Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.     

Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation.     

Das Bewegungsverhalten der Coelomzellen vonPsammechinus miliaris bei der Wundheilung (Echinodermata)

Zusammenfassung 1. Das Bewegungsverhalten der Coelomzellen des EchinoidenPsammechinus miliaris Gmel. wird an kleinen von Stacheln, Füßchen und Pedicellarien befreiten Stellen der Skelettoberfläche in Periproctnähe untersucht.2. Aus dem freiliegenden Bälkchenwerk treten Coelomzellen aus, von denen nur die rotbraunen Amoebocyten auf dem hellen Kalkuntergrund im Auflicht (Ultropak;E. Leitz) sichtbar sind.3. Nach einigen Stunden ist die Wundfläche mit einer dicken rötlichen Zellmasse bedeckt, dem primären Wundverschluß. Außer den Coelomzellen enthält der Wundverschluß noch verschieden große Kalkpartikel, die vom Abschleifen der Versuchsstelle herrühren.4. Bei direkter Beobachtung ist weder an den rotbraunen Amoebocyten noch am Wundverschluß die geringste Bewegung zu erkennen.5. Zeittransformation (Zeitraffung [Z.R.] auf 1/240 und 1/480) zeigt die mit erheblicher Ortsverlagerung und Metabolie verbundene Bewegung der allein wahrehmbaren rotbraunen Amoebocyten auf der Wundfläche. Im scheinbar in Ruhe befindlichen Wundverschluß findet eine ständig hin- und herwogende Bewegung der Zell-Kalkamsse statt.6. Bereits nach 6 bis 7 Stunden ist das Operationsfeld völlig geglättet; die Lücken im Kalkskelett sind kaum noch zu erkennen infolge der neu eingebauten Kalkelemente. Die eigentlichen Heilungsvorgänge (Wiederherstellung der Feinstruktur des Kalkskelettes) erfolgen unterhalb des primären Wundverschlusses, sind also nicht der Beobachtung zugängig.7. Wird der primäre Wundverschluß im ganzen vorsichtig abgehoben und zerzupft, so kann das Bewegungsverhalten der entstandenen kleinen und großen Aggregate im Durchlicht unter Z.R. untersucht werden.8. Die im zerriebenen Explantat erhaltenen Coelomzell-Aggregate aller Größen weisen erhebliche Ortsveränderungen auf; oft breiten sie sich langsam aus unter Auswanderung zehlreicher randlich liegender Zellen. An den Außenzonen mittlerer und großer Aggregate werden plasmatische Netze sichtbar, die ständig ihre Gestalt und Maschenweite ändern.9. Diese Plasma-Netze bilden die Grundlage der Aggregate; ihre Kontraktionen und Dilatationen bewirken die Ortsverlagerungen der Aggregate (Netzbildende Coelomzellen;Kuhl 1937).10. Wenigzellige Aggregate vereinigen sich in den allermeisten Fällen, sobald ein gewisser Abstand überbrückt ist. Mittlere und große Aggregate gehen häufig eine Verbindung ein; meist werden vorher lockere Coelomzell-Brücken hergestellt. In manchen Fällen gleiten die Aggregate aneinander vorbei.11. Im polarisierten Licht lassen sich bei gekreuzten Nicols die ersten kleinen neugebildeten Kalkkristalle in den skelettbildenden Coelomzellen (= netzbildenden Zellen) nachweisen.12. Der Verschluß kleiner Kratzwunden im noch dünnen primären Wundverschluß (die Kratzer dringen bis zur abgeschliffenen Skelettoberfläche vor) wird unter Z.R. im Ultropak-Auflicht untersucht. Die Ergebnisse am explantierten Wundverschluß im Durchlicht führen zum Verständnis der Bewegungsvorgänge im ungewohnten Auflicht.13. Im zunächst verwirrenden Bewegungsgeschehen (die auffälligen rotbraunen Amoebocyten haben bei der Wundheilung keine Funktion) fallen die durch die Operationsnadel herausgerissenen kleinen Kalktrümmer auf; sie werden passiv durch die Plasmanetze bewegt, gelangen auch zufällig in die Kratzer und werden an den Rändern durch neugebildetes Kalkmaterial festgelegt oder eingebaut. Aus der Tiefe der Kratzer können lose Kalkpartikel heraufbefördert werden; auch diese werden häufig eingebaut. Die entstehenden Kalkbrücken werden schließlich untereinander verbunden und dadurch die kleine Wunde verschlossen. Das eingebaute Kalkmaterial zeigt auch unter starker Z.R. keine passive Bewegung mehr.14. In seltenen Fällen kann der Vorgang des schubweisen Aufsteigens der skelettbildenden Zellen aus dem Panzer und ihre Zusammenballung im Z.R.-Laufbild beobachtet werden.15. Ob der Einbau von herausgerissenem Kalkmaterial temporär oder dauernd ist, muß noch geprüft werden.

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The locomotory behaviour of coelom cells ofPsammechinus miliaris (Echinodermata) during wound-healing

In the sea urchinP. miliaris application of time lapse photography allows a study of the very slow movements of coelom cells during the healing process of small wounds on the surface of the calcareous skeleton near the periproct. For observation and time lapse photography LEITZ-Ultropak objectives were used (incident light). Ambulacral feet, spines and pedicellaria were removed, and the animal was fixed in three places in a ring of plexiglass by means of three little screws, which touched the equator of skeleton. The rate of time transformation was 1/240 to 1/480. The film reveals the behaviour of coelom cells, which move out the skeleton to the surface of the small experimental region. Within several hours the white polished surface is covered with hundreds of red-brown amoebocytes; only these are visible on the white lime-ground; they have no function in the healing process, which takes place below the surface of the primäre Wundverschluß and therefore cannot be observed. There are three main types of coelom cells: red-brown amoebocytes, körnchenführende Zellen (white amoebocytes) and leucocytes (netzbildende or skelettbildende Zellen); the flagellated cells may be neglected here. In order to be able to study the behavior of the three main types of coelom cells, the primäre Wundverschluß, i. e. the total cell-covering of the wound, is removed and torn into microscopic fragments. These are studied (time lapse) under normal optical conditions (transmitted light). The slides show many aggregates of different sizes, single cells and little calcareous concrements torn off the skeleton. The aggregates, even the big ones, exhibit slow locomotion and change their positions considerably. If the distance of two aggregates becomes small enough, they fuse. In these cases a loose cell bridge between the two aggregates is formed. Sometimes no union occurs, although the distance is very small. Even big aggregates suddenly show considerable contractions if spreading has preceded. All movements and place changing of cell-aggregates are caused by contractions and dilatations of the plasmatic network which forms the cellular basis. Little wounds in the newly built Wundverschluß scratched with a lancet, heal within several hours. Time lapse shows passive movements of small calcareous fragments, which by chance sometimes enter the small wounds, where they help and accelerate the closing of the injury. The fragments are fixed on the edge of the wound by newly produced lime. Skeleton building coelom cells (netzbildende Coelomzellen) come up in batches from the depth of the sea urchin's skeleton; each cell contains lime crystals.

     

Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups.

The use of liposomes as drug delivery systems has been limited by their rapid clearance from circulation by the mononuclear phagocyte system. Recent studies have found that circulation times can be greatly enhanced by incorporating a small amount of modified lipids whose headgroups are derivatized with a bulky water soluble polymeric chain of poly ethylene oxide. We report here a systematic study using the Surface Forces Apparatus to measure directly the interactions between two phosphatidyl ethanolamine lipid bilayers, exposing this polymeric headgroup at different concentrations in the bilayer. We found that the force becomes repulsive at all separations and that the thickness of the steric barrier could be controlled easily by adjusting the concentration of the modified lipids. Equilibrium force profiles were measured that were reversible and largely insensitive to changes in electrolyte concentration and temperature. The results have enabled the Dolan and Edwards theory for the steric forces of low coverage polymer surfaces and the Alexander de Gennes theory for high coverage surfaces to be tested, and both were found to apply. We conclude that these simple theories can be used to model the interactions of surprisingly short segments and, hence, apply to such systems as lipids with bulky headgroups and liposomes containing a sterically stabilizing polymer.     

Charge displacement in bacteriorhodopsin during the forward and reverse bR-K phototransition.

Dried oriented purple membrane samples of Halobacterium salinarium were excited by 150 fs laser pulses of 620 nm with a 7 kHz repetition rate. An unusual complex picosecond electric response signal consisting of a positive and a negative peak was detected by a sampling oscilloscope. The ratio of the two peaks was changed by 1) reducing the repetition rate, 2) varying the intensity of the excitation beam, and 3) applying background illumination by light of 647 nm or 511 nm. All of these features can be explained by the simultaneous excitation of the bacteriorhodopsin ground form and the K intermediate. The latter was populated by the (quasi)continuous excitation attributable to its prolonged lifetime in a dehydrated state. Least-square analysis resulted in a 5 ps upper and 2.5 ps lower limit for the time constant of the charge displacement process, corresponding to the forward reaction. That is in good agreement with the formation time of K. The charge separation driven by the reverse phototransition was faster, having a time constant of a 3.5 ps upper limit. The difference in the rates indicates the existence of different routes for the forward and the reverse photoreactions.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Assignment of a gene for human quinoid-dihydropteridine reductase (QDPR,EC 1.6.5.1) to chromosome 4

Summary An acrylamide gel electrophoretic procedure is described which allows the separation of human quinoid-dihydropteridine reductase (QDPR), EC 1.6.5.1) from the homologous enzyme expressed in established rodent cell lines. The human enzyme marker segregates exclusively with chromosome 4 in a series of well characterized man-mouse somatic cell hybrid clones from our clone bank. This observation supports the assignment of a structural gene for QDPR to human chromosome 4.