Fluorophore-assisted electrophoresis of urinary carbohydrates for the identification of patients with oligosaccharidosis-and mucopolysaccharidosis-type lysosomal storage diseases

Lysosomal storage diseases result from defects in the activity of the lysosomal enzymes that break down macromolecules in the cell. These enzyme defects contribute to over 30 separate storage diseases that result in nearomuscular and intellectual impairment and, in some cases, early childhood death. This report describes a new method for identifying defects in the lysosomal enzymes and in the metabolic pathway that functions in the degradation of complex carbohydrates. The method involves identifying abnormal carbohydrates in the urine of affected patients using fluorescent carbohydrate tags and polyacrylamide gel electrophoresis. Currently, the method can be used as a simple screen for the identification of at least 12 different lysosomal storage diseases using a single electrophoretic procedure. Both oligosaccharidoses and mucopolysaccharidoses (MPS) can be identified, and in many cases the MPS subtype can be determined. In addition, the method can be used to confirm enzymatically the results of the initial screening test. We believe that this method will become extremely useful not only in the diagnosis of these diseases but in the management of patients on therapy.     

Role of cytosolic free calcium and phospholipase C in leukotriene-B4-stimulated secretion in human neutrophils. Comparison with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 greater than the stereochemical isomer of LTB4, (5S, 12S)-LTB4 much much greater than the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4. This threshold is about an order of magnitude higher than that required for the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). The increase in [Ca2+]i elicited by LTB4 was unaffected by increasing cellular cAMP, while secretion was completely inhibited. These results indicate, that similar to fMet-Leu-Phe, leukotrienes generate other signals in addition to [Ca2+]i elevations. Contrary to previous claims, leukotrienes stimulate polyphosphoinositide hydrolysis, as indicated by the increase in [3H]inositol trisphosphate, InsP3, observed upon stimulation of myo[3H]inositol-labelled neutrophils with LTB4 or (5S, 12S)-LTB4. The two InsP3 isomers [Ins(1,4,5)P3 and Ins(1,3,4P3] were separated by high-pressure liquid chromatographed and, as reported for other cell types, the formation of Ins(1,4,5)P3 precedes that of Ins(1,3,4)P3. Maximal stimulatory doses of LTB4 or (5S, 12S)-LTB4 produce about 50% the amount of InsP3 generated by equimolar concentrations of fMet-Leu-Phe. The present observations suggest that, though the transmembrane signalling systems activated by LTB4 and fMet-Leu-Phe are the same, the different efficacy of these two agonists at stimulating neutrophil functions is due, at least in part, to a different degree of activation of phospholipase C.     

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.     

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin

Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types.     

Oxygen tolerance of anaerobic bacteria isolated from human feces

The large bowel intestinal flora of mammals is made up mostly of O₂-intolerant anaerobic microorganisms which are irreversibly damaged by brief exposure to air. The aim of our work was to investigate the effect of atmospheric O₂ on human intestinal anaerobic microorganisms. Thirty O₂-intolerant bacterial strains that reached 100% mortality after 120 min of air exposure were isolated. Ten of these strains were tested for their atmospheric O₂ sensitivity as a function of air exposure time; all tested microorganisms showed a similar mortality trend on exposure to air. In fact, 50% of cells survive, on the average, after 4–5 min of atmospheric O₂; this percentage decreases to 3–5% after only 20 min, and after 40 min only one cell in a thousand survives; all strains reached 100% mortality in a time range of 100–120 min. The strains examined were identified as belonging to the generaEubacterium, Peptostreptococcus, andCoprococcus.     

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.     

CD94/NKG2-A inhibitory complex blocks CD16-triggered Syk and extracellular regulated kinase activation, leading to cytotoxic function of human NK cells.

The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor zeta subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.     

Ascorbate-dependent hydrogen peroxide detoxification and ascorbate regeneration during germination of a highly productive maize hybrid: Evidence of an improved detoxification mechanism against reactive oxygen species

Ascorbate content and the activities of some key enzymes involved in the detoxification from reactive oxygen species were investigated in germinated embryos of two Zea mays L. inbred lines (B73 and Mo17) and of their heterotic F1 hybrid (B73×Mo17). The F1 hybrid showed a higher ascorbate biosynthetic capability owing to a higher activity of l -galactono- Γ -lactone dehydrogenase (EC 1.6.5.4), the last enzyme in ascorbate biosynthesis. Ascorbate peroxidase (EC 1.11.1.11), ascorbate free radical reductase (EC 1.6.5.4) and dehydroascorbate reductase (EC 1.8.5.1) activities were much higher in the F1 hybrid than in either inbred line, whereas catalase (EC 1.11.1.6) activity was similar in the three genotypes. Native polyacrylamide gel electrophoresis (PAGE) analysis showed three forms of cytosolic ascorbate peroxidase, both in parental lines and in the F1 hybrid. On the other hand, a complex pattern of proteins with dehydroascorbate reductase activity was observed, with the hybrid combining the different dehydroascorbate-reducing proteins expressed by the inbred lines. The possible involvement of the enzymes of the ascorbate system in the phenomenon of hybrid vigour is discussed.     

Ricerche istologiche sull' innervazione del timo dei Sauropsidi

Zusammenfassung Die sehr zahlreichen Nervenfasern für die Thymus der Sauropsiden gehen hauptsächlich vom zervikalen sympathischen Strang, aber zum Teil auch vom Vagus und vielleicht von den ventralen Ästen der zervikalen Nerven aus und erreichen die Thymus, indem sie den Gefäßen entlang laufen.Die Faserbündelchen, in welchen man oft isolierte oder in Gruppen gesammelte sympathische Zellen antrifft, dringen in das Thymusparenchym ein und hier verästeln sie sich sehr stark. Ein kleiner Teil der Nervenfasern sind Vasomotoren, ein anderer ebenfalls kleiner Teil verschwindet innerhalb von Gruppen von epithelioiden Zellen, welche oft mit drüsenähnlichen Höhlungen versehen sind (einige von diesen epithelioiden Anhäufungen erinnern im Aussehen an dieHassall-Körperchen der Säugetiere); echte typische H. K. sind sehr selten in erwachsenen Tieren nachweisbar.Der größte Teil der Nervenfasern erreicht jedoch die myoiden Zellen und verbindet sich mit denselben. Bei Cheloniern und bei Hühnern ist der Nervenanteil, der den myoiden Elementen vorbehalten ist, wirklich übermäßig groß.Die myoiden Zellen sind bekanntlich ein oft sehr ansehnlicher Bestandteil der Thymus der Sauropsiden, wie bei anderen Wirbeltiergruppen. Sie sind regressiven und progressiven Veränderungen unterworfen: je nach den Jahreszeiten (Dustin), ebenso besonderen funktionellen Bedingungen wie Fasten, Winterschlaf (Hammar); sie zeigen beim Huhn eine Hyperplasie-Hypertrophie als Folge der Kastration und des Alters (Terni).In vorliegenden Untersuchungen sind nebenbei einige neue Tatsachen über die Morphologie der myoiden Zellen festgestellt worden, unter anderen folgende: a) ihre histologische Differenzierung während der Entwicklung tritt sehr spät ein; b) sie sind räumlich von dem retikulär-kollagenen Netze des Thymusläppchens unabhängig, und sie besitzen keine retikulosarkolemmale Membran; c) die strahlenförmige (konzentrische) oder regellose Anordnung der Querstreifung der Myofibrillen in den großen myoiden Elementen bildet sich als Resultat der Verschmelzung von vorher unabhängigen Zellen (weshalb die besprochenen Elemente echte Syncytien sind); d) im Protoplasma der myoiden Zellen finden sich Spuren von Glykogen; usw.Die Verbindungen zwischen Nervenfasern und myoiden Elementen und andere Einzelheiten der feineren Verteilung der Nervenelemente im Thymusläppchen wurden bei Cheloniern und Vögeln besonders eingehend untersucht. An der Oberfläche der myoiden Zellen bilden die Nervenfasern Windungen oder spatel-, knopf-, keulchen- oder füßchenförmige Verbreitungen, welche der myoiden Substanz anhängen (neuromyoide Verbindungen).Die Nervenfasern, welche sich durch diese Endigungsweise mit den myoiden Zellen verbinden, gehören sehr wahrscheinlich zu den postganglionären Neuronen, welche entweder im Thymus (intraparenchymale oder perivasale mikroskopische Ganglien) oder im zervikalen sympathischen Gefäßgeflecht oder im sympathischen Grenzstrang liegen.Über Wesen, Zweck und Ziel der Vagusfibern habe ich mir kein bestimmtes Urteil bilden können.Außerdem befinden sich im Thymusläppchen wenige Nervenzellen des gewöhnlichen sympathischen Typus und in größerer Zahl kleine isolierte Nervenzellen, die zweifellos mit den interstiziellen ZellenCajals zu identifizieren sind. Diese interstiziellen Neuronen befinden sich meistensin der Nähe der myoiden Zellen und liegen oft auf der Oberfläche derselben, indem sie sie mit ihren verästelten Fortsätzen umfassen. Manchmal verbindet sich ein langer und feiner Fortsatz der interstiziellen Neuronen mit einer entfernt gelegenen myoiden Zelle. Diese Nervenzellen müssen zum größten Teil alsautonome effektorische Neurone aufgefaßt werden, wegen ihrer innigen Verbindung mit der kontraktilen Substanz. Wenn eine Kontraktionsmöglichkeit der myoiden Zellen auch nicht in Abrede zu stellen ist, ist es nicht recht verständlich, was für eine nützliche Wirkung ihre Kontraktion haben könnte (darum gebrauchen wir den Ausdruck effektorisch und nicht motorisch).Man kann oft beobachten, daß an der Oberfläche einer und derselben myoiden Zelle sich sowohl Fäden von exogenen Nervenfasern, als auch verästelte Fortsätze einer kleinen interstiziellen paramyoiden Zelle ausbreiten.Obwohl in der Thymus (wie auch im Darm;Cajal) das Wesen der Fortsätze der interstiziellen Neuronen zweifelhaft ist, mangels sicherer differentialer Merkmale zwischen Neuriten und Dendriten, ist doch das Aussehen der mit den myoiden Zellen verbundenen Fasern ganz verschieden von demjenigen der Fortsätze der interstiziellen Zellen.In einigen wenigen Fällen ist es möglich, einen dünnen und langen Fortsatz (Neurit?) der interstiziellen Zelle zu verfolgen, welcher ein kleines Blutgefäß erreicht; es ist möglich, daß er längs desselben eine proximale Richtung verfolgt. Dieses Verhalten läßt die Vermutung zu, daß wenigstens einigen dieser Neuronen die Bedeutung vonrezeptorischen Neuronen zuzuschreiben sei.Die Deutung des reichen Zuflusses und der ansehnlichen Verteilung des nervösen Anteils im Thymusparenchym der Sauropsiden ist, vom Gesichtspunkt ihrer möglicherweise endokrinen Funktion, nicht leicht: Sei es, weil die Innervation anderer endokriner Drüsen histologisch nicht genau bekannt ist (mit Ausnahme der Paraganglien); sei es, weil es überhaupt zweifelhaft ist, ob die Thymus eine innere Sekretion besitzt.Es ist möglich, daß die Anwesenheit der neuromyoiden Synapsen in der Thymus (welche hier zum ersten Male hervorgehoben wird), wenn auch die myoiden Zellen nicht kontraktionsfähig sein sollten, trotzdem mit dem Kohlenhydratenstoffwechsel in Zusammenhang steht, ähnlich wie es für die neuromuskularen Synapsen des zerebrospinalen Systems angenommen wird (Roncato).Der beinahe übergroße Reichtum nervöser Verzweigungen und neuromyoider Verbindungen, besonders bei Cheloniern, legt die Vermutung nahe, daß in zyklischen degenerativen Vorgängen des Thymusparenchyms eine Zerstörung und nachfolgende übermäßige Regeneration von Nervenfasern stattfindet; andererseits läßt die Zunahme der Zahl und Verzweigung der Nervenfasern im Kapaun und alten Hahn (Terni) die begründete Vermutung zu, daß es sympathische Neuronen gibt, welche einer auch verspäteten progressiven histologischen Differenzierung ihrer Neuriten fähig sind (eine verspätete histologische Vervollkommnung des Zellenleibes und der Dendriten in sympathischen Neuronen ist schon in menschlichen Ganglien bekannt;Terni).Aus diesen Gründen lassen die voliegenden Beobachtungen über die Thymus der Sauropsiden den Gedanken aufkommen, daß die stark entwickelte autonome Innervation der Thymus in der Funktion dieses Organs eine bedeutende Rolle spielt: sei es als Sitz besonderer Reize, welche sich wahrscheinlich in den neuromyoiden Apparaten entladen, sei es, weil die Nervenfasern mit Vorrichtungen versehen sind, welche auf lokale oder allgemeine Reize mit besonderer Empfindlichkeit morphologisch reagieren.