Agrobacterium-mediated DNA transfer

Summary Agrobacterium tumefaciens naturally transfers DNA into plant cells and is clearly one of the most effective methods of directed DNA transfer presently available. Two kinds of vectors are commonly used. Cointegrative vectors have the foreign genes incorporated directly into the Ti plasmid. Binary vectors carry two plasmids; the main Ti plasmid where most of the T-DNA has been removed, and a second plasmid containing the foreign genes between the usual border sequences. The vir genes on the main plasmid function to mobilize the foreign genes into a plant cell. Most plant transformation methods follow the procedure of cocultivating wounded tissue with vir-gene-induced bacteria. The cocultivation step is followed by transfer to a selective medium containing antibiotics to kill the bacterium and to allow only growth of transformed tissue. Several selectable markers are available that include resistance to antibiotics, herbicides, or drugs. In addition, several scorable markers such as the bacterial glucuronidase, chloramphenicol acetyl transferase, and the Agrobacterium opine genes are used to verify transformation. Southern blotting and inheritance of transferred genes are ultimately used to demonstrate stable transformation.     

Transformationsvorgänge in der Frühphase der Entstehung des arteriellen Thrombus

Zusammenfassung In unserer ultrastrukturell durchgeführten Studie wurden Thromben in der Arteria carotis communis von Ratten nach einer zuerst von Meng und Seuter (1977) beschriebenen Methode experimentell erzeugt. Induktion der Thrombusbildung erfolgte in vivo durch Unterkühlung eines kleinen Gefäßabschnittes unter konstantem Druck und kurzfristiger Stase. Eine Änderung des Blutflusses wurde durch einen Silberclip erzeugt. Die geschädigten Gefäßsegmente einschließlich der Thromben bzw. deren Vorstufen wurden nach 5, 10, 30 min und 1, 4 und 24 h nach der Thrombosestimulation entnommen und fixiert. Semidünnschnitte und Ultradünnschnitte wurden im Licht- und Elektronenmikroskop morphologisch untersucht.Den Transformationsvorgängen im Thrombus konnten exakte Zeitmarken zugeordnet werden. Als wichtigstes histopathologisches Merkmal für die Altersbestimmung arterieller Thromben in der Frühphase der Thrombogenese werteten wir die Querstreifung der Fibrinfasern. Diese trat bereits nach 5 min auf, erreichte nach 30 min ein Maximum und verschwand als Folge der zunehmenden Verdichtung der Fasern nach einer Stunde. Nach 4 h sahen wir eine weitgehende Retraktion der Fibrinfasern, die nach 24 h zur Bildung des Fibrinfasergerüstes mit Einmauerung korpuskulärer Elemente führte. Überdies beobachteten wir zwei Thrombocytenaggregate von differenter Struktur. Wir unterschieden ein fibrinarmes Aggregat, in dem die Thrombocyten dichtgepackt und pseudopodienreich erschienen von einem thrombocytenarmen Aggregat mit reichlich interponierten Fibrinfasern. Die nach 5 min im Zentrum des Thrombus auftretende Agglutination der Plättchen im thrombocytenreichen Aggregat führte nach 30 min zur Thrombocytorrhexis und ergab daher einen weiteren Anhalt für die Altersbestimmung des Coagulum. Der entstandene celluläre Abraum stimulierte mononucleäre Zellen und Leukocyten zur Phagocytose. Daher sahen wir nach 4 h eine massive Leukocytose als Folge der frühen Thrombocytorrhexis. Nach 24 h war die viscöse Metamorphose im fibrinreichen und fibrinararmen Aggregat weitgehend abgeschlossen. Innerhalb des beobachteten Zeitraumes entstand eine Verballung und bizarre Deformierung der Erythrocyten, die bereits nach 5 min vom Zentrum des Thrombus ausging und nach 24 h die Peripherie erreichte. Eine Hämolyse der Erythrocyten war nach dieser Zeit noch nicht erkennbar.

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Evolution in the early changes in the establishment of arterial thrombi

Summary Ultrastructural studies of thrombi were carried out on the common carotid artery of the rat using a method first described by Meng and Seuter (1977). Induction of thrombus formation in vivo was achieved by chilling of a small vessel segment under constant pressure and short-termed stasis. Disturbance of the blood flow was produced by a silver clip. The damaged vessel segments with the thrombotic deposits were removed 5, 10, 30 min, and 1, 4 and 24 h after stimulation of thrombosis. They were fixed and samples were studied as semithin and ultrathin sections morphologically using light and electronmicroscopy.In the maturation of thrombi exact time intervals could be determined. The most important histopathological characteristics for age determination of arterial thrombi in the early period of thrombogenesis were the cross stripes of fibrin fibres. They appeared after 5 min, reaching a maximum after 10 min and disappeared as a result of increasing fibre density after 1 h. After 4 h nearly complete retraction of fibrin fibres was found which led after 24 h to the formation of a corresponding frame walling in the corpuscular elements. Apart from this aggregation of thrombocytes, which were of two different types were observed, one showing a fibrin-poor aggregate in which the thrombocytes appeared densely packed with numerous pseudopods, and one showing a thrombocyte poor aggregate with abundant interposed fibrin fibres. Agglutination of platelets which occurred in the thrombocyte-rich aggregate in the centre of the thrombus after 5 min led to thrombocytorrhexis after 30 min. The resulting cellular waste stimulated phagocytosis by mononuclear cells and leucocytes. Because of this a massive leucocytosis was found as a result of the early thrombocytorrhexis after 4 h. After 24 h the viscous metamorphosis in the fibrin-rich and in the fibrin-poor aggregate was largely completed. Clumping and deformation of erythrocytes was observed in the middle of the thrombus after 5 min and at the periphery of the thrombus after 24 h. Haemolysis did not occur within this time interval.

Frau Antoni, Herrn Ing. grad. Derks und Herrn Rieger sei für ausgezeichnete technische Assistenz herzlichst gedankt.     

Malignant transformation of mature cystic teratoma to squamous cell carcinoma involves altered expression of p53‐ and p16/Rb‐dependent cell cycle regulator proteins

Ovarian mature cystic teratomas (MCT) uncommonly undergo malignant transformation to squamous cell carcinoma (SCC). While alterations in the p53 tumor suppressor gene and protein have been shown, few studies have analyzed other molecular changes leading to this malignant conversion. The purpose of the present study was to investigate 21 samples of SCC arising in MCT for altered expression in known p53‐ and p16/Rb‐dependent cell cycle regulatory proteins, and the association between their expression and cellular proliferation and histological features. Overexpression of the p53 protein was observed in 14 SCC (67%), while four (19%) had point mutations in the p53 gene. Reduced expression of the p16 protein was observed in 18 SCC (86%), while p16 gene alterations (hypermethylation (29%) and point mutation (33%)) were found in 11 (52%). Furthermore, a statistically significant correlation was observed between p53 and Rb overexpression (P = 0.0010), and the overexpression of both p53 and Rb was respectively significantly correlated with increased cellular proliferation. The results indicate that alterations in both the p53 and p16‐Rb pathways are associated with SCC arising in MCT.     

Spatial object representation and its use in planning eye movements

The eye movements we make to look at objects require that the spatial information contained in the objects image on the retina be used to generate a motor command. This process is known as sensorimotor transformation and has been generally addressed using simple point targets. Here, we investigate the sensorimotor transformation involved in planning double saccade sequences directed at one or two objects. Using both visually guided saccades toward stationary objects and objects subjected to intrasaccadic displacements, and memory-guided saccades, we found that the coordinate transformations required to program the second saccade were different for saccades aimed at a new target object and saccades that scanned the same object. While saccades aimed at a new object were updated on the basis of the actual eye position, those that scanned the same object were performed with a fixed amplitude, irrespective of the actual eye position. Our findings demonstrate that different abstract representations of space are used in sensory-to-motor transformations, depending on what action is planned on the objects.     

How the lack of visuomotor feedback affects even the early stages of goal-directed pointing movements

Pointing movements made with a hidden cursor from the center of gaze to a stationary, visible target overshot the actual target location. The systematic error decreased when the final cursor location from the previous trial was shown, which likely led to the creation of an internal sensorimotor model of movement. However, the putative model had a short memory, and could not substitute for on-line visuomotor feedback on subsequent trials. Contrary to common belief, the effect of a lack of visuomotor feedback was seen even in the early acceleration stage of the movement trajectory. Unchecked in the absence of visual monitoring, the acceleration stage of the movement lasted longer, as was evidenced by the significantly larger value of the peak cursor speed. Moreover, the speed peaked much later in the course of the movement. Speed declined more rapidly thereafter. Consequently, the delayed deceleration stage lasted far less than the acceleration stage. In the absence of visual feedback, the shift rightward in time of the peak speed position (PSP) in relation to total movement duration and other changes in the trajectory imply that visual feedback must play a significant role in determining when acceleration ceases (d V/d t=0), and argue against the traditional notion that visuomotor feedback is unavailable until the later stages of movement. Moreover, our data suggest that non-visual modalities, e.g., proprioception, may be too slow to make up for the absence of vision.     

Follicular hyperplasia presenting with a marginal zone pattern in a reactive lymph node lesion

Histologically, the marginal zone pattern of the lymph node is characterized by lymphoid follicles with three distinct layers. The inner layer is composed of follicular center zones, the middle layer of darkly stained mantle zones, and the outer layer of marginal zones. However, the marginal zone pattern is rarely seen in reactive lymph nodes except for mesenteric lymph nodes. We describe the clinicopathologic, immunohistochemical and genotypic findings of six cases of reactive follicular hyperplasia exhibiting the marginal zone pattern. The patients comprised three males and three females (age range 24 to 63 years; medium 56 years). Follow-up data were obtained from five patients. None of them developed malignant lymphomas during the follow-up period of from 5 to 204 months (median 68 months). Histologically, the lesion was characterized by numerous lymphoid follicles and partial distortion of lymph node structure. Varying degrees of progressive transformation of the germinal center (PTGC) were noted in the four cases. The marginal zone pattern was observed in some or most of the lymphoid follicles including PTGC. The marginal zone B cells were small to medium-sized lymphocytes with round or slightly indented nuclei and a broad rim of pale cytoplasm. Some of them had a monocytoid appearance. They were CD20+, CD79a+, sIgM+/-, sIgD-, CD5-, CD10-, CD21-, CD23-, CD43-, CD45RO-, Bcl-6-, cyclin D1-, EMA- and p53-. A portion of them were Bcl-2 positive. Occasional large lymphoid cells with round or indented nuclei and moderate amounts of cytoplasm were observed in the marginal zone in four cases. These large lymphoid cells were usually CD20 positive, but Bcl-6 negative. A small number of them contained polytypic intracytoplasmic immunoglobulins. The polytypic nature of B lymphocytes was demonstrated by immunohistochemistry and polymerase chain reaction. Recognition of unusual marginal zone hyperplasia in reactive lymph node lesions is important to avoid confusion with nodal involvement in various low-grade B cell lymphomas presenting a marginal zone distribution pattern.     

高精度红外光点位置检测分析系统

高精度红外光点位置检测分析系统是基于光电位置敏感器件、立体测量学研制而成的运动物体分析系统。它能快速、准确地测出运动物体各特征点的瞬时位置，并在此基础上进行分析计算，给出三维坐标及针对具体应用领域的特征曲线和参数。该系统可应用于人体运动检测、外科手术定位等领域。     

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.     

Energy state and myosin heavy chain isoforms in single fibres of normal and transforming rabbit muscles

 Energy-rich phosphates, [ATP]/[ADP_free] ratios, and the myosin heavy chain (MHC) complement were determined in single fibres from normal rabbit muscles, and in fibres isolated from tibialis anterior muscle undergoing fast-to-slow conversion by chronic low-frequency stimulation (CLFS). In normal muscles, energy-rich phosphate contents and [ATP]/[ADP_free] ratios could thus be assigned to different MHC-based fibre types. Phosphocreatine (PCr) contents and [ATP]/[ADP_free] ratios differed markedly between fast- and slow-twitch fibres, as well as within the fast fibre subtypes. Both magnitudes were approximately twofold higher in the fastest (type IIB) fibres as compared to the slowest (type I) fibres. According to PCr contents and [ATP]/[ADP_free] ratios pure and hybrid fibres were aligned in an order similar to that determined by their contractile properties and myofibrillar ATPase activities. CLFS for up to 30 days induced pronounced decreases in PCr and [ATP]/[ADP_free] which attained levels twofold lower than in normal slow-twitch fibres. In both normal and stimulated muscles, PCr and [ATP]/[ADP_free] ratios were correlated, indicating their equilibrium in the different fibre types. The relationship detected between MHC isoform expression and the [ATP]/[ADP_free] ratio suggests that the drastic and persistent depression of the cellular energy state may act as an important signal initiating fast-to-slow transformation processes in muscle fibres. Received: 26 June 1998 / Accepted: 31 July 1998