it compared to controls. The magnitude of the effect was comparable in intact cells and erythrocyte lipid vesicles enriched in dipalmitoylphosphatidylcholine, while the influence of dilinoleoylphosphatidylcholine was more marked in the intact cells. These results demonstrate that changes in the molecular species composition of the phosphatidylcholine pool can influence the rate of exchange of cholesterol but not necessarily the cellular content of sterol in the human erythrocyte. The influence of this phospholipid appears to be expressed independently of the presence of membrane protein or an underlying cytoskeleton.     

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.