Cholera Toxin B-Mediated Targeting of Lipid Vesicles Containing Ganglioside GM1 to Mucosal Epithelial Cells

Purpose. To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1. Methods. Sonicated lipid vesicles containing ganglioside GM1 were prepared. Inter-vesicle cross-linking due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicles was analyzed with continuous sucrose gradient centrifugation. CTB-mediated binding of GM1 vesicles to human mucosal epithelial cells (Caco-2 and HT-29), mucous membranes of mouse trachea, and nasal tissues were detected with fluorescent labeled vesicles. Results. An increase in lipid particle size due to binding of CTB to lipid vesicles and inter-vesicles cross-linking was detected. At a 30-to-1 mole ratio of membrane-bound GMl-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were associated with GM1 vesicles. Time course analysis showed that inter-vesicles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal epithelial cells HT-29 and Caco-2 with similar affinity [K_d = 7.8 × 10^–4 M lipid (Caco-2) and 7.6 × 10^–4 M lipid (HT-29)]. GM1 mediated binding specificity was demonstrated by blocking with anti-GMl antibody and the insignificant degree of CTB-associated GM1 vesicle binding to GM1 deficient C6 cells. Conclusions. The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 vesicles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Fas receptor expression on B-lineage cells

Mice homozygous for the lpr mutation have B and T cell defects and develop autoantibodies, suggesting that lpr plays a role in their genesis. The lpr defect has been identified as a mutation in the apoptosis-associated Fas receptor (FasR) gene. To begin to define the role of FasR in B cells, we have surveyed FasR expression on B-lineage cells from early progenitors in the bone marrow through their maturation in the periphery. Contrary to some reports, we found that FasR is expressed on B cells at all stages of their development and is highest on germinal center B cells. FasR is not expressed on lpr/lpr-derived cells. These data are consistent with the idea that lpr/lpr mice have an intrinsic B cell defect that may be manifested in developing as well as peripheral B cells. An unexpected finding is that B-1 (CD5) B cells do not constitutively express FasR: FasR becomes detectable on B-1 B cells only after activation.     

气管与主支气管肿瘤的影像诊断（附32例报告）

作者对３２例经病理证实的气管与主支气管良、恶性肿瘤的影像学表现进行分析，归纳为３型：腔内型、壁增厚型和腔内外型。后２种类型提示恶性，而无蒂的腔内型表现者不能鉴别良，恶性。作者还评价了常规体层、ＣＴ及ＭＲＩ诊断气管与主支气管肿瘤的优、缺点，讨论了影像诊断的作用。     

Ultrastructural and immunohistochemical study of Ewing's sarcoma and related tumors: Morphological neural markers suggesting neural histogenesis in 32 small round-cell sarcomas

The purpose of this study was to validate the hypothesis of neural histogenesis of Ewing's sarcoma of bone and related tumors by light microscopic, electron microscopic, and immunohistochemical analysis. We studied 32 round-cell sarcomas (19 cases of Ewing's sarcoma of bone, 3 extraskeletal Ewing's sarcomas, 5 peripheral primitive neuroectodermal tumors (PNET) and 5 cases of unclassified small round-cell type of neurogenic sarcoma (NS). Immunoreactivity for MIC2 was observed in all cases of Ewing's sarcoma and PNET, and in 1 cases of NS. Positive immunoreactivity for neural markers (NSE, synaptophysin, S-100) was found frequently in some tumors. Ultrastructurally, some specific features of neural differentiation, such as a fragmented basal lamina and neurosecretory granule-like particles, were found even in typical cases of Ewing's sarcoma of bone, which presented without a rosette arrangement and were almost negative for neural immuno-markers, but positive for MIC2. These ultrastructural neural features were observed less frequently in Ewing's sarcoma of bone than in PNET and NS. However, no significant correlation was demonstrated between the immunoreactivity for neural markers and the ultrastructural and histological neural features. These results support the hypothesis of a neural origin of Ewing's sarcoma and related neoplasms, and suggest that some overlapping entity could persist in PNET and Ewing's sarcoma and that this entity could be seen in histological and immunohistochemical studies of both tumors.     

Dipeptidyl peptidase IV is down-regulated in rat hepatoma cells at the mRNA level

Dipeptidyl peptidase IV (DPP-IV) is a cell surface ectopeptidase that has been implicated in cell-extracellular matrix interactions, lymphocyte growth and the regulation of biological peptides. Previous studies have shown that immunostaining for DPP-IV and DPP-IV enzyme levels is decreased in hepatoma cells and levels have been correlated with the ability of such cells to adhere in vitro. The aim of this paper was to measure DPP-IV enzyme levels in rat hepatoma cells and to examine whether changes were associated with alterations at the mRNA level. The results indicate a greater than 90% reduction in DPP-IV enzyme levels in two rat hepatoma cell lines, HTC and H35, compared with rat hepatocytes. Enzyme levels of the control enzyme leucine aminopeptidase (LAP) were not decreased. mRNA studies indicated that these changes were associated with similar reductions in rat DPP-IV mRNA. It is concluded that DPP-IV is markedly reduced at the protein, enzyme and mRNA levels in rat hepatoma cells. The significance of these changes is unclear but may lead to decreased extracellular matrix interactions by such cells.     

Antisperm antibodies and lymphocyte subsets in semen—not a simple relationship

The significance of white blood cells in the ejaculate remains a matter of controversy. Several authors have suggested that such cells are important in the modulation of an antisperm antibody response, i.e. a predominance of suppressor/cytotoxic to helper/inducer T cells may prevent the development of antisperm antibodies. In order to examine this relationship further we have documented the white blood cell types, with emphasis on the T-lymphocyte populations, in the ejaculates of men from infertile couples with and without antisperm antibodies; the latter group was divided further into two groups--vasovasostomized men and idiopathic men. All seven of the men without antisperm antibodies had a predominance of suppressor/cytotoxic T cells to helper/inducer T cells in the ejaculate. However, only in some of the men with antibodies was there a predominance of T-helper/inducer cells. It is clear that the relationship between antisperm antibodies and seminal leucocytes is therefore not as straightforward as has been proposed.     

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside-specific plant lectin, Viscum album agglutinin (VAA-I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA-I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA-I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA-I induced a significant increase in the cytokine-dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non-metastatic breast cancer using fluorescence-activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence-conjugated VAA-I. Co-incubation with d -galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin-3, granulocyte/monocyte colony-stimulating factor and granulocyte colony-stimulating factor. VAA-I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA-I increased the cytokine-dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA-I binds to haematopoietic progenitor cells and has a co-stimulatory effect on their proliferation.     

人酪氨酸羟化酶基因真核表达载体的构建及在哺乳动物细胞中的表达

用限制性内切酶EcoRI从pKS(-)HTH_1切下全长为1.9 kb的人酪氨酸羟化酶基因,在T_4DNA连接酶的作用下连接在真核表达载体pCDNA_3的EcoR Ⅰ位点,构建成重组质粒pcD-NA_3HTH_1,该质粒转染COS-7细胞,免疫荧光组织化学染色证实酪氨酸羟化酶在其中的表达。