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K Smetana

Publications and source records attributed to K Smetana.

At least 19 recordsLinked to original sources

Nucleolar coefficient of granulocyte precursors and granulocytes after visualization of nucleoli by two different methods.

To clarify differences in the incidence and number of nucleoli in the granulopoietic lineage, these nuclear components were studied in human and rabbit granulocyte precursors and granulocytes after visualization by 2 widely employed cytochemical procedures, i.e. a procedure for the demonstration of RNA and the silver reaction for the demonstration of nucleolar silver stainable proteins (SSPs). In early stages of the granulocyte proliferating compartment, substantial differences were not found between specimens in which nucleoli were visualized by both procedures. However, in contrast to specimens stained with the silver reaction, the number of cells without nucleoli was substantially larger in advanced stages of granulocyte development in specimens stained for RNA. The number of nucleoli per cell as expressed by the nucleolar coefficient was generally larger in specimens stained with the silver reaction for nucleolar SSPs. These differences were significant starting with the stage of myelocytes. Moreover, in specimens stained with the silver reaction, most of human mature granulocytes did not contain nucleoli but nucleoli were present in all mature granulocytes of rabbits. Such differences were not observed in specimens stained for RNA in which most granulocytes were without RNA-containing nucleoli. Thus, the evaluation of the presence or absence of nucleoli in specimens depended on the visualization procedure. It is likely that in micronucleoli which are characteristic for terminal differentiation of the granulocytic lineage, RNA-containing structures may be lost or are below the detection limit of the light microscope. In addition, differences in the presence of nucleoli exist apparently between human and rabbit granulocytes in specimens stained for SSPs but not in those stained for RNA.

Animals↗

Endogenous lectins (galectins-1 and -3) as probes to detect differentiation-dependent alterations in human squamous cell carcinomas of the oropharynx and larynx.

Expression of glycan determinants for in situ binding is the prerequisite for a productive protein (lectin)-carbohydrate recognition. Labeled tissue lectins as tools are preferable to plant lectins to assess this parameter, because plant and animal lectins with identical saccharide specification can well differ in their profiles of oligosaccharide binding pattern. Due to their relevance in growth control and matrix adhesion the family of galectins (galactoside-binding metal ion independent animal lectins) is receiving increasing utilization in human biology. Employing biotinylated galectin-1 and galectin-3 we studied the expression of binding sites for these galectins in normal human squamous epithelium and human carcinomas from the oropharyngeal region and larynx in relation to the expression of LP-34+ cytokeratins by the procedure of double labeling. Tissue sites accessible for galectin-1 were located in all layers of normal epithelium and in tumor cells. In contrast, galectin-3 binding was suprabasal in the normal epithelium and in tumor cells exhibiting signs of keratinization. These results reveal differences in the localization of accessible sites for the two galectins. Relating to cell development galectin-3 appeared to display affinity to areas with increased extent of differentiation.

Antigens, Differentiation↗

Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells.

Galectin-3 is an immunomodulatory protein with binding capacity for various glycoconjugates including IgE. It has been shown to be produced by epidermal keratinocytes and is present on the surfaces of skin Langerhans cells (LC). Therefore, it may have a role in the pathogenesis of various skin diseases, such as atopic dermatitis. To study the expression of galectin-3 in LC, we used, in addition to specific antibodies, a panel of synthetic, carrier-immobilized, specific oligosaccharides of the A- and B-histo-blood group, which are recognized by this lectin. In the mean time, Birbeck granules were visualized with an anti-Lag antibody. The double labeling experiments showed a remarkable colocalization of signals for Lag antigen (Birbeck granules) and galectin-3, as well as the binding sites for A- and B-histo-blood group trisaccharides. The specificity of the oligosaccharide binding was demonstrated by the lack of binding by Le(c), Le(d) (H blood group antigen), and sLe(x), which are not recognized by galectin-3. These results suggest that galectin-3 is present in Birbeck granules, where it retains reactivity for its glycoligands.

ABO Blood-Group System↗

The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations.

The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.

Cell Nucleolus↗

Binding sites for carrier-immobilized carbohydrates in the kidney: implication for the pathogenesis of Henoch-Schönlein purpura and/or IgA nephropathy.

BACKGROUND: Henoch-Schönlein purpura is a common vasculitis of childhood affecting the skin, joints, gastrointestinal tract, and kidney. The mesangial deposition of IgA1 is the most critical factor for the prognosis of patients with this disease. The aberrant glycosylation of the IgA1 subclass with the absence of terminally located galactose and presence of only alpha-N-acetylgalactosamine in O-linked oligosaccharides in the hinge region of IgA1 represents a prominent difference from the normal IgA1. These alterations prompt the supposition that the sugar part may guide IgA deposition by recognition of endogenous lectins on the mesangium. METHODS: Owing to the limited knowledge about the expression of carbohydrate-binding sites in the human kidney we initiated the study of this aspect with a class of tools which are suitable to map the lectinome of cells. Employing biotinylated neoglycoconjugates, glycosaminoglycans, and sulphated polysaccharides we monitored the presence of accessible carbohydrate-binding sites in control kidneys represented by tumour-free areas of kidneys with Grawitz tumour and in biopsies from patients with Henoch-Schönlein purpura-associated IgA nephropathy. RESULTS: Using frozen sections, no expression of any tested carbohydrate-binding site(s) was observed in the endothelial and the mesangial cells in glomeruli of the control kidneys as well as in the biopsies from Henoch-Schönlein purpura IgA nephropathic kidneys, in contrast to the tubules. The N-acetylgalactosamine-binding sites were expressed only in the inner layer of Bowman's capsule of 20% of glomeruli of the control kidney from one patient with Grawitz tumour and one biopsy from a patient with Henoch-Schönlein purpura-associated IgA nephropathy. However, the macrophages in the glomeruli of patients with IgA nephropathy and interstitial macrophages from both studied groups, i.e. without and with IgA nephropathy, harbour capacity to recognize carrier-immobilized alpha-N-acetylgalactosamine. Access to this binding site for the neoligand conjugate can be blocked by the monoclonal antibody MEM-18 recognizing CD14 antigen. CONCLUSION: The possibility for a participation of macrophage deposition of IgA1 in mesangium via a lectin mechanism involving this binding capacity warrants further studies.

Acetylgalactosamine↗

Incidence of nucleoli in erythroblasts in patients suffering from refractory anemia of myelodysplastic syndrome.

Nucleoli of erythroblasts have been studied in patients suffering from refractory anemia (RA) of myelodysplastic syndrome (MDS) and in control patients without a disturbed erythropoiesis in order to provide information on the incidence of nucleoli and micronucleoli in these cells. Nucleoli in erythroblasts were visualized by a simple cytochemical procedure for the demonstration of RNA which facilitated the visualization not only large nucleoli but also micronucleoli in advanced stages of the erythroblastic maturation. In control patients nucleoli were detected in all stages of erythroblastic development. In patients suffering from RA of MDS, a relatively large population of polychromatic and orthochromatic erythroblasts was characterized by a loss of nucleoli accompanied by the decreased incidence of micronucleoli characteristic of these cells. In contrast to control patients, in patients suffering from RA of MDS the number of nucleoli expressed by the values of the nucleolar coefficient of erythroblasts was smaller, particularly in both the early and terminal stages of erythroblastic development. Thus in patients with RA of MDS both the abnormal loss of nucleoli and decreased number of nucleoli in erythroblasts apparently represent and reflect a further abnormality of disturbed erythropoiesis.

Anemia, Refractory↗

A note on nucleoli in granulocytic precursors of the granulopoietic proliferating compartment in patients suffering from the chronic phase of chronic myeloid leukemia treated with two different drugs with different mode of action.

The incidence of main nucleolar types in granulocytic precursors was studied in the granulopoietic proliferating compartment (GPC) of patients suffering from chronic phase of the chronic myeloid leukemia (CML) who were treated by the widely used therapy with two different drugs with different mode of action - hydroxyurea (HU) and interferon alpha (IFN-alpha). In comparison with IFN early stages of GPC, i.e. myeloblasts and promyelocytes in patients treated with HU possessed more frequently micronucleoli which are known to reflect the direct as well as indirect inhibition of the nucleolar biosynthetic activities. On the other hand, the incidence of micronucleoli in these cells of a small percentage of patients treated with IFN also reached the average values of these nucleoli which were noted in patients treated with HU.

Cell Nucleolus↗

Experimental lesion of medial frontal cortex mediates inhibition of expression of carbohydrate-binding sites in the spleen macrophages in rat.

The fundamental concept of a neuroimmunological network is well appreciated although detailed description of the individual mechanisms has not yet been attained. In an effort to close this gap, the effect of electrolesion of the frontal medial cortex, a structure with the known "immunoreactivity", on selected phenotypic features of spleen macrophages was studied. Since sugar receptors (lectins) are pivotal for recognition, custom-made tools termed neoglycoproteins were employed to delineate any injury-induced changes of their expression. The total number of macrophages in the spleen red pulp was assessed using the ED-1 monoclonal antibody. The results showed that after lesion of the medial frontal cortex, the extent of expression of carbohydrate-binding sites in red pulp spleen macrophages significantly decreased without affecting the total number of these cells. These data intimate that distinct brain regions are involved in the control of the phenotype of macrophages in the central lymphoid organs by currently elusive biochemical mechanisms.

Animals↗

The asymmetric distribution of interphasic silver-stained nucleolus organizer regions in human and rat proerythroblasts.

The distribution of SSPs representing AgNORs was studied in human as well as rat proerythroblasts to provide information on the distribution of these nucleolar components in highly immature and proliferating non-neoplastic cells. The distribution of SSPs was asymmetric and most of the cells contained one nucleolus which possessed a larger number of these nucleolar components than the remaining nucleoli. Such nucleolus might be functionally dominant, since the number of nucleolar SSPs is apparently related to the nucleolar biosynthetic activity. On the other hand, when a proerythroblast possessed only one nucleolus, the number of SSPs in such a cell was very similar to the sum of SSPs in a polynucleolar cell. The asymmetric distribution of SSPs characteristic for most proerythroblasts disappeared in the terminal stages of the erythroblastic development. Cells in such stages, as described previously, were characterized by the presence of a limited number of single SSPs.

Animals↗

Detection of alpha(beta)-N-acetyl-D-galactosamine-binding sites in kidney--relation to Henoch-Schönlein-associated IgA nephropathy.

Henoch-Schönlein purpura is the most common vasculitis of childhood, accompanied by the deposition of IgA1 immunoglobulins into the glomerular mesangium. The actual molecular mechanism of IgA deposition is not clear, but the altered glycosylation of O-linked oligosaccharides of the hinge region of IgA1 is generally considered as the crucial etiopathogenic factor. The oligosaccharides of this glycoprotein from healthy persons are principally of mucin-type Galbeta1,3GalNAcalpha-O-glycan core structure, frequently sialylated. The patient's IgA hinge region saccharide is an incomplete GalNAcalpha-O-glycan only. This study investigates the presence of binding sites for alpha-GalNAc and beta-GalNAc in frozen sections of kidney with and without nephropathy prompted by the possibility for a lectin mechanism of IgA deposition to mesangium. Neoglycoproteins prepared as conjugates with derivatized alpha- or beta-GalNAc moieties as histochemically crucial ligands and biotinylated bovine serum albumin as a carrier were employed for this purpose. The result of the experiments demonstrated expression of specific and accessible binding sites for both alpha- and beta-GalNAc in tubules but not in glomeruli of kidney samples both with and without nephropathy. These findings imply no involvement of a lectin mechanism of IgA1 binding to mesangium, unless a temporary alteration of accessibility of binding sites for probes in glomeruli occurs or the linkage region beyond the monosaccharide is pivotal for a receptor whose binding site may accommodate a peptide epitope in addition to the O-linked alpha-GalNAc residue.

Acetylgalactosamine↗

Simultaneous detection of endogenous lectins and their binding capacity at the single-cell level--a technical note.

Endogenous lectins are proteins/glycoproteins which selectively recognize distinct saccharide ligands and are different from immunoglobulins and carbohydrate-utilizing enzymes. Expression of these molecules can be detected immunohistochemically using nonblocking monoclonal antibodies (A1D6: anti-galectin-3, MR-15-2-2: anti-175 kD mannose receptor). Alternatively, biotinylated (neo)glycoconjugates which are recognized by a studied lectin can be employed as convenient probes to demonstrate specific binding of sugar epitopes by carbohydrate recognition domains (CRD) of endogenous lectins. In this study, we describe a new procedure for immunocytochemical visualization of the expression of endogenous lectins and glycochemical visualization of the reactivity of carbohydrate recognition domain(s), performed simultaneously at the single-cell level.

Animals↗

Attempts to induce melanosome degradation in vivo.

Contradiction between repeatedly reported electron microscopic and histochemical observation of melanosome disintegration in the presence of lysosomal enzymes in vivo and failing attempts to induce such degradation by biochemical means in vitro with the aim to explain chemical mechanism(s) of this process belongs to chronically challenging problems in biochemistry of melanin structures. Attempts have been made to bring about melanosome disintegration in vivo by inoculating melanosomes isolated from dog hair into the tissue of amelanotic Bomirski melanoma and into peritoneal cavities of DBA/2 and C57BL/6J mice, and by repeated injection of melanosomes isolated from Bomirski pigmented melanoma into Syrian hamster foot pads. Both histological and electron microscopic observations demonstrated that melanosomes were phagocytized by macrophages and sporadically by fibroblasts. In peritoneal cavities the injected foreign melanosomes remained mostly extracellularly, were surrounded by foreign body multinuclear cells and formed granulomas. There were no convincing signs of degradation of the hair melanosomes, which behaved like inert foreign bodies. Only some phagocytized Bomirski hamster melanoma melanosomes tended to lose their integrity. Our data suggest that melanosomes devoid of their limiting membranes are not necessarily prone to extensive disintegration in vivo. The earlier reported association of lysosomal enzymes with disintegrating melanosomes does not constitute an evidence for their participation in melanosome degradation. Considering the structure of melanins, redox mechanisms (analogous with the metabolism of polycyclic hydrocarbons (DePierre and Ernster, 1978)) seem to be more probably involved in pigment degradation than hydrolytic reactions.

Animals↗

The brain lesion influences the numbers of peripheral blood leukocytes in the rat.

The mutual relationship between the central nervous and immune system are intensively studied. The lesion of distinct structures of the rat brain such as septum influence the model immune response such as lymphocyte proliferation and delayed skin hypersensitivity. Employing the model of the damage of septum in the rat brain by electrolesion we demonstrated the decrease of the number of peripheral blood leukocytes, mainly cells exhibiting CD25 and CD45RA antigens in the rat. The striatum destruction has much lower influence on the studied parameters, which suggests a specific effect of the septum on these hematological parameters.

Animals↗

The nucleolar functional asynchrony--imbalance--in the granulopoietic proliferating compartment of patients suffering from chronic myeloid leukemia.

Nucleoli were studied in all stages of the granulopoietic proliferating compartment in the bone marrow of patients suffering from chronic myeloid leukemia to provide an information on the incidence of the nucleolar functional asynchrony (imbalance) in these cells. The nucleolar functional asynchrony is morphologically expressed by the presence of "active" large nucleoli with a relatively uniform distribution of ribonucleic acid (RNA) and "resting" ring shaped nucleoli with RNA only in their peripheral part in one and the same cell. This phenomenon was noted in a small but constant percentage of myeloblasts and decreased in myelocytes regardless of the phase of the disease and therapy. In addition, the nucleolar functional asynchrony was also noted in all stages of the granulopoietic proliferating compartment of control not-leukemic persons.

Bone Marrow Cells↗

Preferential silver reaction of nucleolar regions adjacent to fibrillar centers in ring shaped nucleoli of leukemic lymphocytes.

Silver stained proteins (SSPs) characteristic for interphasic nucleolus organizer regions (NORs) associated with fibrillar centers (FCs) and adjacent nucleolar regions of ring shaped nucleoli in leukemic lymphocytes exhibit a different sensitivity to the mild acid extraction including that with HCl. Such extractions permit a preferential visualization of fibrillar centers adjacent regions (FCARs) which are believed to represent sites of the ribosomal RNA (rRNA) transcription. The resistance of SSPs in FCARs to the extraction with HCl seems to be due to their binding to other components present in these regions. The extractibility of SSPs with HCl was influenced by the fixatives used. The largest resistance of SSPs to the extraction with HCl was noted after fixation with glutaraldehyde. In contrast, the largest extractibility of these proteins was observed after fixation with unbuffered formaldehyde.

B-Lymphocytes↗

Fusion of macrophages on an implant surface is associated with down-regulated expression of ligands for galectin-1 and -3 in the rat.

Galectins have a wide range of biological activities which are elicited by binding to appropriate glycoligands. Besides regulation of the expression of the galectins the extent of the presence of suitable binding sites will be relevant to infer the cellular responsiveness to this class of sugar receptors. Thus ligand presentation requires monitoring by the tissue lectin. We demonstrate the expression of galectin-3 by macrophages and foreign-body giant multinucleate cells colonizing a cellophane implant in the rat by the A1D6 monoclonal antibody. The extents of ligand presence are visualized in the same cells by biotinylated galectin-3 and also by galectin-1 which is produced by diverse mammalian cell types and widely distributed. Labeled mistletoe (VAA) and tomato (LEA) lectins are used as tools to assess the degree of similarity of the binding profile between endogenous and exogenous proteins. The presentation of alpha-galactosides is monitored with a natural immunoglobulin G subfraction obtained by two consecutive affinity chromatography steps. The binding of labeled galectins and plant lectins was significantly lower to foreign-body giant multinucleate cells than to mononuclear macrophages. The application of the alpha-galactoside-specific probe yielded no significant staining. The potential problem of epitope accessibility could be excluded by the concomitant positivity obtained with an IgG subfraction with selectivity to beta-galactosides also obtained by affinity chromatography. These results provide no evidence for a role of alpha-galactosides for the binding of galectins in the rat macrophages colonizing the implant. The reduced level of expression of glycoligands for galectin-1 and -3 in foreign-body giant multinucleate cells in contrast with the mononuclear macrophages suggests an inhibitory influence of macrophage fusion on the expression of galectin-reactive molecules.

Animals↗