Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin

The conformation of the sialyl Lewis ^x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewis^x in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewis^x/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewis^x/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewis^x/E-selectin complex.     

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.     

Accelerating proton spin diffusion in perdeuterated proteins at 100 kHz MAS

Fast magic-angle spinning (>60 kHz) has many advantages but makes spin-diffusion-type proton–proton long-range polarization transfer inefficient and highly dependent on chemical-shift offset. Using 100%-HN-[²H,¹³C,¹⁵N]-ubiquitin as a model substance, we quantify the influence of the chemical-shift difference on the spin diffusion between proton spins and compare two experiments which lead to an improved chemical-shift compensation of the transfer: rotating-frame spin diffusion and a new experiment, reverse amplitude-modulated MIRROR. Both approaches enable broadband spin diffusion, but the application of the first variant is limited due to fast spin relaxation in the rotating frame. The reverse MIRROR experiment, in contrast, is a promising candidate for the determination of structurally relevant distance restraints. The applied tailored rf-irradiation schemes allow full control over the range of recoupled chemical shifts and efficiently drive spin diffusion. Here, the relevant relaxation time is the larger longitudinal relaxation time, which leads to a higher signal-to-noise ratio in the spectra.     

The wheat durable,multipathogen resistance gene Lr34 confers partial blast resistance in rice

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain‐of‐function mutations in an ATP‐binding cassette transporter gene. An Lr34‐like fungal disease resistance with a similar broad‐spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34‐expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence‐based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad‐spectrum disease resistance against the most devastating fungal disease of rice.     

Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis

Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.     

Umsatz,Proliferation und Phagozytosetätigkeit der freien Zellen im Peritonäalraum der Maus nach Injektion von Polystyren-Partikeln

Zusammenfassung Bei Mäusen wurden die Art, die absolute Zahl, der Phagozytosegrad und der in DNS-Synthese befindliche Anteil der Zellen bestimmt, die sich zu verschiedenen Zeiten nach intraperitonäaler Injektion einer Suspension kleiner Polystyren-Partikeln (Durchmesser: 0,796 ) aus der Bauchhöhle auswaschen ließen. Diese Methode gestattet eine gut reproduzierbare, quantitative Analyse der zellulären Reaktion auf einen nicht-antigenischen Stimulus. An dem Geschehen beteiligt sich eine heterogene Population von Phagozyten, die neben Granulozyten zahlreiche sog. mononukleäre Elemente umfaßt. Die letzteren lassen sich morphologisch, färberisch und auf Grund ihres kinetischen Verhaltens in mehrere Untergruppen unterteilen, nämlich: monozytoide Zellen mit nierenförmigem Kern, die weitgehend den Blutmonozyten gleichen; ferner monozytoide Zellen mit rundem Kern sowie lymphomonozytoide Elemente und größere lymphoide Zellen. Zu geringfügiger Phagozytose der Partikeln ist auch ein Teil der kleinen Lymphozyten befähigt.Beurteilt am Anstieg der absoluten Zellzahl, treten nach Stimulation die neutrophilen Granulozyten am raschesten in die freie Bauchhöhle aus, gefolgt von den kleinen Lymphozyten, dann den monozytoiden Zellen mit nierenförmigem Kern und andern Zellarten. In der Zeit zwischen 16 und 24 Std nach Injektion der Partikelsuspension nahm die Zahl der kleinen Lymphozyten signifikant ab, während in derselben Periode diejenige der größeren lymphoiden Zellen und der monozytoiden Elemente mit nierenförmigem Kern steiler anstieg.Mit Ausnahme der kleinen Lymphozyten fanden sich unter allen Gruppen sog. mononukleärer Zellen solche, die sich initial mit Thymidin-³H markieren ließen. Ein signifikanter Anstieg des initialen Markierungsindex (Maximalwert: 20%) über die Kontrollwerte hinaus zeigte sich indessen nur bei größeren lymphoiden Zellen, und dies nur während der ersten Stunden nach Stimulation. Die lymphomonozytoiden Zellen ließen um den 2. Tag nach Stimulation herum eine angedeutete relative Vermehrung der DNS-synthetisierenden Zellen erkennen. Bei den ändern sog. mononukleären Zellformen fand sich während 10 Tagen nach Stimulation keine verwertbare Änderung des initialen Markierungsindex.Die durchschnittliche Partikelbeladung pro Zelle (Phagozytosegrad) war zu allen Zeiten nach Stimulation bei den monozytoiden Zellen mit rundem Kern am stärksten. Einen etwas niedrigeren Phagozytosegrad wiesen die lymphomonozytoiden Zellen und die monozytoiden mit nierenförmigem Kern auf, die im Durchschnitt pro Einzelzelle eine vergleichbare Phagozytoseleistung vollbrachten. Ein noch geringerer Phagozytosegrad fand sich bei den größeren lymphoiden Zellen, die im Mittel ungefähr gleich viele Partikeln enthielten wie die neutrophilen Granulozyten. Die aus dem Produkt aus mittlerem Phagozytosegrad und absoluter Zellzahl geschätzte totale Phagozytosearbeit ergab während der ersten 8–12 Std nach Stimulation für die neutrophilen Granulozyten die höchsten Werte. Später wurden sie von den monozytoiden Zellen mit nierenförmigem Kern abgelöst, die während der restlichen Versuchsdauer von 10 Tagen die Führung beibehielten. Eine erhebliche Phagozytosearbeit wurde in spätem Stadien nach Injektion der Partikelsuspension auch von den übrigen sog. mononukleären Zellen geleistet, mit Ausnahme der kleinen Lymphozyten. Die am stärksten mit Partikeln beladenen Zellen besaßen in der Regel einen Kern von mittlerer Größe. Viele der DNS-synthetisierenden Zellen enthielten auch reichlich Partikeln, d. h. Proliferation und Phagozytose schließen sich gegenseitig nicht aus; sehr wahrscheinlich handelt es sich hierbei um differenziertere Vertreter der Makrophagenvorläufer.Die Befunde werden im Hinblick auf Probleme der Makrophagenvorläufer, des Zellaustritts aus der Blutbahn, der Transformation lymphoider Elemente und der ortständigen Proliferation der sog. mononukleären Zellen diskutiert.

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Response of free cells in the peritoneal cavity of the mouse after injection of polystyrene particles

Summary Young adult female mice (Charles River strain) were given a single intra-abdominal injection of 1.12×10¹⁰ Polystyrene (Latex) particles with a mean diameter of 0.796 , suspended in 3 ml of 0.9% saline. Animals received a single i.v. injection of thymidine-³H one hour prior to sacrifice. Free peritoneal cells were harvested by peritoneal washings at various time intervals following injection of the particle suspension. This method gives reproducible quantitative results with regard to absolute cell numbers involved in the response to this non-antigenic stimulus. Differential counts, grading of phagocytic particle loading and autoradiographic analysis were made on smear preparations of the peritoneal exudate. The phagocytic reaction comprised granulocytes and a heterogeneous population of mononuclear elements. The latter could he separated on the basis of morphological, tinctorial and kinetic characteristics into various subgroups, i.e.: monocytoid cells with kidney-shaped nuclei comparable to blood monocytes; monocytoid cells with round nuclei; lymphomonocytoid cells; large lymphoid cells; and small lymphocytes, a fraction of which showed a slight phagocytic capacity.As judged from the changes in absolute cell counts, the neutrophilic granulocytes showed the fastest entry rate, followed by small lymphocytes, then monocytoid cells with kidneyshaped nuclei and other cell types. In the period between 16 and 24 hours following stimulation the absolute number of small lymphocytes dropped significantly while during the same time interval the counts of large lymphoid cells and monocytoid cells with kidney-shaped nuclei rose more steeply.Except for small lymphocytes, cells incorporating thymidine-³H into their DNA were found among all types of mononuclears. The only significant rise of the initial labeling index up to 20% following injection of this radioactive precursor was seen in large lymphoid cells and occurred during the first 24 hours following stimulation. An abortive, non-significant rise of the initial labeling index was observed around the second day in lymphomonocytoid cells. All other mononuclear cell types exhibited initial labeling indices comparable to control values throughout the observation period.Mean particle loading per cell (grade of phagocytosis) was heaviest in monocytoid cells with round nuclei throughout the experiment. A moderate mean phagocytic grade was found in lymphomonocytoid elements and monocytoid cells with kidney-shaped nuclei. Lower phagocytic grades were seen in neutrophilic granulocytes and large lymphoid cells. The highest percentage of the total phagocytic performance (mean phagocytic grade per cell X absolute number of cells of the same type) was observed for neutrophilic granulocytes during the first 8 to 12 hours following stimulation, later for monocytoid cells with kidney-shaped nuclei. Except for small lymphocytes all types of mononuclears were engaged to a considerable extent in phagocytosis, particularly in later stages after injection of the particles. Cells showing high grades of phagocytosis usually had medium-sized nuclei. Many DNA-synthesizing cells were heavily loaded with particles indicating that proliferation may still go on while differentiation towards phagocytic capacity has already reached a high degree.The findings are discussed in relation to problems of macrophage precursors, cell emigration from the blood stream, transformation of lymphoid elements and local proliferation of mononuclear cells.

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Die Untersuchungen wurden mit Unterstützung durch den Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung durchgeführt.     

Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes

We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.     

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.