RESUMO
The early effects of deafferentation on the postsynaptic membrane beneath the end bulb of Held in the anteroventral cochlear nucleus (AVCN) were studied with the freeze-fracture technique. Three distinct responses were seen on the external membrane leaflet after cochlear ablation. Within 12 h the number of nonaggregate particles increased 147% by the addition of new particles to the membrane. The increase in number of nonaggregate particles continued until 4 days after cochlear ablation. The other responses occurred later, after degenerative changes were present in the end bulb. Between 1 and 2 days after cochlear ablation, the number of perisynaptic aggregates surrounding the postsynaptic active zone decreased to 10% of normal numbers. By 4 days, all perisynaptic aggregates had disappeared from the membrane. Coated vesicles may be involved in removing these aggregates. Between 1 and 3 days, the number of junctional aggregates decreased, but the size of the aggregates increased, apparently as a result of coalescence of nearby junctional aggregates. The total number of particles in junctional aggregates in the membrane was not altered during the first 6 days after cochlear ablation. The three separate responses suggest the existence of at least three different types of intramembranous particles on the external leaflet of the principal cell membrane, with each type dependent upon different cues for its maintenance in the membrane.
Assuntos
Nervo Coclear/fisiologia , Sinapses/ultraestrutura , Membranas Sinápticas/ultraestrutura , Vias Aferentes , Animais , Membrana Celular/ultraestrutura , Denervação , Técnica de Fratura por Congelamento , Cobaias , Fatores de TempoRESUMO
The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesis N-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated with N-butyldeoxynojirimycin, the accumulation of GM2 in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (beta-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Encéfalo/metabolismo , Inibidores Enzimáticos/uso terapêutico , Gangliosídeo G(M2)/metabolismo , Lisossomos/metabolismo , Doença de Tay-Sachs/tratamento farmacológico , 1-Desoxinojirimicina/farmacocinética , 1-Desoxinojirimicina/uso terapêutico , Animais , Barreira Hematoencefálica , Modelos Animais de Doenças , Gangliosídeo G(M2)/biossíntese , Camundongos , Microscopia Eletrônica , Neurônios/metabolismo , Neurônios/ultraestrutura , Doença de Tay-Sachs/metabolismoRESUMO
In order to gain insight into the metabolism of keratin intermediate filaments (KIF) as well as the ability of KIF degradation products to interact with the immune system, we performed enzymatic degradation of purified KIF and examined their interaction with anti-KIF autoantibodies and their ability to act as immunogens. Aliquots of KIF aggregates were exposed to 3 different enzymes, that is, alpha-chymotrypsin, plasmin, and trypsin, in dose- and time-dependent experiments. The effect of the digestion was monitored sequentially by polyacrylamide gel electrophoresis and simultaneously by transmission electron microscopy. Furthermore, the KIF degradation proteins were then examined for their ability to bind anti-KIF autoantibodies by immunoblot and for their immunogenicity. In addition, preincubation of KIF with anti-KIF autoantibodies prior to the digestion procedure was performed to investigate a possible protective effect of this treatment against proteolytic degradation. The experiments demonstrated that: (1) KIF are degraded by serine proteinases, (2) with prolonged incubation time intact KIF are progressively replaced by more granular-amorphous material in transmission electron microscopy, (3) anti-KIF autoantibodies bind to KIF degradation proteins, (4) preincubation of KIF with anti-KIF autoantibodies does not exert any major protective effect for KIF against proteolytic degradation, and (5) the enzymatic degradation products of KIF can function as effective immunogens causing the formation of high-titer anti-KIF antibodies.
Assuntos
Proteínas de Filamentos Intermediários/imunologia , Queratinas/metabolismo , Autoanticorpos/imunologia , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Fibrinolisina/metabolismo , Humanos , Técnicas Imunológicas , Proteínas de Filamentos Intermediários/metabolismo , Microscopia Eletrônica , Serina Endopeptidases , Tripsina/metabolismoRESUMO
Ultraviolet radiation of murine skin in vivo or epidermal cells (EC) in vitro dramatically inhibits the antigen-presenting capacity of EC in vitro and results in the inhibition of immune responses to antigen challenge. In humans, UV exposure in vivo markedly inhibits alloantigen presentation by EC in the EC-lymphocyte reaction (ELR) when EC are harvested immediately after the administration of 4 times the minimal erythema dose (4 MED), whereas EC harvested 72 h after 4 MED (UV-EC) exhibit enhanced allostimulatory capacity in the ELR. This enhanced ELR reactivity is due to the appearance, in the epidermis, of bone marrow-derived OKT6- DR+ cells which are distinct from Langerhans cells (LC) in their lack of surface OKT6 and in their ultrastructural morphology. This report focuses on the phenotype and function of T6- Dr+ UV-EC and on their relationship to known human antigen presenting cell (APC) subsets. Approximately 60% of T6- Dr+ UV-EC bore the monocyte marker defined by monoclonal antibody OKM5, but lacked determinants recognized by OKM1, Leu M1, Leu M3, Leu M4, Leu M5, and Mac1. All T6- Dr+ UV-EC bore the class II MHC antigen HLA-DQ (DC/DS), which is associated with a specialized subset of antigen-presenting monocytes capable of stimulation in the autologous mixed leukocyte reaction (AMLR). Panning of OKM5+ UV-EC resulted in a population of cells which was markedly enriched in melanophages and which exhibited potent alloantigen-presenting capacity in the ELR. Since OKM5+ T6- Dr+ UV-EC were similar to the specialized APC minor subset of OKM1- OKM5+ blood monocytes both in phenotype and in apparent phagocytic function, we examined other APC functions of UV-EC to assess the extent of this analogy. Relative to control EC (containing only LC as APC), UV-EC (containing functionally inactivated LC but many T6- Dr+ APC) induced significantly greater degrees of T-cell proliferation in the presence of either tetanus toxoid antigen or the mitogen concanavalin A. UV-EC, as well as panning-purified OKM5+ UV-EC, were also able to induce autologous T-cell proliferation in the absence of added antigen (autologous ELR), in contrast to control EC which were poor stimulators of an autologous ELR. Thus, although human EC 72 h after UV exposure are numerically and functionally depleted of LC, at least 2 additional subsets of T6- Dr+ APC appear in the epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Células Apresentadoras de Antígenos/efeitos da radiação , Antígenos de Superfície/análise , Melanócitos/efeitos da radiação , Monócitos/efeitos da radiação , Células Apresentadoras de Antígenos/classificação , Células Apresentadoras de Antígenos/imunologia , Antígenos de Diferenciação de Linfócitos T , Epiderme/imunologia , Antígenos HLA-DR , Antígenos de Histocompatibilidade Classe II , Humanos , Ativação Linfocitária/efeitos da radiação , Teste de Cultura Mista de Linfócitos , Melanócitos/classificação , Melanócitos/imunologia , Monócitos/classificação , Monócitos/imunologia , Fenótipo , Linfócitos T/imunologia , Raios UltravioletaRESUMO
We have studied various tissues from 10 patients with cicatricial pemphigoid using direct and indirect immunofluorescence, mechanical suction blister induction, and immunoelectron microscopy. In 8 of the 10 patients, direct immunofluorescence of buccal mucosa showed a linear deposition of immunoreactants, IgG and C3 being those most commonly detected. Direct immunofluorescence of skin was positive in only 4 patients. Only 1 patient had a detectable circulating anti-basement membrane zone antibody. Substitution of normal human oral mucosa for adult skin as the tissue substrate for indirect immunofluorescence did not prove useful in the detection of circulating autoantibodies. Immunoelectron microscopy was performed in the skin or mucosa (buccal or ocular) of 6 patients, revealing lamina lucida localization of in vivo-bound immunoreactants. Indirect immunofluorescence studies on mechanically induced suction blisters in skin of 2 patients with in vivo-bound IgG suggest that the lamina lucida antigen involved in cicatricial pemphigoid may be distinct from the bullous pemphigoid antigen.
Assuntos
Complemento C3/análise , Imunoglobulinas/análise , Penfigoide Mucomembranoso Benigno/imunologia , Dermatopatias Vesiculobolhosas/imunologia , Adulto , Idoso , Bochecha , Túnica Conjuntiva/imunologia , Feminino , Imunofluorescência , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/análise , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mucosa Bucal/imunologia , Pele/imunologiaRESUMO
The basement membrane zone (BMZ) of human skin is a complex structure which contains several well-defined components including bullous pemphigoid antigen, laminin, type IV collagen, and proteoglycan. Characterization of additional basement membrane (BM) constituents has been limited by their relative inaccessibility, insolubility, and low tissue concentration. We have produced a murine monoclonal antibody that has enabled us to define a unique constituent of the BMZ of human stratified squamous epithelia. The monoclonal antibody (KF-1) was raised by standard techniques using suction blister-derived trypsinized human epidermal cells as the antigen. Indirect immunofluorescence and immunoperoxidase staining of human and rhesus monkey tissues with KF-1 produced linear BMZ staining of stratified squamous epithelia. Glandular and vascular BMs were not stained. Immunoelectron microscopic studies of normal human skin and esophagus showed specific binding of KF-1 to the lamina densa of the BMZ, a localization identical to that of type IV collagen. However, unlike type IV collagen, which is not species specific and is found in all BMs, the antigen defined by KF-1 is collagenase-resistant and is specific for primate stratified squamous epithelia. These findings confirm the existence of regional variation in BM composition, and demonstrate for the first time that the lamina densa of stratified squamous epithelial BMs contains a constituent other than type IV collagen.
Assuntos
Anticorpos Monoclonais/imunologia , Membrana Basal/imunologia , Pele/imunologia , Animais , Galinhas , Colágeno/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Cobaias , Humanos , Técnicas Imunoenzimáticas , Macaca mulatta , Camundongos/imunologia , Camundongos Endogâmicos BALB C/imunologia , RatosRESUMO
Between 10 and 60 days of age in the waltzing guinea pig, there is a genetically induced loss of all hair cells in the organ of Corti. About 43% of the spiral ganglion cells degenerate between 30 and 60 days of age. After 90 days of age, there is no further loss of spiral ganglion cells. Both Type I and II ganglion cells remain and are without afferent input. The terminals of these ganglion cells in the rostral AVCN, the end bulbs of Held, are normal until 30 days of age. During the period of ganglion cell loss degenerating end bulbs are seen. After 60 days of age, when most ganglion cell degeneration is complete, the remaining end bulbs have fewer synaptic vesicles and their synaptic junctions are flattened. The channels of enlarged extracellular space, which normally surround each synaptic junction or small groups of junctions, are only infrequently present. In freeze-fracture replicas of the rostral AVCN of waltzing guinea pigs after hair cell loss, the number of large, non-aggregate particles on the external leaflet of the principal cell opposite the end bulb is increased, and the number of perisynaptic aggregates is decreased compared to waltzing guinea pigs 10 days of age. The junctional aggregates are unaltered. These changes in the presynaptic terminal and postsynaptic membrane may be related to the loss of afferent input to the spiral ganglion cells, suggesting that activity is important for maintaining the synapse.
Assuntos
Cóclea/ultraestrutura , Nervo Coclear/ultraestrutura , Células Ciliadas Auditivas/fisiologia , Mecanorreceptores/fisiologia , Gânglio Espiral da Cóclea/ultraestrutura , Sinapses/ultraestrutura , Fatores Etários , Animais , Vias Auditivas/fisiologia , Contagem de Células , Denervação , Cobaias , Degeneração Neural , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Patients with xeroderma pigmentosum (XP) have more than a 1000-fold increased risk of cutaneous melanoma. To determine if the XP DNA repair defect is present in cutaneous pigmentary cells, nevus cells and melanocytes from four large, pigmented nevi were cultured from a 12-year-old girl with XP. Cultured melanocytes showed dendritic morphologic features, contained mature melanosomes, and reacted with monoclonal antibody to tyrosinase. Nevus cells were spindle shaped and expressed nevus cell-associated antigens. Melanocytes, nevus cells, and dermal fibroblasts from the patient with XP all had a similar reduction in DNA repair: unscheduled DNA synthesis was 30% to 50% of that in normal fibroblasts following a 30 J/m2 ultraviolet dose. After a 6 J/m2 ultraviolet dose, the proliferative ability of XP nevus cells and fibroblasts was reduced to 10% of that of normal fibroblasts. This study indicates that cultured melanocytes and nevus cells express the characteristic XP DNA repair defect.
Assuntos
Reparo do DNA , Melanócitos/ultraestrutura , Nevo/genética , Xeroderma Pigmentoso/genética , Sobrevivência Celular , Criança , Feminino , Fibroblastos/ultraestrutura , Humanos , Microscopia Eletrônica/métodos , Nevo/ultraestrutura , Células Tumorais CultivadasRESUMO
Two morphological differences distinguish the membranes of the end bulb-spherical cell synapse in rats and mice from those in guinea pigs and chinchillas. First, in freeze-fracture replicas, the membranes of rat and mouse spherical cells lack perisynaptic aggregates which are present in the other species. Second, small gap junctions are present between the end bulb and spherical cell soma of rats and mice. These interspecies differences are not reflected in thin-sectioned material. This observation points out the difficulty in attempting to generalize about the significance of intramembrane specializations in synaptic membranes.
Assuntos
Cóclea/ultraestrutura , Membranas Sinápticas/ultraestrutura , Animais , Técnica de Fratura por Congelamento , Cobaias , Camundongos , Terminações Nervosas/ultraestrutura , RatosRESUMO
The maturation of the end bulb of Held was studied in serial thin sections through the rostral pole of the rat anteroventral cochlear nucleus (AVCN) at 2-day intervals from birth through 16 days of age. In the neuropil of newborn rats primary auditory terminals occasionally synapse with large dendritic processes of spherical cells. Between 6 and 10 days of age, the spherical cell has numerous, long, fingerlike appendages at the base and proximal part of its primary dendrite which extend into the neuropil and envelope a single primary terminal. Small processes from the terminal extend along the appendages toward the cell body. At 10 days, these processes reach the dendritic pole of the soma and fill the interstices between the appendages. Synaptic contacts are present between the end bulb processes and the cell body. At 12 days, the end bulb covers most of the dendritic pole of the spherical cell. The remaining somatic appendages are small and are clustered beneath the end bulb. Synapses between the end bulb and soma are frequent. At 16 days, the end bulb resembles that seen in the mature rat. Nonprimary terminals appear on the soma at 10 days. These terminals are randomly distributed, with fine processes which extend from the end bulb over the remaining regions of the cell body.
Assuntos
Nervo Coclear/ultraestrutura , Envelhecimento , Animais , Animais Recém-Nascidos , Nervo Coclear/crescimento & desenvolvimento , Dendritos/ultraestrutura , Microscopia Eletrônica , Ratos , Sinapses/ultraestruturaRESUMO
The freeze-fracture technique was used to study the maturation of the postsynaptic membrane of spherical cells of the anteroventral cochlear nucleus (AVCN) of the developing rat brain. Observations were made at 2-day intervals from birth to 16 days of age. At all ages, the external leaflet (E-face) of the spherical cell membrane has aggregates of particles opposite presynaptic active zones. From birth to 6 days of age, these particle aggregates were found only on dendritic processes, not on the cell body. At 8 days of age, the aggregates were found on small somatic appendages and on the cell body adjacent to somatic appendages. In animals from 10 to 12 days of age, particle aggregates became increasingly common on the cell body. Morphometric analysis demonstrated a decrease in size but not packing density of the particles in the aggregates. These changes in the location and size of the aggregates correspond to the location of asymmetrical synapses seen in thin section material at these same time intervals (Neises et al., 1982). At 14 days the fracture plane no longer followed the postsynaptic membrane at the active zone; instead it shifted to the presynaptic membrane and the postsynaptic specializations were rarely seen. This fracture pattern is typical of adult animals.
Assuntos
Nervo Coclear/ultraestrutura , Sinapses/ultraestrutura , Envelhecimento , Animais , Animais Recém-Nascidos , Nervo Coclear/crescimento & desenvolvimento , Técnica de Fratura por Congelamento , Microscopia Eletrônica , Ratos , Membranas Sinápticas/ultraestruturaRESUMO
Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge.
Assuntos
DNA Recombinante/imunologia , DNA Viral/genética , Infecções por Herpesviridae/imunologia , Serina Endopeptidases/genética , Simplexvirus/genética , Animais , Sequência de Bases , DNA Recombinante/administração & dosagem , Feminino , Deleção de Genes , Infecções por Herpesviridae/prevenção & controle , Camundongos , Dados de Sequência Molecular , Simplexvirus/imunologiaRESUMO
The imino sugar deoxynojirimycin and its alkylated derivatives are inhibitors of the N-linked oligosaccharide processing enzymes alpha-glucosidase I and II. These compounds are glucose analogues and have the potential to inhibit both glucosidases and glucosyltransferases. However, to date there has been no report of deoxynojirimycin or similar analogues inhibiting a mammalian glucosyltransferase. We have investigated the effects of deoxynojirimycin and its alkylated derivatives on the biosynthesis of glycolipids in HL-60 cells. We have found that the N-butyl and N-hexyl derivatives of deoxynojirimycin, but not deoxynojirimycin itself, are novel inhibitors of the glucosyltransferase-catalyzed biosynthesis of glucosylceramide. This results in the inhibition of biosynthesis of all glucosylceramide-based glycosphingolipids. We have investigated the ability of one of these compounds, N-butyldeoxynojirimycin, to offset glucosylceramide accumulation in an in vitro Gaucher's disease model. This compound prevents lysosomal glycolipid storage and offers a novel therapeutic approach for the management of this and other glycolipid storage disorders.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Glicolipídeos/biossíntese , Inibidores de Glicosídeo Hidrolases , 1-Desoxinojirimicina/farmacologia , Animais , Linhagem Celular , Doença de Gaucher , Glucosiltransferases/antagonistas & inibidores , Glicolipídeos/antagonistas & inibidores , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Modelos Biológicos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
There is substantial evidence supporting the role of aspartate or glutamate as the neurotransmitter of the auditory nerve. The concentration of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an enzyme associated with the metabolism of these amino acids, is high in axons and terminals of the auditory nerve. Antibodies were raised against aspartate aminotransferase and used in immunocytochemical studies to determine its localization in the cochlear nucleus of the guinea pig. Indirect immunofluorescence techniques were used for light microscopic localization of aspartate aminotransferase-like immunoreactivity in normal guinea pigs and guinea pigs with auditory nerve lesions. Fluorescent rings of aspartate aminotransferase-like immunoreactivity were seen around spherical cells in the anteroventral cochlear nucleus. In animals with auditory nerve lesions, rings were no longer seen in the ipsilateral cochlear nucleus. Immunoreactivity was also seen on cells in the posteroventral cochlear nucleus and in auditory nerve fibers. Ultrastructural studies were done in the rostral anteroventral cochlear nucleus, using the peroxidase-antiperoxidase technique. Aspartate aminotransferase-like immunoreactivity was seen at axosomatic synapses on large spherical cells in terminals with the morphological characteristics of auditory nerve terminals. Other classes of terminals on the soma of large spherical cells showed no immunoreactivity. It was concluded that aspartate aminotransferase-like immunoreactivity is present in axons and terminals of the auditory nerve. These findings indicate that aspartate aminotransferase-like immunoreactivity may serve as a marker at terminals where aspartate or glutamate is a neurotransmitter.
Assuntos
Aspartato Aminotransferases/metabolismo , Nervo Coclear/enzimologia , Animais , Nervo Coclear/ultraestrutura , Cobaias , Histocitoquímica , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Basic principles of production of monoclonal antibodies are discussed in this paper. In order to illustrate the usefulness of such production, studies of a new monoclonal antibody designated KF-2 which defines a unique cell-surface antigen found exclusively in the stratum spinosum of man and lower primates are recounted.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Epiderme/imunologia , Acantólise/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Antígenos de Superfície/análise , Sítios de Ligação de Anticorpos , Ligação Competitiva , Humanos , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Primatas , Especificidade da EspécieRESUMO
We have previously reported that the imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits glycolipid biosynthesis, in addition to its known activity as an inhibitor of the N-linked oligosaccharide processing enzyme alpha-glucosidase I. In an attempt to dissociate these two activities and identify an inhibitor which was more selective for the glycolipid biosynthetic pathway, several imino sugars have been N-alkylated and tested for inhibitory activity. The galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) was found to be a potent inhibitor of glycolipid biosynthesis but in contrast to NB-DNJ had no effect on the maturation of N-linked oligosaccharides or on lysosomal glucocerebrosidase. The effect of increasing N-alkyl chain length on glycolipid inhibition was investigated. Nonalkylated DGJ, the N-methyl and N-ethyl derivatives, were noninhibitory. However, N-propylation resulted in partial inhibition while the N-butyl and N-hexyl derivatives resulted in maximal inhibition. Increasing alkyl chain length also resulted in increased potency of glucosyltransferase inhibition. In an in vitro Gaucher's disease model NB-DGJ was as effective as NB-DNJ in preventing glycolipid storage and may represent a more selective potential therapeutic agent than NB-DNJ for the management of this and other glycosphingolipidoses.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Glicolipídeos/biossíntese , Oligossacarídeos/biossíntese , 1-Desoxinojirimicina/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Doença de Gaucher/metabolismo , Glucosilceramidase/efeitos dos fármacos , Glucosiltransferases/antagonistas & inibidores , Humanos , Camundongos , Relação Estrutura-Atividade , alfa-Glucosidases/efeitos dos fármacosRESUMO
The imino sugar N-butyldeoxynojirimycin inhibits the N-linked oligosaccharide processing enzymes alpha-glucosidases I and II, and the ceramide specific glucosyltransferase which catalyses the first step in glucosphingolipid biosynthesis. We have studied the effects of this compound on the ultrastructure of HL-60 cells to identify novel activities of this compound. Treatment of HL-60 cells with this imino sugar results in several morphological changes within the cell, none of which result in cytotoxicity. The plasma membrane stains heavily with potassium ferrocyanide within 30 min following addition of the compound to the medium, and there is then a time dependent involvement of all other intracellular membranes. Secretory granules become enlarged and lose their dense core morphology and appear either empty and vacuolated or have low density contents. However, the most striking effect of NB-DNJ treatment is on the Golgi apparatus. The Golgi exhibits a time-dependent change from typical Golgi morphology to a structure almost completely devoid of cisternae and consisting predominantly of vesicles. All the observed changes are fully reversible on withdrawal of the compound.