Regulation of S100A8/A9 (calprotectin) binding to tumor cells by zinc ion and its implication for apoptosis-inducing activity

S100A8/A9 (calprotectin), which is released by neutrophils under inflammatory conditions, has the capacity to induce apoptosis in various cells. We previously reported that S100A8/A9 induces apoptosis of EL-4 lymphoma cells via the uptake of extracellular zinc in a manner similar to DTPA, a membrane-impermeable zinc chelator. In this study, S100A8/A9-induced apoptosis was examined in several cell lines that are weakly sensitive to DTPA, suggesting S100A8/A9 is directly responsible for apoptosis in these cells. Since zinc inhibits apoptosis of MM46, one of these cells, the regulation by zinc of the capacity of S100A8/A9 to bind MM46 cells was studied. When MM46 cells were incubated with S100A8/A9 in standard or zinc-depleted medium, the amounts of S100A8/A9 bound to cells was markedly lower at 3 h than at 1 h. In contrast, when MM46 cells were incubated with S100A8/A9 in the presence of high levels of zinc, binding to cells was the same at 1 and 3 h. When the cells were permeabilized with saponin prior to analysis, a larger amount of cell-associated S100A8/A9 was detected at 3 h. The amount was further increased in cells treated with chloroquine, suggesting that S100A8/A9 was internalized and degraded in lysosomes. Although it has been reported that S100A8/A9 binds to heparan sulfate on cell membranes, the amount of S100A8/A9 bound to MM46 cells was not reduced by heparinase treatment, but was reduced by trypsin treatment. These results suggest that S100A8/A9 induces apoptosis by direct binding to MM46 cells, and that this activity is regulated by zinc.     

Cadmium transport by a Cd2+-sensitive and a Cd2+-resistant strain of Bacillus subtilis

Cadmium uptake by a Cd2+-sensitive (1A1) and a Cd2+-resistant mutant (1A1r) strain of Bacillus subtilis was investigated. Uptake of 109Cd2+ was determined for cells of both strains grown in tryptone broth and in broth containing tryptone, yeast extract, and glucose (TYG). The extent of 109Cd2+ uptake by cells of 1A1r was less than by cells of 1A1 under both growth conditions. In both growth media, 109Cd2+ uptake by 1A1 cells demonstrated saturation kinetics and was energy dependent. In both TYG and tryptone broth, 109Cd2+ uptake by 1A1 cells was inhibited by the addition of unlabeled Mn2+. Although lower in magnitude, the kinetics of 109Cd2+ uptake by 1A1r cells were similar to those of 1A1 cells when grown in tryptone broth. However, no obvious saturation kinetics, energy dependence, temperature sensitivity, or inhibition of 109Cd2+ uptake by the addition of unlabeled Mn2+ was observed in 1A1r cells grown in TYG. Differential Mn2+ accumulation by 1A1r cells in TYG and tryptone broth correlated with differential 109Cd2+ uptake by 1A1r cells in these media.     

环状RNA Circ_0001073在恶性黑素瘤中的表达及其对A375细胞恶性表型的影响

目的:通过检测环状RNA Circ_0001073在恶性黑素瘤细胞与正常表皮黑素细胞中的表达差异,及其对恶性黑素瘤A375细胞恶性表型的影响,阐明Circ_001073在恶性黑素瘤细胞中的表达及功能。方法:采用聚合酶链式反应(Real-time quantitative polymerase chain reaction, RT-QPCR)方法检测正常表皮黑素细胞和恶性黑素瘤细胞中Circ_0001073的表达水平;应用小干扰RNAs沉默恶性黑素瘤A375细胞中Circ_0001073表达,5-乙炔基-2'-脱氧尿苷(5-Ethynyl-2'-deoxyuridine,EdU)实验检测沉默Circ_0001073对A375细胞增殖的影响,平板克隆实验检测沉默Circ_0001073对A375细胞克隆形成的影响,细胞划痕实验评估沉默Circ_0001073对A375细胞迁移能力的影响,Transwell实验评估沉默Circ_0001073对A375细胞侵袭能力的影响。结果:qRT-PCR结果显示Circ_0001073在恶性黑素瘤细胞中的表达低于正常表皮黑素HEMa-LP细胞(P0.05)。应用Circ_0001073si RNA转染A375细胞后,Circ_0001073的表达水平较转染Circ_0001073 NC的细胞显著降低(P0.05)。EdU实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞增殖能力显著增强(P0.05);平板克隆实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞克隆形成能力显著增强(P0.05);细胞划痕实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞迁移能力显著增强(P0.05);Transwell实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞侵袭能力显著增强(P0.05)。结论:Circ_0001073在恶性黑素瘤细胞中低表达,并可抑制A375细胞的增殖、克隆形成、迁移及侵袭。     

Chromogranins A and B and secretogranin II in hormonally identified endocrine cells of the gut and the pancreas

Chromogranins A and B and secretogranin II have been localized in a wide spectrum of gastroenteropancreatic endocrine/paracrine cells. Chromogranin A immunoreactivity showed the widest distribution and was displayed by glucagon-, PP-, gastrin-, gastrin-CCK-, secretin-immunoreactive cells, the most intense stainings being peculiar of enterochromaffin cells. Chromogranin B immunoreactivity was detected in gastrin- and glucagon cells and in some enterochromaffin cells containing also chromogranin A. Secretogranin II was paired to chromogranin A in glucagon cells of pancreatic islets or occurred alone in glycentin/PP cells of colonic mucosa. Neither of the chromogranins nor secretogranin II have been so far detected in somatostatin-, GIP-, or motilin-immunoreactive cells. Chromogranin A but not chromogranin B or secretogranin II has been detected in the gastric argyrophilic ECL cells.     

Different mRNAs induced by interferon in cells from inbred mouse strains A/J and A2G

Treatment of cells from inbred mouse strains A/J and A2G with interferon resulted in the development of different antiviral states for influenza viruses. A2G mice-derived cells that carry the resistance gene Mx were efficiently protected by interferon against influenza viruses, whereas the interferon protection against the same viruses in wild-type A/J mice-derived cells was only marginal. The two cell types, however, were equally protected by interferon against vesicular stomatitis virus and other non-orthomyxoviruses. The interferon-induced mRNAs of mouse embryonic fibroblast cells that carried either homozygous wild-type alleles or homozygous Mx alleles were compared. The isolated polysome-bound mRNAs from A/J (+/+) and A2G (Mx/Mx) cells were translated in a cell-free translation system, and the translation products were analyzed after two-dimensional gel electrophoresis. New mRNAs coding for at least eight proteins with molecular weights (MW) ranging from 30,000 to 80,000 were found in interferon-treated cells but not in control cells. Differences in the interferon-induced mRNAs from A/J and A2G cells were also found. An mRNA coding for a 72,000-MW protein was found in interferon-treated A2G cells but not in interferon-treated A/J cells. Interferon-treated A/J cells, on the other hand, contained an mRNA coding for a 65,000-MW protein that was not found in interferon-treated A2G cells. The in vitro-synthesized 65,000-MW protein efficiently bound to GMP. Cytoplasmic extracts prepared from interferon-treated A/J cells also contained a GMP-binding 65,000-MW protein that was undetectable in similarly treated A2G cells.     

Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by approximately 80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.     

Identification and characterization of a diamine exporter in colon epithelial cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.     

Preparation of stable target cells for anti-herpes simplex virus cytotoxic T lymphocytes

Using an avirulent strain of herpes simplex virus (HSV), SKa, and a methylcholanthrene induced sarcoma cell line, Meth A cells, we have developed a reliable target cell system for detection of cell-mediated cytotoxicity directed against HSV-infected cells. SKa-infection in Meth A produced no progeny virus but induced HSV-specific surface antigens as revealed by radioimmunoassay using 125I-labeled HSV antibody. Spontaneous release of 51Cr from the SKa-infected Meth A cells was no more than that from uninfected control cells but a strong spontaneous 51Cr release was produced in Meth A cells infected with KOS, a virulent strain which produced a progeny virus in Meth A and was lytic for the cells. When used as a target, SKa-infected Meth A cells could detect HSV-specific cytotoxicity by spleen and lymph node lymphocytes of mice immunized with SKa and KOS. This system also detected effector cytotoxic lymphocytes stimulated in vitro by mixed cultures of immune spleen cells and KOS-infected Meth A cells. Thus, the system should be valuable in studies of cell-mediated cytotoxicity directed against HSV-infected cells.     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

Pathway of taurolipid B formation from exogenous taurolipid A by Tetrahymena thermophila

When Tetrahymena thermophila was incubated with taurolipid A isolated from T. pyriformis NT-1, the exogenously added taurolipid A was deacylated rapidly, and taurolipid B content in the cells was increased. The deacylated taurolipid A (lysotaurolipid A) content reached a maximum early on during incubation, and then declined. Taurolipid B and lysotaurolipid B contents in the cells were increased continuously during the incubation. These observations suggest that lysotaurolipid A was an intermediate for taurolipid B formation. When cells were incubated with lysotaurolipid A, newly formed lysotaurolipid B and taurolipid B were observed. Furthermore, when cells were incubated with lysotaurolipid B, only taurolipid B was newly formed. In contrast, newly formed lysotaurolipid B was observed when cells were incubated with exogenous taurolipid B. From the results, we have postulated the biosynthetic pathway of taurolipid B from exogenous taurolipid A in cells of Thermophila.     

Localization of neurokinin A and chromogranin A immunoreactivity in the developing porcine adrenal medulla

Summary The presence of neurokinin A immunoreactivity was studied in the chromaffin cells of the porcine adrenal medulla and in the nerve fibres innervating the adrenal gland during ontogenic development. For comparison, chromogranin A immunoreactivity was used as a marker for chromaffin cells.Whereas chromogranin A was found in chromaffin cells through all steps in embryonic development, three developmental stages of neurokinin A immunoreactivity could be distinguished. In the first and second trimester of gestation, neurokinin A was observed in some groups of chromaffin cells, but no neurokinin-immunoreactive nerve fibres could be detected. In the last trimester of gestation, neurokinin A-reactive chromaffin cells and nerve fibres were both found in adrenal glands. However, in adrenal glands of neonatal piglets, neurokinin A was found only in nerve fibres and not in chromaffin cells. From these results a hypothesis is proposed that neurokinin A might act as a neurotrophic factor in the early stages of the developing porcine chromaffin cells. Biochemical studies are being performed in order to confirm these morphological results and to study the possible role of neurokinin A as a neurotrophic factor in the adrenal gland.On leave from Xian Medical University of China.     

Transfer of concanavalin A between responding lymphocytes and syngeneic stimulating cells in cell-mediated mitogenic response.

We have studied the mechanism of the cell-mediated mitogenic response (CMMR), in which proliferative responses are generated in mouse T lymphocytes co-cultivated with syngeneic mitomycin C-treated spleen cells (Mito-SP), when either responder or stimulator cells are briefly pretreated with concanavalin A (Con A) under nonmitogenic conditions. We present evidence that an intact membrane of the stimulator cells is required in CMMR, since the response was abolished by fixation, or by freezing and thawing of stimulator cells. The fate of cell-bound Con A was studied by tracing I-Con A bound to either stimulator or responder. A lymphoblast that developed from untreated CRT stimulated by I-Con A-coated syngeneic Mito-Sp carried about 105 molecules of 125I-Con A. The amount of Con A released into the culture medium was not sufficient for inducing a mitogenic response by itself, nor to bind to cells at the level found in CMMR, suggesting that 125I-Con A was transferred directly from labeled cells to unlabeled cells. Transferred 125I-Con A found in lymphoblasts was undergraded intact Con A, as demonstrated by gel electrophoresis. Autoradiography allowed visualization of the movement of 125I-Con A from stimulator to responder cells within 60 min of cell contact.     

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

Elevated expression of hexokinase II protects human lung epithelial-like A549 cells against oxidative injury

Increased glucose utilization and hexokinase (HK)-II expression are adaptive features of lung cells exposed to hypoxia or hyperoxia. HK-II is the most regulated isoform of HK. Whether its overexpression could be protective against oxidative stress was explored in human lung epithelial-like (A549) cells. HK-II was overexpressed in A549 cells in a tetracycline-repressible retroviral vector system. Elevated expression of HK-II was confirmed by Western blot and activity measurements. Cell death caused by exposure to hyperoxia was decreased in HK-II-overexpressing cells. This effect was reversed when HK-II expression was suppressed with doxycycline. A similar protective effect was observed in HK-II-overexpressing cells after treatment with 1 mM hydrogen peroxide for 48 h. At baseline, fluorescence microscopy showed that overexpressed HK-II was localized to mitochondria. Electron microscopic studies showed that hyperoxia-exposed HK-II overexpressors had better-preserved and quantitatively smaller mitochondria than those in which the HK-II expression was suppressed or in the nontransduced A549 cells. Mitochondrial membrane potential was increased in HK-II-overexpressing cells exposed to hyperoxia compared with the nontransduced control cells under similar conditions. The present study demonstrates that HK-II protects human lung epithelial-like A549 cells against oxidative insults by protecting the mitochondria.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

Lectin target cells in human central nervous system and the pituitary gland

Summary Peanut lectin (PNL), Concanavalin A (Con A) and Ulex europaeus lectin I (Ulex) were chosen to map their binding sites in different regions of formalin fixed and paraffin embedded human central nervous system tissue and pituitary gland tissues. An extended PaP method was used for PNL and Ulex, whereas a direct peroxidase technique was employed for Con A. In astrocytes, the cytoplasm as well as the delicate processes were stained by PNL and Con A; the most conspicious binding of PNL was seen in the ependymal cells and on the surface of plexus epithelial cells; in the anterior part of the pituitary gland a selective population was PNL positive. Intracytoplasmic Con A acceptors could be demonstrated in neurons, in ependymal cells, and in plexus epithelial cells. Intracytoplasmic Con A receptors were finely granular in astrocytes, oligodendrocytes, and in some cells in the pituitary gland. Ulex binding was restricted to the vascular endothelial cells and a selective population of cells in the pituitary gland. Our results suggest that lectins may be good tools for the evaluation of their respective target cells in the central nervous system and in the pituitary gland.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.