In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

Postembedding immunogold histochemistry for the localization of laminin and its E4 and P1 fragments in mouse kidney embedded in LR-White and LR-Gold

Summary A method is presented which permits the ultrastructural localization of laminin and its E4 and P1 subunits in the renal cortex of the mouse embedded in LR-White or LR-Gold. It was performed with postembedding immunogold histochemistry using polyclonal antibodies against either the entire laminin molecular or the E4 fragment or with a monoclonal antibody against the P1 fragment. Localization of laminin was achieved in LR-White and in LR-Gold embedded kidney. Using polyclonal antibodies against the entire laminin molecule, laminin could be localized with direct as well as with indirect immunogold histochemistry with a gold labelled IgG as secondary antibody. In contrast, immunostaining for the E4 or the P1 fragments was possible only with antibodibodies directly labelled with gold.     

小鼠膜型抗衰老蛋白Klotho基因特异片段的克隆、原核表达及多克隆抗体制备

目的克隆小鼠膜型抗衰老蛋白Klotho基因特异片段,制备小鼠Klotho多克隆抗体。方法以小鼠基因组为模板进行PCR,克隆了小鼠膜型抗衰老蛋白Klotho基因外显子Ⅳ部分序列,经BamH I和Nhe I双酶切后定向克隆到质粒pET-GST中,构建原核表达质粒pET-GST-Klotho,转化大肠埃希菌BL21（DE3）,用IPTG诱导表达。以重组GST-Klotho融合蛋白免疫家兔,制备Klotho多克隆抗体。结果表达产物经SDS-PAGE检测表明,在大肠埃希菌中成功表达了GST-Klotho融合蛋白,GST-Klotho融合蛋白表达量占菌体总蛋白的15％左右;另外通过ELISA法测得抗血清抗体效价约为1：10000,Western印迹分析验证了抗体特异性。结论GST-Klotho融合蛋白的表达和Klotho多克隆抗体的制备为进一步研究Klotho蛋白在小鼠体内的表达模式以及相关抗衰老药物的研制奠定了基础。     

Localization of transforming growth factor-beta 1 in mitochondria of murine heart and liver.

Using both electron microscopic immunohistochemistry and cell fractionation techniques, we show that transforming growth factor-beta 1 (TGF-beta 1) is found in mitochondria of rat and mouse cardiac myocytes and rat hepatocytes. Four different polyclonal antibodies, raised against various epitopes encompassing the mature portion of the TGF-beta 1 molecule as well as the pro-region of its precursor, were used for the electron microscopy studies. The localization of TGF-beta 1 in mitochondria was confirmed by detection of the native peptide in mitochondria isolated from rat heart and liver; the majority of native TGF-beta 1 found in liver homogenates was recovered in highly pure mitochondrial fractions. The functional role of TGF-beta in the mitochondrion is unknown at present.     

The expression of ceruloplasmin, an angiogenic glycoprotein, by mouse embryonic fibroblasts

Balb/c 3T3, Swiss 3T3 and Rous sarcoma virus transformed Balb/c 3T3 mouse embryonic fibroblasts produced ceruloplasmin in vitro, whereas primary cultures prepared from the Balb/c mouse embryos did not produce ceruloplasmin. The amount of ceruloplasmin synthesis by the Balb/c 3T3 cell line is enhanced by Rous sarcoma virus-transformation (1.5-3 fold) and by treatment with dexamethasone (about 2.4 fold). The protein was identified as ceruloplasmin by immunoprecipitation with ceruloplasmin-specific polyclonal antibody, and by similarity of peptide maps, and subunit molecular weight (135,000 dalton) to that of authentic ceruloplasmin from primary cultures of mouse hepatocytes.     

Protective effects of HFE7A,mouse anti-human/mouse Fas monoclonal antibody against acute and lethal hepatic injury induced by Jo2

HFE7A is a mouse anti-human/mouse Fas monoclonal antibody which, protects mice from fulminant hepatitis induced by Jo2. Herein, we report on the mechanism of the protective effect of HFE7A against Jo2-induced acute and lethal hepatic injury. HFE7A reduced the serum aminotransferase level which was elevated after Jo2 injection. HFE7A also inhibited caspase activation and mitochondrial depolarization in hepatocytes derived from apoptosis induced by Jo2 injection. The protective effect of HFE7A against Jo2-induced apoptosis in mouse hepatocytes was reproducible in vitro. The cell death and caspase activation in isolated mouse hepatocytes were induced by incubating these cells with Jo2 in vitro, and HFE7A inhibited the cell death and caspase activation in mouse hepatocytes in a dose-dependent manner. The affinity of HFE7A to mouse Fas was lower than that of Jo2. The binding of Jo2 to neither recombinant mouse Fas nor mouse hepatocytes was inhibited by an excessive amount of HFE7A. Interestingly, HFE7A bound to hepatocytes isolated from Fas knockout mice. From these results, it is suggested that HFE7A may exert a protective effect against Jo2-induced hepatitis not by competitively inhibiting the binding of Jo2 to Fas on hepatocytes, and that a distinct molecule other than Fas may possibly be involved in the protective effect of HFE7A against Jo2-induced hepatic injury.     

小鼠骨保护素配基胞外片段的表达、纯化及生物活性分析

骨保护素配基(OPGL)是调节破骨细胞分化和成熟的核心细胞因子。由小鼠骨组织提取总RNA,RTPCR扩增得到小鼠OPGL胞外片段(sOPGL)cDNA,以特定策略克隆人表达载体pET-42a( ),以便使未来表达产物的融合标签序列能够完全被因子Xa切除。重组载体在大肠杆菌中诱导表达可获得高水平的47kD产物,Western blotting证实它可被OPGL抗体识别。经Glutathione-sepharose 4B亲和层析,除融合蛋白外,还有一约30 kD蛋白与层析柱发生了特异性亲和。该30kD蛋白可被GST-IGF-I多克隆抗体识别,但不能被OPGL抗体识别,提示它的产生乃由于融合蛋白在融合位点附近发生裂解。融合蛋白经Xa因子裂解和进一步纯化,得到分子量约17．5kD的sOPGL。生物活性分析证明,重组sOPGL可以促进OLC的生成,并呈现剂量依赖关系。     

Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.     

A new embryonic antigen,p67, present during mouse preimplantation development

An antibody prepared against nullipotential teratocarcinoma stem cells (A-N₁) detects cell surface antigens expressed by early mouse embryos and inhibits in vitro development of embryos in the absence of complement [Calarco and Banka, 1979]. Here we report the immunoprecipitation and electrophoretic characterization of A-N₁-detected antigens from preimplantation mouse embryos. Predominant antibody activity is directed against a 67,000-dalton glycoprotein (p67) with a mean pI of 5.3, which has not been previously described. This protein is not detected, at least as p67, after culture of embryos in tunicamycin. The p67 antigen is also expressed by pluripotential PSA1 teratocarcinoma cells but not by several different differentiated mouse cell types.     

小鼠KCTD10特异性抗体制备及其在小鼠背神经上皮和背根神经节中的表达

KCTD10蛋白是一个TNF-α诱导表达的蛋白,能与DNA聚合酶δ的小亚基和PCNA相互作用。但其具体功能尚不清楚。为了研究KCTD10的功能,用小鼠KCTD10融合蛋白免疫新西兰大白兔获得兔抗鼠KCTD10多克隆抗体。由于小鼠KCTD10蛋白与小鼠PDIP1蛋白和TNFAIP1蛋白有较高的蛋白序列相似性,KCTD10抗体同PDIP1蛋白、TNFAIP1蛋白有抗体交叉反应。将同源蛋白PDIP1和TNFAIP1的部分降解片段与抗KCTD10血清在4℃下孵育3h从而消除非特异性抗体,成功地消除了KCTD10多克隆抗体对PDIP1蛋白和TNFAIP1蛋白的交叉反应。用此抗体做小鼠胚胎及胚胎切片的免疫组化,发现KCTD10蛋白在E12.5d的小鼠胚胎背根神经节以及神经管神经上皮中有明显的表达。该结果提示KCTD10可能在小鼠的神经管神经上皮和背根神经节发育中发挥作用。     

Plasma cell antigen PC-1 and the transferrin receptor in mouse, rat, and hamster: serologic and biochemical analysis

The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.     

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development

Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

Preparation of an antibody recognizing both human and rodent MRP1.

Based on the high level of identity among human, mouse, and rat MRP1 protein sequence, we produced a specific polyclonal antibody (MRP1-A23) against a synthetic polypeptide covering the C-terminus of the human protein. Western blot analysis showed a reactivity against human MRP1 similar to that obtained with the monoclonal QCRL1 antibody. Differently from other available antibodies against human MPR1, MRP1-A23 also detected both rat and mouse MRP1. No cross-reactivity was observed with either human or mouse MRP2 while MRP1-A23 weakly cross-reacted with rat MRP2 in the protein region ranging from 1512 to 1533 amino acids. These data indicate that MRP1-A23 allows specific MRP1 detection in both human and rodent tissues and may provide an important tool in the study of MRP1 expression and function in both experimental and clinical materials.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

唐氏综合症决定域蛋白1泛素化分析

利用Fm oc固相多肽合成的方法合成DSCR1羧基端一个多肽片段(55-70AA),经HPLC纯化后偶联到匙孔槭血蓝蛋白,免疫新西兰雄兔后采血检测、纯化、经W estern b lotting、免疫沉淀证实得到的抗体为抗DSCR1的特异抗体。该抗体即能检测人源DSCR1蛋白,又能检测小鼠的DSCR1蛋白。运用获得的DSCR1多克隆抗体进行功能研究,发现DSCR1广泛存在泛素化,参与泛素化-蛋白酶体途径。