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.     

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.     

A three-constituent damage model for arterial clamping in computer-assisted surgery

Robotic surgery is an attractive, minimally invasive and high precision alternative to conventional surgical procedures. However, it lacks the natural touch and force feedback that allows the surgeon to control safe tissue manipulation. This is an important problem in standard surgical procedures such as clamping, which might induce severe tissue damage. In complex, heterogeneous, large deformation scenarios, the limits of the safe loading regime beyond which tissue damage occurs are unknown. Here, we show that a continuum damage model for arteries, implemented in a finite element setting, can help to predict arterial stiffness degradation and to identify critical loading regimes. The model consists of the main mechanical constituents of arterial tissue: extracellular matrix, collagen fibres and smooth muscle cells. All constituents are allowed to degrade independently in response to mechanical overload. To demonstrate the modularity and portability of the proposed model, we implement it in a commercial finite element programme, which allows to keep track of damage progression via internal variables. The loading history during arterial clamping is simulated through four successive steps, incorporating residual strains. The results of our first prototype simulation demonstrate significant regional variations in smooth muscle cell damage. In three additional steps, this damage is evaluated by simulating an isometric contraction experiment. The entire finite element simulation is finally compared with actual in vivo experiments. In the short term, our computational simulation tool can be useful to optimise surgical tools with the goal to minimise tissue damage. In the long term, it can potentially be used to inform computer-assisted surgery and identify safe loading regimes, in real time, to minimise tissue damage during robotic tissue manipulation.     

Porcine E. coli: Virulence-Associated Genes,Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.