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.

     

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-l-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.Abbreviations BMT S-adenosyl-l-methionine: bergaptol O-methyltransferase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the acyltransferase family.     

Vitamin k-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype

Background

Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE^−/− model of atherosclerosis.

Methodology/Principal Findings

A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE^−/− mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K₁ (VK₁, 1.5 mg/g) or vitamin K₁ and warfarin (VK₁&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden.

Conclusions/Significance

VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE^−/− mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.     

Mechanisms of cell differentiation: Affinity of a morphogenetic factor to DNA

Summary A morphogenetic factor which induces inTriturus gastrula ectoderm tissues which are derived from mesoderm and endoderm has been extracted from chicken and amphibian embryos. The factor which is protein in nature has been obtained from chicken embryos in a highly purified state.The biological activity of the chicken factor is partially inhibited when the factor is combined with chicken DNA or sonicated chicken DNA.When the 3H-labelled factor is combined with sonicated DNA and then centrifuged on a sucrose gradient the factor migrates in part with the DNA. This indicates that the factor is bound to DNA.The inferences from these results are discussed with regard to the possible mechanism of action of the factor and the molecular mechanism of differentiation.     

Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on Fetuin-A controlled calcification inhibition

Clinical studies and animal experiments have shown that the serum protein fetuin-A is a highly effective inhibitor of soft tissue calcification. This inhibition mechanism was elucidated on the basis of an in vitro fetuin-A-mineral model system. In a previous study, we found that in a two-stage process ∼100-nm sized calciprotein particles (CPPs) were formed whose final stage was stabilized by a compact outer fetuin-A monolayer against further growth. Quantitative small-angle neutron scattering data analysis revealed that even at a fetuin-A concentration close to the stability limit, only approximately one-half of the mineral ions and only 5% of the fetuin-A were contained in the CPPs. To uncover the interplay of the remaining supersaturated mineral ion fraction and of the 95% non-CPP fetuin-A, we explored the fetuin-A monomer fraction in solution by contrast variation small-angle neutron scattering. Our results suggest that the mineral ions coalesce to subnanometer-sized clusters, reminiscent of Posner clusters, which are stabilized by fetuin-A monomers. Hence, our experiments revealed a second mechanism of long-term mineral ion stabilization by the fetuin-A that is complementary to the formation of CPPs.