Investigation of the anisotropy of glycocalyx stained with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride (author's transl)]

In the present study the anisotropic staining of the erythrocyte membrane with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride was studied and simultaneously compared with the toluidine blue topo-optical staining. The difference between anisotropic toluidine blue and 1.9-dimethyl methylene blue staining, except after KMnO4-oxidation, was only of quantitative nature. On the contrary, striking differences were observed between N,N'-diethylpseudoisocyanine chloride staining, and toluidine blue or 1.9-dimethyl methylene blue staining. Enzymatic and chemical degradation resulted the disappearance of N,N'-diethylpseudoisocyanine chloride staining. Following these treatment membrane birefringence could be restored by aldehyde bisulfate and/or KMnO4-oxidation, while the N,N'-diethylpseudoisocyanine chloride staining was restored only after KMnO4-oxidation. After methylation or acetylation the membrane birefringence disappears, while after KMnO4-oxidation both topo-optical reactions return. The digitonin reaction brought about a rearrangement of the glycocyalyx components. The results draw attention to the spatial orientation of the glycoprotein of the erythrocyte membrane. The role of glycocalyx in the three topo-optical reactions was thus clearly demonstrated.     

The suitability of further thiazine and quinoline dyes for topo-optical reactions on the plasmalemma (author's transl)]

The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, AZURE C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N'-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.     

Optical polarization reveals different ultrastructural molecular arrangement of polysaccharides in the yeast cell walls.

The topo-optical aldehyde bisulfite-toluidine blue (ABT) reaction of vicinal OH and amino-OH groups offers new ways to study the ultrastructure of polysaccharides in different biological substrates. Through oriented dye binding on the reacting groups, the ABT reaction induces strong birefringence on the linearly ordered polysaccharides, which is negative with respect to their chain length. Using this method, two types of molecular order of the polysaccharides could be distinguished in the cell walls and capsules of yeasts. (1) The optically negative spherulitic character of the yeasts after the ABT reaction indicated that the toluidine blue molecules were bound tangentially (in a surface-parallel pattern) while the polysaccharide chains of the cell walls and capsules were oriented mainly radially. This structural pattern may be explained as resulting from a helicoid conformation of the polysaccharide component. (2) Acid or alkali hydrolysis removed the radially oriented polysaccharide component of the cell wall. The remaining, resistant polysaccharides showed up in the form of optically positive spherulites indicating radially oriented dye molecules on a circularly ordered, micellar polysaccharide texture.     

Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments

Summary Using topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS.The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.     

Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments.

Using topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS. The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.     

Zur Eignung weiterer Thiazin- bzw. Chinolinfarbstoffe für die topo-optische Reaktion am Plasmalemm

Zusammenfassung Die hier durchgeführten Untersuchungen erwiesen, daß die Thiazinfarbstoffe Azur B, Azur C und Thionin sowie der Chinolinfarbstoff Pinacyanol bzw. sein Hydrochlorid für die topooptische Reaktion am Plasmalemm geeignent sind.-Die an der Membranoberfläche orientiert gebundenen Farbstoffmoleküle werden durch eine nachträgliche Präzipitation stabilisiert und gleichzeitig wird der anisotrope Effekt verstärkt.-Die Thiazinfarbstoffmoleküle (Azur B, Azur C, Thionin) sind nach der optischen Analyse radiär zur Membran ausgerichtet.-Gegenüber dem früher untersuchten Chinolinfarbstoff N,N-Diäthylpseudoisocyaninchlorid, dessen Farbstoffmoleküle parallel zur Membran lagen, sind das Pinacyanol bzw. sein Hydrochlorid in gleicher Weise, wie die Thiazinfarbstoffe radiär zur Membran ausgerichtet.

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The suitability of further thiazin and quinolin dyes for topo-optical reactions on the plasmalemma

Summary The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, azure C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.

     

Polarization optical investigation by topo-optical reactions of various blood platelet aggregates

Phases I and II (early and advanced) of platelet aggregation could readily be followed and studied by various topo-optical reactions. Our studies indicate that in the early stage (monitored by an aggregometer) aggregates show no alteration in membrane structure of platelet. In the advanced stage (phase II), in addition to membrane alteration, platelets 2-3 times larger than in the control were observed. These were stained selectively in the pH 1-2 range with thiazine dyes. After hyaluronidase digestion they lost the ability to stain and became birefringent. The elucidation of this phenomenon requires further study.     

Polarization microscopic evidence for an oriented cytoplasmic structure in the "dark" variants of adrenalin-storing cells.

A diffuse cytoplasmic birefringence confined to "dark" adrenalinstoring cells has been described. The main optical characteristics of the birefringence factor include: regular orientation of birefringence relative to the base-apex axis of cells; additive anisotropic staining with methods based on the principle of topo-optical staining reactions; dependence of birefringence on labile morphologic properties. On the basis of the capacity of the macromolecular matrix of chromaffin granules to form lamellar liposomal structures in vitro it has been proposed that a reorientation of molecular organization in the matrix of chromaffin cells is responsible for the observed optical phenomenon. The direction of birefringence was explained by a preferential direction of contractile forces acting during "dark" cell formation.     

Topo-optical reactions of the human blood platelet membrane

The polarization optical analysis of human blood platelets was carried out by means of topo-optical staining reactions. Similar studies have not been performed so far. With this approach we were able to demonstrate the spatially oriented nature of glycoprotein components in the platelet membrane. Using a sialic acid specific topo-optical reaction the sialic acid component of human platelet membrane was selectively demonstrated and the even distribution of sialic acid residues on the membrane surface was also suggested. Polarization optical analysis has shown a membrane-parallel orientation of oligosaccharide chains carrying sialic acids.     

Membrane microviscosity differences in normal and leukaemic human lymphocytes.

A study of the fluorescence polarization and fluorescent lifetimes of 1,6 diphenyl hexatriene in human normal and leukaemic lymphocytes, lymphocyte plasma membranes and liposomes from the plasma membranes failed to reveal any fluidity differences which could be attributed to the leukaemic transformation. The plasma membranes were more viscous than the whole cells, and on average the liposomes were only 57% as viscous as the plasma membranes from which they were prepared. The average fluorescent lifetime of DPH in the liposomes was 7.9 nanoseconds as opposed to 9.7 in the plasma membrane. The polarization degree of DPH in the lymphocytes was much lower and more variable than that of DPH in platelets, polymorphonuclear leucocytes or erythrocyte membranes.     

Aldehyde bisulfite-toluidine blue (ABT) staining as a topo-optical reaction for demonstration of linear order of vicinal OH groups in biological structures.

The aldehyde-bisulfite-toluidine blue reaction followed by poststaining stabilization with potassium ferricyanide (ABT) is described as a topo-optical, oriented staining reaction of the vicinal OH groups of complex carbohydrates in biological structures such as polysaccharides, glycoproteins and glycolipids. The birefringence as induced by the oriented dye binding as a result of ABT is indicative of linear order of the vicinal OH groups and, in turn, provides information on the ultrastructural pattern of carbohydrate moieties in biological substances, which pattern is often not demonstrable by other ultrastructural methods. The possibilities of this new approach to the ultrastructural analysis of complex carbohydrates with ABT in a great number of biological substances is demonstrated and its practical value in histopathology discussed.     

Annular self-assembly of DNA molecular chains occurring in natural dry process of diluted solutions

The molecular orientation of DNA membranes, which were prepared by subjecting diluted solution of salmon sperm DNA to a natural drying process, was examined. X-ray diffraction and polarization microscopy revealed that the dried membranes have zonal structures. Each zone shows different features in molecular orientation. In the outer zones formed at the early stage in drying, DNA molecular chains were found to be aligned along the annular curve of the peripheral edge. Both the uneven thickness and disproportional molecular weight distribution in a membrane suggested that the radial flow transported DNA molecules to the edge and that they formed the accumulated phase. Above the critical concentration for the formation of liquid crystalline, the DNA chains would be aligned onto the preformed solid--liquid interface. The membranes with the highest birefringence were obtained from DNA samples with the relatively high molecular weight of around 29 kbp. In the innermost zone, on the other hand, the DNA molecules were radially oriented. This alignment can be attributed to the hydrodynamic effect caused by the rapid translation of the edge interface, which overcomes the Brownian motion of the chain segments. A transitive balance of physical effects was revealed in the dry process, which provided quite orthogonal molecular orientations in a single droplet.     

Demonstration of microorganisms in tissues by the ABT and KOH-ABT topo-optical reactions.

The aldehyde-bisulphite-toluidine blue (ABT) reaction, as a selective topo-optical test of vicinal OH and amino-OH groups is suited for the selective demonstration in tissues of microorganisms of polysaccharide containing cells. Alkaline pretreatment of the polysaccharide cell walls, releases, by splitting the O-acyl radicals, further vicinal OH groups for the ABT reaction, thus actually increases the sensitivity of the method. The topo-optical reactions are characterized by a strong birefringence induced by oriented dye-binding, due to the linear arrangement of polysaccharides composing the cell wall. Differences in the character of birefringence have made it possible to work out a new method for the analysis of the cell wall ultrastructure as well as to demonstrate microorganisms in tissues. The practical value of the reactions is illustrated by examples.     

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes

Summary The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the gluycophorin A.     

Polarization optical analysis of blood cell membranes

The present study deals with investigations of membrane structure using polarization topo-optical reactions. Polarization microscopy is a special field of biological submicroscopic morphology. It represents a powerful tool well able to reveal the features of organization of biological structures, and the regularity of macromolecules building cells and tissues - properties that cannot directly be studied by other approaches to complex biological systems. Only in "pure" systems can X-ray diffraction, or the analysis of circular dichroism and the dispersion of optical rotability provide data equivalent to those obtained by polarization microscopy in complex systems. One of the main drawbacks of molecular biology is that most information is relevant to isolated, purified particles or macromolecules. Thus, no conclusions can be drawn concerning the original arrangement of molecules. The gap between biochemical-biophysical and morphological approaches to molecular arrangement in complex structures is bridged by the polarization optical technique. As was pointed out in the introduction, polarization microscopy became a routine biological research method following the pioneering work of Romhányi. His enlightening topo-optical reactions (Romhányi 1960, 1963, 1966) were based on the oriented dye binding of the original charge carriers of regularly arranged tissue constituents. The second group of Romhányi's topo-optical reactions comprised procedures such as sulfation (Romhányi et al. 1973, 1974), the aldehyde-bisulfite-toluidine blue (ABT) reaction (Romhányi et al. 1974, 1975), the permanganate-bisulfite-toluidine blue (PBT) reaction (Fischer 1979, 1979a), and the sialic acid-specific reaction (Makovitzky 1980) all of which operate with induced dye-binding groups; i.e. dye-binding moieties on biological macromolecules are produced by specific chemical reactions.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

Proteins of marsupial erythrocyte membranes

The proteins of erythrocyte membranes from the red kangaroo, western grey kangaroo, eastern grey wallaroo (euro), red-necked wallaby, Tammar wallaby, and brush-tail possum have been fractionated on acrylamide gels in the presence of sodium dodecyl sulfate. The pattern of proteins was remarkably similar between the different marsupial species. The pattern of Coomassie blue-staining proteins in the membranes of these species was also very similar to that of the human erythrocyte membrane. However, the glycoproteins in the marsupial erythrocyte membranes were markedly less conspicuous than those of the human erythrocyte membrane. Furthermore, the mobilities of the glycoproteins from the marsupials were different from those of the human erythrocyte membrane. The erythrocytes of the western grey kangaroo, the eastern wallaroo and the red-necked wallaby showed pronounced resistance to hypotonic lysis compared with those of the Tammar wallaby and the human. This effect seems to be related to the size of the erythrocytes rather than to differences in their protein composition.     

Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Topo-optische Färbung der Erythrocytenmembran mit Thiazin- und Chinolinfarbstoffen

Zusammenfassung Die Untersuchungen erwiesen, daß die Farbstoffe 1:9-Dimethyl-Methylenblau, Azur A und N,N-Diäthylpseudoisozyaninchlorid für topo-optische Reaktionen an der Membran von Erythrocyten geeignet sind. Die Farbstoffmoleküle werden an der Membran orientiert gebunden. Ihre Bindung kann durch Behandlung mit Präzipitationslösungen stabilisiert werden, und zugleich wird die Anisotropie verstärkt. Die optische Analyse ergab, daß 1:9-Dimethyl-Methylenblau und Azur A radiär zur Membran ausgerichtet sind, während sich N,N-Diäthylpseudoisozyaninchlorid membranparallel anlagert.

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Tope-optical staining with thiazin and quinolin dyestuffs of the erythrocyte membrane

Summary The present studies prove the dyestuffs 1.9-dimethyl methyleneblue, azure A and N,N-diethylpseudoisocyanine chloride suitable for topo-optical reactions with the membrane of the red blood cell. The dye molecules are bound in orientated fashion. Treatment with precipitants stabilizes the binding of dye molecules and, in addition, it enhances the birefringence. Optical analysis revealed 1.9-dimethyl methyleneblue and azure A bound in radial position, however, N,N-diethylpseudoisocyanine chloride was bound parallel to the membrane's plane.