Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Cell Surface Carbohydrates in Tritrichomonas foetus1

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid-thiosemicarbazide-silver proteinate, concanavalin A-horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.     

Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1β-treated human peritoneal mesothelial cells

The peritoneal mesothelium acts as a regulator of serosal responses to injury, infection, and neoplastic diseases. After inflammation of the serosal surfaces, proinflammatory cytokines induce an “activated” mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1β for 48 h, respiratory activity of suspended cells was analyzed by high-resolution respirometry. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were determined by spectrophotometry. Treatment with IL-1β resulted in a significant decline of respiratory capacity (p<0.05). Respiratory control ratios (i.e., uncoupled respiration at optimum carbonyl cyanide p-trifluoromethoxyphenylhydrazone concentration divided by oligomycin inhibited respiration measured in unpermeabilized cells) remained as high as 11, indicating well-coupled mitochondria and functional integrity of the inner mitochondrial membrane. Whereas respiratory capacities of the cells declined in proportion with decreased CS activity (p<0.05), LDH activity increased (p<0.05). Taken together, these results indicate that IL-1β exposure of peritoneal mesothelial cells does not lead to irreversible defects or inhibition of specific components of the respiratory chain, but is associated with a decrease of mitochondrial content of the cells that is correlated with an increase in LDH (and thus glycolytic) capacity. Contributed equally to this work. For papers with multiple authorship, the asterisk identifies the author to whom correspondence and reprint requests should be addressed.     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae

Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.     

Carbonate precipitates and bicarbonate secretion in the intestine of sea bass, <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>

The aim of this paper was to study the chemical composition of the precipitates found in the intestine of Dicentrarchus labrax and the source of HCO₃ ⁻ secreted into the intestinal lumen. The chemical analysis was performed by employing the potentiometric double titration method and by means of an electron microscope coupled with a spectrometer and X-ray powder diffraction. The results obtained suggest the presence of very insoluble intestinal precipitates, presumably formed by a mixture of CaCO₃ and MgCO₃, with a higher quantity of the former with respect to the latter. HCO₃ ⁻ secretion rate was investigated with the aid of the pH stat method in isolated tissues mounted in Ussing chamber, where the transepithelial electrical parameters were also measured. When the serosal surface of the intestinal mucosa was bathed in HCO₃ ⁻-Ringer bubbled with 1% CO₂ in O₂ while the serosal surface was bathed in HCO₃ ⁻ free Ringer solution bubbled with pure O₂, bicarbonate secretion proceeded at an almost stable rate of 0.9 ± 0.05 μeq cm⁻² h⁻¹ for about 3 h while I _sc maintained a constant value of 38 ± 1.5 μA cm⁻². The carbonic anhydrase inhibitor ethoxyzolamide elicited a progressive reduction of HCO₃ ⁻ secretion that was about 75% of the initial value after 80 min. When serosal HCO₃ ⁻–CO₂ saline was substituted with Hepes–O₂ saline base secretion progressively declined reaching a value of about 20% of the initial value. It was also strongly inhibited when Na⁺ was substituted with the impermeant cation choline and when either DIDS or ouabain were added to the basolateral side. These results suggest that most of the bicarbonate secreted is of extracellular source and is probably transported across the basolateral membrane by both Na⁺ independent mechanism and Na⁺ dependent transporter, presumably a NaHCO₃ cotransport.     

Enzymatic properties of sialidase from the ovary of the starfish, Asterina pectinifera

A sialidase [EC 3.2.1 18] was isolated and highly purified from the ovary of the starfish, Asterina pectinifera, and its enzymatic properties were compared with those of human placental sialidase. The final preparation gave one broad protein band corresponding to sialidase activity on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 360 000 by HPLC on Sigma Chrome GFC-1300 and Sephadex G-150 column chromatography, and 55 000 by SDS-PAGE, suggesting the presence of a hexamer in the native protein. The optimum pH was between 3.0 and 4.0, and the enzyme liberated sialyl residues from the following compounds: α(2-3) and α(2-6) sialyllactose, colominic acid, fetuin, transferrin, gangliosides GM₃, GD_1a and GD_1b. The enzyme was strongly inhibited by 4-aminophenyl and methyl thio-glycosides of sialic acid, but not by those glycosides of 5-amino sialic acid or sialic acid methyl ester. The enzyme was also highly inhibited by sulfated glucan and glycosaminoglycans. The substrate specificity and the effects of inhibitors on starfish sialidase were very similar to those of human placental sialidase.     

Colloidal alpha-stannic acid and negative iron colloid as differential electron stains for surface proteins.

Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.     

Nonrandom distribution of sialic acid over the cell surface of bristle- coated endocytic vesicles of the sinusoidal endothelium cells

Previous studies with protein tracers have shown that the luminal surface of the vascular endothelium of the bone marrow is endocytic. The endocytosis occurs through the formation of large bristle-coated vesicles (LCV). The anionic charge distribution in this process was examined at the luminal surface of the endothelial cell, At pH 1.8, colloidal iron (CI), native ferritin, and polycationic ferritin (PCF) are bound by the luminal surface of the endothelial cell, but not at the sites of LCV formation. PCF used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels) revealed LCV binding of this agent in increasing manner from pH 3.5 upwards. PCF binding at low pH (1.8) at the endothelial cell surface was markedly reduced by neuraminidase. Neuraminidase did not reduce PCF binding by the endothelial cell surface nor by the LCV at higher pH levels. It is concluded that the luminal surface of the endothelial cell has exposed sialic acid groups which are absent or significantly diminished at endocytic sites. The free surface of the endothelial cells as well as the sites of endocytosis have, in addition, anionic material with a pKa higher than that of sialic acid (pKa 2.6). These anionic materials may be different at the sites of endocytosis as compared to those present at the free cell surface.     

Elektronenmikroskopisch-histochemische Untersuchungen zur Darstellung saurer Mucopolysaccharide und sialinsäurehaltiger Glycoproteine in Nierenrinde und innerem Nierenmark der Ratte

Zusammenfassung Saure Mucopolysaccharide und sialinsäurehaltige Glycoproteine wurden elektronenmikroskopisch-histoohemisch mit der kolloidalen Eisenreaktion in der Niere dargestellt. Alle Zellmembranen in Rinde und Mark lagern an der Oberfläche positive kolloidale Eisenteilchen an. Die Dicke der Eisenschicht ist bei den Zellen unterschiedlich und variiert auch zwischen luminaler, lateraler und basaler Seite einer Zelle. Bei Tubuli und Capillaren besteht eine Parallele zwischen der Dicke des Außenblattes der Plasmalemm-Unit membrane und der Dicke der kolloidalen Eisenschicht. Auf Grund biochemischer Analysen von Zellmembranen und negativer histochemischer Eisenreaktion nach Sialidasebehandlung ist anzunehmen, daß diese kolloidale Eisenschicht sialinsäurehaltigen Glycoproteinen entspricht. Dieser Glycoproteinbesatz ist ein integrierender Bestandteil der Zellmembran.In mehreren Zellen war das endoplasmatische Retikulum eisenpositiv. Auch hier wurde schon biochemisch Sialinsäure ermittelt.Die positive kolloidale Eisenreaktion im extrazellulären Raum beruht in der Nierenpapille auf dem Vorkommen von sauren Mucopolysacchariden und Sialinsäure und in der Lamina rara interna und externa der glomerulären Basalmembran auf dem Sialinsäuregehalt.

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Localization of acid mucopolysaccharides and sialic acid containing glycoproteins in the renal cortex and inner medulla by a combined electron microscopic histochemical method

Summary Acid mucopolysaccharides and sialic acid containing glycoproteins are localized in the kidney by the colloidal iron method. All cell membranes in cortex and medulla are coated by positive colloidal iron particles. This iron-coat variies in thickness at different cells and at the luminal, lateral and basal surface of one cell, too. Tubules and capillaries show a parallelism between the thickness of the outer leaflet of the plasmalemm unit-membrane and the thickness of the colloidal iron coat. Biochemical analyses of cell membranes and the negative result of the colloidal iron reaction after sialidase-treatment allow to conclude, that this colloidal iron coat represents sialic acid containing glycoproteins. This glycoprotein-coat is part of the whole cell membrane.In some cells the endoplasmatic reticulum reacts with colloidal iron. Here, too, sialic acid was found by biochemical analysis.The positive colloidal iron reaction in the extracellular space depends on acid mucopolysaccharides and sialic acid. In the lamina rara interna and externa of the glomerular basement membrane the positive colloidal iron reaction correlates to the sialic acid content.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Teilbefunde von A. Nolte auf der 51. Tagung der Deutschen Gesellschaft für Pathologie, Göttingen 1967, vorgetragen.     

Lectin-gold cytochemistry of the Golgi apparatus in rabbit luteal cells,with special emphasis on the formation of a lysosomal-type membrane

Summary In rabbit luteal cells embedded in glycolmethacrylate and stained with PTA at low pH highly glycosylated membrane patches can be observed after vesiculation of the trans-Golgi network. As these membranes could be prelysosomal, their sialic acid content was investigated by postembedding labeling with Limax flavus agglutinin (LFA)/fetuin-Au. Additional labeling of the Golgi apparatus was performed with Wheat germ agglutinin (WGA)/ovomucoid Au, Ricinus communis agglutinin_I (RCA_I)/Au and Helix pomatia agglutinin (HPA)/Au. The sections were then counterstained with PTA at low pH, which allows a clear distinction between the elements of the trans-Golgi network (G₂-G₁) and the saccules of the stack (g).With WGA, LFA and RCA_I the trans-Golgi network was observed to be clearly more reactive than the stack. After vesiculation most intense labeling was found over the highly glycosylated vacuolar membranes derived from the G₂-element. The limiting membrane of lysosomes, the MvB's and the plasma membrane also reacted strongly. Colloidal gold particles were also found over the membranes of the vacuoles derived from G₁. The Golgi stack showed a lower reactivity and label for all three lectins could be found over three to four saccules of the stack (g₃-g₄). The matrix of the lysosomes was slightly labeled. Labeling with HPA was absent from the trans saccules and was consistently found in the cis and cis-most (g₄-g₅) saccules of the stack. Some cytoplasmic vesicles near the cell border were also labeled. With our procedure the Golgi apparatus can easily be detected and it is apparent that in rabbit luteal cells the highest lectin reactivity is found in the trans-Golgi network. A striking similarity is observed between the highly glycosylated membrane structures derived from G₂ and the border of the lysosomes.     

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of<Emphasis Type="Italic"> Fonsecaea pedrosoi</Emphasis>

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing 2,3- and 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.     

The histochemical demonstration of sialic acid residues in pancreatic islets

Summary Using a modified colloidal iron reaction in connection with neuraminidase extraction test 3 different sialic acid-containing components have been demonstrated in pancreatic islets comprising golgi region and glycocalyx layer of islet cells and intrainsular capillary walls. The colloidal iron positive cationophilia increased markedly after treatment with alkali; an effect which might be due to deesterification, thus exposing additional free carbonyl groups of sialic acid residues.     

The histochemical demonstration of sialic acid residues in pancreatic islets.

Using a modified colloidal iron reaction in connection with neuraminidase extraction test 3 different sialic acid-containing components have been demonstrated in pancreatic islets comprising golgi region and glycocalyx layer of islet cells and intrainsular capillary walls. The colloidal iron positive cationophilia increased markedly after treatment with alkali; an effect which might be due to deesterification, thus exposing additional free carbonyl groups of sialic acid residues.     

Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell

Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H₂PO₄⁻ groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.     

The Surface Charge of Tritrichomonas foetus

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of ?1.03 μmμs^?1μV^?1μcm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (> 9.0), there is an increase in the surface charge reaching an EPM of ?2.5 μmμs^?1μV^?1μcm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.     

Mucosal acidification and an acid microclimate in the hen colon in vitro

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63±0.25 Eq·cm^-2·h^-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10^-3 mol·l^-1), and serosal ouabain (5x10^-4 mol·l^-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10^-3 mol·l^-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59±0.13 Eq·cm^-2·h^-1, P<0.002 versus controls) and addition of glucose (15 mmol·l^-1), but not galactose, partially restored acidification to control levels. Anoxia (N₂ gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1–7.4 was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH. Histochemical staining for carbonic anhydrase localized this enzyme to cytoplasm and lateral margins of one subfraction of colonic cells, and to cytoplasm in a second subpopulation Intense staining was also evident in subepithelial capillaries. These results suggest that a large part of mucosal acidification and maintenance of the acid microclimate in hen colon may be dependent on glycolysis and metabolic acid production, although a smaller, electrogenic and acetazolamidesensitive component also appears to exist. This latter component may become more prominent under conditions of cellular acidification.Abbreviations CA carbonic anhydrase - I _SC short circuit current - NFM N-ethylmaleimide - PD transepithelial potential - SCFA short chain fatty acids     

Oligosaccharide-derivatized dendrimers: defined multivalent inhibitors of the adherence of the cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1

Poly(propylene imine) dendrimers having four or eight primary amino groups and a Starburst^TM (PAMAM) dendrimer having eight primary amino groups were used as core molecules, to which phenylisothiocyanate derivatized (PITC) galβ1-3galNAcβ1-4[sialic acidβ2-3]-galβ1-4glc (oligo-GM1) residues were covalently attached to yield multivalent oligosaccharides. The synthesis of the oligo-GM1-PITC derivatized dendrimers was monitored using high performance thin layer chromatography, infrared spectroscopy, sialic acid content, and mass spectroscopy. The ability of multivalent oligo-GM1-PITC dendrimers to inhibit the binding of ¹²⁵I-labeled cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1-coated microtiter wells was determined. IC₅₀s obtained for the oligo-GM1-PITC dendrimers, GM1, and the oligosaccharide moiety of GM1 indicated that the derivatized dendrimers inhibited binding of the choleragenoid and the heat labile enterotoxin to GM1-coated wells at a molar concentration five- to 15-fold lower than native GM1 and more than 1,000-fold lower than that of the free oligosaccharide. This revised version was published online in November 2006 with corrections to the Cover Date.     

Kinetics of ferrous iron oxidation by batch and continuous cultures of thermoacidophilic Archaea at extremely low pH of 1.1–1.3

The extreme acid conditions required for scorodite (FeAsO₄·2H₂O) biomineralization (pH below 1.3) are suboptimal for growth of most thermoacidophilic Archaea. With the objective to develop a continuous process suitable for biomineral production, this research focuses on growth kinetics of thermoacidophilic Archaea at low pH conditions. Ferrous iron oxidation rates were determined in batch-cultures at pH 1.3 and a temperature of 75°C for Acidianus sulfidivorans, Metallosphaera prunea and a mixed Sulfolobus culture. Ferrous iron and CO₂ in air were added as sole energy and carbon source. The highest growth rate (0.066 h⁻¹) was found with the mixed Sulfolobus culture. Therefore, this culture was selected for further experiments. Growth was not stimulated by increase of the CO₂ concentration or by addition of sulphur as an additional energy source. In a CSTR operated at the suboptimal pH of 1.1, the maximum specific growth rate of the mixed culture was 0.022 h⁻¹, with ferrous iron oxidation rates of 1.5 g L⁻¹ d⁻¹. Compared to pH 1.3, growth rates were strongly reduced but the ferrous iron oxidation rate remained unaffected. Influent ferrous iron concentrations above 6 g L⁻¹ caused instability of Fe²⁺ oxidation, probably due to product (Fe³⁺) inhibition. Ferric-containing, nano-sized precipitates of K-jarosite were found on the cell surface. Continuous cultivation stimulated the formation of an exopolysaccharide-like substance. This indicates that biofilm formation may provide a means of biomass retention. Our findings showed that stable continuous cultivation of a mixed iron-oxidizing culture is feasible at the extreme conditions required for continuous biomineral formation.     

Surface pH at the Basolateral Membrane of the Caecal Mucosa of Guinea Pig

Since the major mechanisms responsible for regulation of intracellular pH of enterocytes are located in the basolateral membrane, respective effects may be expected on pH in the compartment near the basolateral membrane. A method was established to estimate the pH at the basolateral membrane (pH _b ) of isolated caecal epithelia of guinea pig using pH-sensitive fluorescein attached to lectin (lens culinaris). In the presence of bicarbonate and a perfusion solution-pH of 7.4, pH _b was 7.70 ± 0.15. In the absence of bicarbonate or chloride as well as by inhibition of the basolateral Cl⁻-HCO⁻ ₃ exchange with H₂-DIDS, pH _b was reduced near to solution-pH. Inhibition of the basolateral Na⁺-H⁺ exchanger by adding a sodium- and bicarbonate-free, low-buffered solution increased pH _b . Decrease of pH of serosal perfusion solution to 6.4 provoked a similar decrease of pH _b to solution pH. Short-chain fatty acids (SCFA) added to the mucosal solution caused a slight decrease of pH _b . SCFA added to the serosal side alkalized pH _b . However, in the presence of bicarbonate pH _b returned quickly to the initial pH _b , and after removal of SCFA a transient acidification of pH _b was seen. These responses could not be inhibited by MIA or H₂-DIDS. We conclude that no constant pH-microclimate exists at the basolateral side. The regulation of the intracellular pH of enterocytes reflects pH _b . The slightly alkaline pH _b is due to the bicarbonate efflux. Data support the presence of an SCFA⁻-HCO⁻ ₃ exchange. Received: 17 December 1998/Revised: 24 February 1999