Biomedical subjects

E Skutelsky

Publications and source records attributed to E Skutelsky.

At least 19 recordsLinked to original sources

PubMed2003

Ceramide mediates growth inhibition of the Plasmodium falciparum parasite.

In mammalian cells, ceramide mediates death by chemotherapeutic drugs. We analysed, for the first time, the role of ceramide in inhibiting growth of the malaria-causing parasite Plasmodium falciparum. Added exogenously, ceramide significantly decreased the number of parasites, and this effect was abolished by sphingosine-1-phosphate, a biological antagonist of ceramide action. Ceramide can induce death of cancer cells by decreasing glutathione levels, and in our work it induced dose- and time-dependent depletion of glutathione in P. falciparum parasites. N-acetylcysteine, a precursor of glutathione, abrogated the cytotoxic effect of ceramide. Thus, ceramide can mediate growth inhibition of P. falciparum parasites by decreasing glutathione levels. The antimalarial drugs artemisinin and mefloquine induced the death of P. falciparum parasites by sphingomyelinase-generated ceramide and by decreasing parasite glutathione levels. Altogether, ceramide was identified as a signalling molecule capable of inducing growth inhibition of P. falciparum malarial parasites.

Animals↗

PubMed2001

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.

Animals↗

PubMed2001

Changes in xylosyltransferase activity and in proteoglycan deposition in bleomycin-induced lung injury in rat.

Several lines of evidence support the hypothesis of the involvement of altered proteoglycan deposition in the development of lung diseases. UDP-D-xylose: core protein beta-D-xylosyltransferase (UDP-xylosyltransferase; EC 2.4.2.26) is a key enzyme for the glycosylation of proteoglycan core proteins. This study examined the catalytic activity of UDP-xylosyltransferase in lung tissue and in isolated fibroblasts, as well as the deposition of the proteoglycans versican, biglycan and decorin in rat lung tissue during bleomycin-induced lung injury. Rats were given, endotracheally, a single dose of bleomycin. Deposition of proteoglycans in lung tissue was assessed by immunohistochemistry and the catalytic activity of xylosyltransferase was determined with an acceptor peptide of the sequence Q-E-E-E-G-S-G-G-G-Q-G-G as a substrate. The results show coincidence of increasing xylosyltransferase activities in lung tissue with accumulation of versican at alveolar entrance rings and in fibrotic regions in close proximity to alpha-smooth muscle actin-positive cells. In contrast, no changes in biglycan and decorin deposition in fibrotic lungs were observed, except for decorin in alveolar type II pneumocytes and alveolar macrophages. Bleomycin treatment of isolated rat lung fibroblasts resulted in a concentration-dependent increase of xylosyltransferase activity up to 2 mU bleomycin x mL(-1). The data suggest a participation of myofibroblasts with increased xylosyltransferase activities in accumulation of versican in fibrotic foci of injured lung tissue at the early stages of development of lung fibrosis.

Animals↗

PubMed2000

Macrophage-recognized molecules of apoptotic cells are expressed at higher levels in AKR lymphoma of aged as compared to young mice.

While a direct relation between aging and tumorigenesis is well established, a slower tumor progression rate was reported in old as compared to young cancer patients. The mechanisms responsible for the less aggressive behavior of tumors in the aged, are largely unknown. We have recently shown an increase in apoptotic cell death in tumors derived from aged as compared to young animals in the AKR lymphoma. This was shown by DNA flow cytometry and by the ladder type DNA fragmentation in agarose gel electrophoresis. Analysis of the expression of genes involved in apoptosis in tumors derived from young and old animals showed a lower bcl-2 expression in those from the aged. The Fas antigen, on the contrary, displayed higher expression levels on lymphoma cells derived from old than on those from young mice. Apoptotic cells are recognized and phagocytosed mainly by macrophages. One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues. While comparing the "eat me status" phenotype of the tumor cells derived from young and aged animals, by the use of lectins recognizing sialic acid and galactose residues, FACS analysis showed a decrease in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis showed that a 130 Kda sialylated membrane glycoprotein was expressed at a lower level in tumors from the old as compared to young mice. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested, one of the mechanisms of the age-related decrease in tumor progression rate.

Aging↗

PubMed2000

Sensitivity to macrophages decreases with tumor progression in the AKR lymphoma.

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-1), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified 3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, inMT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found moreover that WGA-treated tumor cells were more sensitive than nontreated cells to macrophages in vitro.

Animals↗

PubMed2000

Glomerular basement membrane polyanion distribution and nitric oxide in spontaneous hypertensive rats: effects of salt loading and antihypertensive therapy with propranolol.

Cationic colloidal gold (CCG), a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, mainly heparan sulfate proteoglycan in spontaneous hypertensive rats (SHR) with or without salt loading and antihypertensive treatment with propranolol. The changes of mean GBM width and anionic sites distribution were assessed by electron microscopy. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO2) + nitrate (NO3), stable metabolites of NO. SHR had decreased NO production and increased GBM width (27%) compared with the control Wistar-Kyoto (WKY) rats. The chronic high dietary salt intake resulted in a significant increase in blood pressure, proteinuria, and renal function in the SHR rats. The chronic high salt dietary intake resulted in a decrease in NO in the WKY and a further reduction in NO production in the SHR. The GBM anionic sites count was similar in the SHR and WKY nonsalt-loaded groups, 13.5 +/- 0.5 and 12.8 +/- 0.4 CCG counts/microm GBM, respectively, but significantly lower in both salt-loaded SHR and WKY, 9.9 +/- 0.55 (P < .01) and 9.6 +/- 0.55 (P < .01) CCG counts/microm GBM, respectively. Antihypertensive treatment with propranolol in the salt-loaded SHR group resulted in lower blood pressure, a further decrease in NO production, but no significant changes in GBM width and anionic sites count. It is concluded that chronic high salt intake may be deleterious to the permselectivity of the GBM. A low NO production state that results from chronic salt loading in already hypertensive rats will result in more severe organ (renal) damage, most probably by the addition of the loss of GBM permselectivity to the existing pathomorphologic changes.

Animals↗

PubMed2000

Glomerular basement membrane anionic sites in adriamycin nephropathy: effect of saline loading and nitric oxide modulation.

BACKGROUND: In previous studies we found that experimental Adriamycin (ADR) nephropathy is associated with the loss of glomerular basement membrane (GBM) anionic sites provided by heparan sulfate proteoglycans. Chronic saline loading in normal rats resulted in a similar effect on the GBM anionic sites. The L-arginine-nitric oxide synthase-nitric oxide system is involved in the pathogenesis of experimental chronic renal failure. The present study was performed to determine the combined effect of nitric oxide (NO) modulation and chronic saline loading in ADR nephropathy. The modulation of NO was done by chronic administration of L-arginine (NO donor) or N(w)-nitro-L-arginine, a known nitric oxide synthase inhibitor. METHODS: Systolic blood pressure was measured in awake rats by a tail-cuff method. Renal function was assessed by creatinine clearance, FeNa%, and daily protein excretion. The change of mean GBM widths and anionic sites distribution were assessed by electron microscopy. The localization of anionic sites was carried out by cationic colloidal gold. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO(2)) + nitrate (NO(3)), stable metabolites of NO. RESULTS: Two weeks after the ADR administration (3.5 mg/kg BW iv) the rats had severe renal failure (creatinine clearance 134 +/- 31 microl/min/100 g BW vs. initial values 670 +/- 29 microl/min/ 100 g BW, p < 0.001), high FeNa%, severe proteinuria, increased GBM width, significant reduction of GBM anionic sites and low urinary NO(x) excretion. The saline loading resulted in further reduction of GBM anionic sites count and blood pressure elevation. The inhibition of NO did not change the course of ADR nephropathy. The main finding of the present study is that chronic administration of L-arginine significantly alleviates the renal failure in the ADR (+/- saline loading) nephropathy. The L-arginine-treated rat had higher creatinine clearance, lower FeNa% and protein excretion and complete normalization of GBM anionic sites distribution. CONCLUSIONS: Sodium loading has a deleterious effect on GBM permselectivity. L-Arginine prevents the reduction of GBM anionic sites, decreases proteinuria and alleviates the renal insufficiency in ADR nephropathy.

Animals↗

PubMed2000

S-nitrosoderivative of a recombinant fragment of von Willebrand factor (S-nitroso-AR545C) inhibits thrombus formation in guinea pig carotid artery thrombosis model.

Antiplatelet drugs are the mainstays of therapy for acute and chronic cardiovascular diseases. S-nitroso-AR545C - an S-nitrosoderivative of a recombinant von Willebrand factor fragment AR545C spanning Ala 444 to Asp 730 and containing an Arg 545 Cys mutation, was previously found to inhibit ristocetin- and ADP-induced platelet aggregation and the interaction of platelets with extracellular matrix (ECM). In the current study we tested the antithrombotic properties of S-nitroso-AR545C on guinea pig platelets and in a platelet-rich thrombosis model in the guinea pig. Preincubation of guinea pig platelets with 0.1 microM of S-nitroso-AR545C decreased ristocetin-induced agglutination by 40% (p = 0.009) and completely abolished ADP-induced aggregation (p <0.0001). At concentration of 1.0 microM, S-nitroso-AR545C completely inhibited platelet adhesion (represented by surface coverage - SC) and decreased aggregate formation (represented by average aggregate size - AS) by more than 50%. Treatment of guinea pigs with 1.0 mg/kg S-nitroso-AR545C resulted in a significantly delayed time to arterial occlusion (31.7+/-6.0 min vs. 13.9+/-3.2 min, p <0.02). Similarly, total patency time was longer in the group injected with S-nitroso-AR545C compared to the control group. However, the difference was not statistically significant (33.8+/-6.3 min vs. 20.2+/-3.3 min, p = 0.07). No change in platelet count, hematocrit and bleeding time was observed 60 min after injection compared to baseline. In contrast, a significant decrease in SC (p <0.0001) and AS (p <0.01) were observed 60 min after the injection of S-nitroso-AR545C, whereas no change in these parameters was observed in the control group. These observations indicate that S-nitroso-AR545C exhibits significant antiadhesive and antiaggregating effects in-vitro and inhibits clot formation in-vivo suggesting that this compound may have potential therapeutic advantages.

Animals↗

PubMed1999

Inability of encapsulated Klebsiella pneumoniae to assemble functional type 1 fimbriae on their surface.

We screened phase variants of Klebsiella pneumoniae isolates for the expression of capsule and type 1 fimbriae and found that all of the 22 blood isolates were encapsulated and did not express type 1 fimbriae while 10 of 11 urinary tract isolates expressed type 1 fimbriae but were unencapsulated. Phase variants from selected isolates were found to be either unencapsulated and fimbriated or lacked both structures. Variants expressing both structures were not detected. Fimbrial subunits FimH and FimA were localized in the periplasmic space of the parent strain and on the surface of the unencapsulated variants. The results suggest that capsule formation impedes assembly of pre-formed fimbrial subunits on the bacterial surface.

Bacteremia↗

PubMed1998

Mechanisms leading to cortical reaction in the mammalian egg.

Activation of the mammalian egg results in cortical reaction (CR), which is correlated with an increase in intracellular Ca2+ concentration and PKC activation. The CR is a gradual rather then an "all or none" response, and can be regulated by different concentrations of parthenogenetic activators. To evaluate the biological significance of parthenogenetic induced CR, rat eggs were fertilized or activated by different concentrations of ionomycin and TPA. Cortical granules (CG) were monitored by electron microscopy, while the CG exudate was visualized by Lens culinaris lectin and Texas Red, using light and confocal microscopy. The ability of the CR to trigger a full block to polyspermy was examined in an IVF system. Our study demonstrates the existence of light and dark CG, which differ by number, distribution in the egg cortex, and sensitivity to parthenogenetic activators. Sperm penetration or high concentration of activators, trigger depletion of both light and dark CG, leading to a full CR. Low concentration of activators altered the CG density, the ratio of dark/light CG, and induced partial CR that was sufficient to cause a block to polyspermy. The results imply that Ca2+ rise or PKC activation have different effects on light and dark CG. In recently fertilized or parthenogenetically activated eggs, CG exudate appeared as evenly distributed spots, whereas in more advanced stages of fertilization the exudate was scattered as patchy aggregates. This observation suggests a difference in the dispersion of CG exudate after fertilization as compared to parthenogenetic activation.

Animals↗

PubMed1998

Protein F1 is required for efficient entry of Streptococcus pyogenes into epithelial cells.

It was recently reported that strains of Streptococcus pyogenes are capable of inducing entry of the bacterium into epithelial cells; however, nothing is known regarding the gene(s) and the underlying mechanism(s) involved. Using isogenic mutants of S. pyogenes JRS4 strain that are defective in the expression of each of the surface proteins F1 and M6, it was demonstrated that both are required for efficient internalization. Expression of F1 on the surface of a poorly invading S. pyogenes strain significantly enhances its internalization efficiency. Protein F1-mediated internalization is inhibited by UR, the nonrepetitive fibronectin-binding domain of this protein, and to a lesser extent, by the repetitive fibronectin-binding domain, RD2. Polyclonal anti-human fibronectin antibodies completely abolish F1-mediated internalization; increasing fibronectin concentrations result in a significant enhancement of bacterial uptake. The findings shown here suggest that protein F1 mediates streptococcal internalization and that the M6 protein is required for more efficient entry of the bacterium.

Adhesins, Bacterial↗

PubMed1997

Glomerular basement membrane polyanionic sites and nitric oxide in genetically salt-sensitive and resistant hypertensive rats.

Cationic colloid gold, a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, including heparan sulfate protoglycan in genetic salt-sensitive (SBH/Y) and resistant (SBN/Y) hypertensive rats, with or without high dietary salt intake. GBM morphology, renal function and nitric oxide, as measured by plasma and urine nitrite (NO2) and nitrate (NO3) were also determined. In the salt-sensitive rats the high-salt dietary intake resulted in severe hypertension, proteinuria and decreased glomerular filtration rate. After 1 month of high-salt intake, the average width of the GBM of salt-sensitive rats was higher by 27% than that of salt-resistant rats. The number of GBM anionic sites (lamina rata externa and interna) was much lower in both salt-sensitive and salt-resistant groups after 1 month of salt loading, 8.04+/-0.36 and 7.8+/-0.25 counts/cm, respectively, compared to the respective values of non-salt-loaded animals, 20.58+/-1.08 counts/cm in the SBH/Y (p < 0.001) and 21+/-1.86 counts/cm in the SBN/Y (p < 0.001). A decreased nitric oxide production was found in the salt-sensitive rats before and after salt loading compared with the salt-resistant group. No correlation was found between the nitric oxide changes and the GBM modifications. It is concluded that high-salt intake may be deleterious to the permselectivity of the GBM. It is suggested that salt restriction in hypertension may have a beneficial effect in preventing GBM permselectivity changes and proteinuria.

Animals↗

PubMed1997

Distribution of glycosaminoglycans in rat renal tubular epithelium.

Polyanionic constituents of the glomerular capillary wall have been previously shown to have a primary role in the control of glomerular filtration. In the study presented here, the distribution and biochemical nature of polyanionic constituents in proximal (PT) and distal (DT) tubules have been investigated as possible determinants of tubulointerstitial function. For histochemical localization of sialic acid, paraffin sections were treated with Arachis hypogaea lectin (PNA) before and after neuraminidase treatment. Electron microscopic characterization of glycosaminoglycans (GAG) was performed on thin LR-white sections, using cationic colloidal gold (CCG) as an histochemical probe, and GAG-degrading enzymes. Without neuraminidase, PNA binded to collecting ducts but not to PT or DT. Neuraminidase pretreatment resulted in intense PNA binding to the tubulointerstitial blood vessels but only in mild apical tubular binding, which implies a lack of sialoglycoconjugates in the tubular basolateral membranes. In contrast, all PT and DT showed intense CCG binding to basolateral, but not to apical, membranes. All basement membranes showed CCG labeling, with considerable variations in labeling densities between PT (124 +/- 8.8/micron 2) and DT (52 +/- 1.8/micron 2), as well as between tubules and Bowman's capsule (P < 0.0001). Heparinase III treatment induced an almost complete loss of CCG binding in all basement and basolateral membranes, whereas chondroitinase ABC treatment led to a lesser but significant loss (P < 0.0001). The results indicate that rat tubulointerstitium expresses polyanionic constituents, consisting mainly of heparan and chondroitin sulfate. The role of these anionic sites in tubular function has yet to be clarified.

Animals↗

PubMed1997

Human mucopolysaccharidosis IIID: clinical, biochemical, morphological and immunohistochemical characteristics.

Mucopolysaccharidosis IIID (MPS IIID) is one of the rarest of the MPS-III syndromes. To date, the clinical manifestations of 10 patients have been reported, the deficient N-acetylglucosamine 6-sulfatase (G6S) enzyme has been purified, and the G6S gene has been cloned, sequenced and localized. However, morphological manifestations of this condition have not been reported and the pathogenesis of the severe neurological deficits remains an enigma. In this paper we describe and correlate the clinical, biochemical and pathological observations for 2 cases of MPS IIID. We used monoclonal antibodies against heparan sulfate (HS) and GM2-ganglioside, thin layer chromatography, mass spectrometry, and morphological techniques to demonstrate the nature and the distribution of the uncatabolized substrates. The majority of the cells in various tissues showed morphological changes expected with lysosomal storage of HS. The central nervous system (CNS) was most severely affected because of the secondary storage of GM2 and GM3 gangliosides in addition to the primary accumulation of HS. The extent as well as the distribution of the diverse storage materials varied within and among different neurons as observed in MPS-III A, B, and C syndromes. This study supports the hypothesis that the neurological dysfunction and neurodegeneration common to the Sanfilippo syndromes is, in part, due to the secondary metabolic perturbations induced by HS accumulation.

Adolescent↗

PubMed1996

Post-fertilization changes in the zona pellucida glycoproteins of rat eggs.

The zona pellucida (ZP) is the extracellular coat surrounding the mammalian egg. Numerous evidence supports the role of ZP carbohydrate residues as the specific sperm receptors. In this study we used lectins to study different distribution patterns of carbohydrate residues in the rat ZP, and to follow changes at fertilization. ZP were collected from follicular, ovulated, and fertilized eggs, incubated with one of 11 different biotin-labeled lectins, followed by avidin-fluorescein isothiocyanate (FITC) complex, and visualized by epifluorescent microscopy. For electron microscope (EM) histochemistry, eggs were embedded in LR white and ultrathin sections were stained with the complex Ricinus communis lectin (RCA-1)-colloidal gold. Some lectins (RCA-I, Glycine max) bound to the entire ZP while others were restricted to the inner or outer zones [Griffonia simplicifolia, Concanovalia ensiformis, Triticum vulgaris (WGA), succinyl-WGA]. Other lectins (Lens culinaris, Ulex europhaeus) were totally excluded. The RCA-1 binding pattern changed following sperm penetration, from homogeneous in ZP of ovulated eggs (57%) to uneven in ZP of fertilized (71%) or activated (68%) eggs. Our results demonstrate an uneven distribution of different sugar residues in the rat ZP, and a post-fertilization change in the distribution of beta-galactose, which is specifically recognized by RCA-I, presumably correlated with other changes in the ZP that lead to the block to polyspermy.

Animals↗

PubMed1996

Translocation of Shiga toxin across polarized intestinal cells in tissue culture.

Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [3H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.

Animals↗

PubMed1996

Lectin histochemistry of the submandibular and sublingual salivary glands in rats.

Tissue sections from rat submandibular and sublingual glands were studied with lectin probes to identify terminal sugars of the glycoconjugates in various cell types of the salivary glands. The lectins used in the study were Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Succinyl WGA (S-WGA) Ricinus communis I (RCA-I), Arachis hypogaea (PNA), and Ulex europeaus (UEA-I). The cytoplasm and cell membrane of both the serous and mucous acinar cells present high similarity in the distribution of some sugar residues, but differ considerably in the expression of specific sugars which appear either in the serous or in the mucous cells. The cytoplasm and cell membrane of the serous and mucous acinar cells express Mannose (Man) and Glucose (Glc), but lack Galactose (Gal), and N-acetylgalactosamine (GalNAc). Fucose (Fuc) is present only in the mucous acinar cytoplasm. The moderate to intense binding of WGA to the acinar and ductal cells and the lack of binding of S-WGA, indicate the presence of sialic acid rather than N-acetylglucosamine (GlcNAc). These sialic acid residues are not associated with PNA-binding sugar sequences as pretreatment with neuraminidase is not associated with exposure of additional PNA receptors.

Acetylglucosamine↗

PubMed1995

An histochemical approach to characterization of anionic constituents in mast cell secretory granules.

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO4, and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500-900 gold particles/microns2), while in other peritoneal cell granules it was about 10 times less (ca. 40-80 gold particles/microns2). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.

Animals↗