大肠菌素Colicin S4的重组表达、分离纯化和性质

传统抗生素的广谱抗菌特性是诱导细菌普遍产生耐药性重要原因之一。细菌素由于具有窄谱抗菌活性而可能被开发为新一代抗菌药物。大肠菌素因被认为对人体安全而倍受关注。本研究利用PCR方法获得了大肠菌素S4及其抑制蛋白基因并将其克隆到pQE30质粒中,构建了表达质粒pQE30-Col S4。大肠菌素S4在该表达体系中以可溶形式高表达,产量达到30~50 mg/L。活性分析发现,带有6His标签的重组大肠菌素S4与天然大肠菌素S4特性一致,仍具有对大肠杆菌的选择性抗菌活性,是一种有潜力的抗菌物质。     

Characterization of CD44 antigen during lymphoid ontogeny

We studied immunohistological and biochemical aspects of the CD44 molecule with a mAb produced in our lab: GRHL-1. The characteristic expression of this antigen in cells of B lineage was analyzed. This mAb showed identical immunohistological patterns of reactivity to other mAbs included in CD44 cluster, on a variety lymphoid and nonlymphoid human tissues, and demonstrated similar bands on SDS-PAGE of 125I labeled lymphocyte lysates. This antigen is limited to cells of mature phenotype, and disappears in proliferating B cells in the germinal centers of the lymphoid follicles. CD44 is absent in pre-B and Burkitt cell lines. PKC activation mediate in vitro differentiation of pre-B cell lines. However, it is not involved in up-regulation of CD44 antigen expression.     

Soluble CD44 splice variants and pelvic lymph node metastasis in ovarian cancer patients

Alternative splicing of CD44 and aberrant levels of soluble CD44 protein in the serum of cancer patients has been correlated to tumor progression and metastasis. To examine the clinical value of CD44 serum levels (sCD44) in ovarian cancer we determined concentrations of the soluble, variable isoforms sCD44std, sCD44v5 and sCD44v6 with a sensitive ELISA. Pre-operative serum samples from 66 patients with histologically diagnosed invasive disease as well as sera taken from 40 healthy blood donors were analyzed. In sera of ovarian cancer patients we detected elevated concentrations of overall CD44 serum levels represented by sCD44std (p=0.001), but decreased levels of the specific isoforms CD44v5 (p=0.0002) and v6 (p=0.0001). This is the first report demonstrating that ovarian cancer patients with pelvic lymph node metastasis at the time of diagnosis showed specifically elevated sCD44v6 (p=0.073) serum concentrations in comparison to patients without lymph node involvement, whereas overall sCD44 serum levels did not differ. Decreased serum levels of sCD44v5 were found in progesterone receptor-positive tumors (p=0. 059) and postmenopausal patients (p=0.032). Increased concentrations of sCD44v6 were detectable in estrogen receptor-positive tumors but not significantly (p=0.138). Serum CD44v5 levels were associated with shortened relapse-free survival time. No association was found between serum CD44 isoforms and the classical clinicopathological parameters stage and grading or overall survival. CD44 splice variants are possibly involved in a complex interaction with the hormonal environment during tumorigenesis and metastasis of ovarian cancer.     

Soluble Recombinant Neutral Endopeptidase (CD10) as a Potential Antiinflammatory Agent

Several endogenous peptides, including bradykinin and sustance P, have potent inflammatory effects in the joint. Levels of these peptides are regulated by plasma and cell-associated peptide degrading enzymes. One of these peptidases, neutral endopeptidase-24.11 (NEP-24.11), is expressed constitutively and in high density on human synovial cells and is presumed to play a critical role in local regulation of peptide levels in the joint. We examined the role of endogenous NEP-24.11 in regulating bradykinin-mediated effects in an articular model, and investigated the ability of soluble, recombinant human NEP-24.11 to augment the effects of the endogenous enzyme. Our studies demonstrate that endogenous synovial NEP-24.11 does not significantly modulate inflammatory peptide effects on cells when competing with colocalizing peptide receptors expressed in high density. Administration of excess, soluble recombinant NEP-24.11 can overcome this problem, however. Furthermore, the activity of the recombinant enzyme was not compromised in the presence of oxidants or inflammatory joint fluids. Recombinant NEP-24.11 holds promise as a novel therapeutic strategy for the treatment of inflammatory arthritis.     

Diagnostic utility of CD44 standard, CD44v6, and CD44v3-10 expression in adenocarcinomas presenting in serous fluids.

CD44 is an 85 to 90 kd integral transmembrane protein encoded by a single 20-exon gene located on the short arm of chromosome 11. In the standard form (CD44s), 10 of the 20 exons are transcribed. Multiple variant isoforms exist (CD44v1-10) which arise from alternate mRNA splicing of the remaining 10 exons. In contrast to the standard form of CD44, which is almost ubiquitously expressed, splice variants are highly restricted in their expression in normal or malignant tissues. The purpose of this study was to evaluate the extent to which metastatic adenocarcinomas in effusions express CD44s, CD44v6, and CD44v3-10 and to assess their diagnostic utility in distinguishing reactive mesothelial cells from adenocarcinomas. Archival paraffin-embedded cell blocks of serous fluids from 23 cases of benign effusions containing reactive mesothelial cells and 45 cases of malignant effusions with metastatic adenocarcinoma (18 ovarian, 11 pulmonary, 9 gastrointestinal, and 7 breast) were retrieved from the surgical pathology files. The cytopathology of all cases was reviewed to confirm the diagnosis. Immunohistochemistry was performed on all cases using antibodies for CD44s, CD44v6, and CD44v3-10 (Bender MedSystems, CA). Positive staining was defined as distinct linear membrane staining. Strong staining in at least 10% of the tumor cells was required to consider the case positive for the particular marker. In benign effusions mesothelial cells expressed CD44s in 22 cases (96%), CD44v6 in 1 cases (4%) and CD44v3-10 in 0 cases (0%). In contrast neoplastic cells in malignant effusions expressed CD44s in 11 cases (24%), CD44v6 in 21 cases (47%), and CD44v3-10 in 39 cases (87%). We concluded that CD44s and CD44v3-10 are useful markers that can be applied to cytologic specimens. CD44s immunostaining can be used as a reliable marker to identify reactive mesothelial cells, meanwhile CD44v3-10 immunostaining can detect majority of adenocarcinomas in malignant effusions.     

Expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in benign and malignant breast lesions: correlation and colocalization with hyaluronan

AIMS: To examine the expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in breast lesions, and to correlate it with the expression of hyaluronan (HA). Methods and results: CD 44 expression was studied in 75 breast tissue samples, consisting of benign, premalignant and malignant breast lesions, using immunohistochemistry. CD 44 s, but not CD 44 v 3 or CD 44 v 6, was found in the stromal cells, and it was similar in benign and malignant tumours. In benign lesions CD 4 v 6 was detected in 20-30% of the ductal epithelial cells, while C 44 v 3 and CD 44 s were not expressed. CD 44 s, CD 44 v 3 and CD 44 v 6 were all up-regulated in the in situ carcinomas and invasive carcinomas. The level of CD 44 expression in carcinoma cells did not correlate with the type or differentiation of the tumours. CD 44 and HA expression levels were not closely linked in the benign or malignant breast lesions, because HA was overexpressed later in breast cancer progression than CD 44. However, in breast carcinomas CD 44 and HA positivity was often found in the same areas of the sections, and the dual staining confirmed actual colocalization of CD 44 s and HA in the same cells. Conclusions: CD 44 s, CD 44 v 3 and CD 44 v 6 are up-regulated earlier than HA in breast carcinoma progression, and in later stages they often colocalize with cell surface HA.     

Characterization of green fluorescent protein-expressing retinal cells in CD 44-transgenic mice

Sensory information in the retina is transferred from rod and cone photoreceptors to higher visual centers via numerous parallel circuits that sample the photoreceptor mosaic independently. Each circuit consists of a unique combination of ganglion cell, bipolar and amacrine cell types. The morphology and physiological responses of many amacrine cells have been characterized. However, the synaptic connections and retinal circuits in which they participate are only rarely understood. A major problem that has prevented fuller characterization of retinal circuitry is the need for specific cellular markers for the more than 50 inner retinal cell types. One potential strategy for labeling cells is to use transgenic expression of a reporter gene in a specific cell type. In a recent study of cluster of differentiation 44 (CD44)-enhanced green fluorescent protein (EGFP) transgenic mice, we observed that the green fluorescent protein (GFP) was expressed in a population of amacrine and ganglion cells in the inner nuclear layer (INL) and the GCL. To characterize the morphology of the GFP-labeled cells, whole mount preparations of the retina were used for targeted iontophoretic injections of Lucifer Yellow and Neurobiotin. Furthermore, immunocytochemistry was used to characterize the antigenic properties of the cells. We found that many GFP-expressing cells were GABAergic and also expressed calretinin. In addition to the somatic staining, there was a strong GFP(+)-band located about 50-60% depth in the inner plexiform layer (IPL). Double labeling with an antibody to choline acetyltransferase (ChAT) revealed that the GFP-band was located at strata 3 inner retina. The best-labeled GFP-expressing cell type in the INL was a wide-field amacrine cell that ramified in stratum 3. The GFP-expressing cells in the GCL resemble the type B1, or possibly A2 ganglion cells. The CD44-EGFP mice should provide a valuable resource for electrophysiological and connectivity studies of amacrine cells in the mouse retina.     

Expression of CD44s, CD44v6, and hyaluronan across the spectrum of normal-hyperplasia-carcinoma in breast.

BACKGROUND: The interaction between transmembrane receptors on epithelial tumor cells and the surrounding extracellular matrix molecules is important in tumor progression and metastasis. This interaction is best exemplified by the relationship of the receptor CD44 and the extracellular matrix component hyaluronan (HA). This study seeks to evaluate the expression and the correlation of CD44s, CD44v6, and HA in normal, hyperplastic, and malignant breast epithelium and stroma. MATERIALS AND METHODS: Archival paraffin-embedded tissue from cases of normal breast tissue (n=10), intraductal hyperplasia without atypia (n=13), ductal carcinoma in situ (DCIS) (n=24), stage I infiltrating ductal carcinoma (n=28), stage II infiltrating ductal carcinoma (n=31), and their corresponding positive lymph nodes were retrieved from the surgical pathology files. Tissue sections were evaluated for the expression of CD44s, CD44v6, and HA in the epithelial and stromal cells by immunohistochemistry. RESULTS: Ductal epithelial cells and myoepithelial cells expressed CD44s in all cases of normal and benign breast tissue. The expression of CD44s in breast epithelium progressively decreased with increasing deviation from normal histology: 83% in DCIS, 46% in stage I ductal carcinoma and 26% in stage II ductal carcinoma. The reverse trend was observed for CD44v6 in ductal epithelium: 0% in normal breast, 15% in intraductal hyperplasia, 100% in DCIS, 82% in stage I infiltrating ductal carcinoma, 94% in stage II carcinoma, and 100% of metastatic carcinoma in the lymph nodes. HA was noted exclusively in the stroma but not in the epithelial cells. HA was faintly expressed in the intralobular stroma of normal breast tissue, confined to a narrow faint band adjacent to intraductal hyperplasia and localized to a broad well-defined band around DCIS. Stromal HA staining was more diffuse and intense in infiltrating carcinomas and was particularly pronounced surrounding the metastatic deposits in lymph nodes. CONCLUSIONS: This study demonstrates decreased expression of CD44s accompanied by increased expression of CD44v6 and increased stromal HA in breast cancer. These findings suggest that CD44s, CD44v6, and HA play complementary roles in the development and progression of breast cancer.     

Expression of hyaluronic acid and its receptors, CD44s and CD44v6, in normal, hyperplastic, and neoplastic endometrium

The interaction between epithelial tumor cells and their surrounding stroma is important in tumor progression and metastasis. This is accomplished through a number of transmembrane receptors that interact with stromal extracellular matrix molecules. One of these receptors, CD44, binds to extracellular matrix component hyaluronic acid (HA). The purpose of this study was to evaluate the significance of HA, CD44s, and CD44v6 in benign, hyperplastic, atypical, and malignant endometrial epithelia. Archival paraffin-embedded cell blocks from proliferative endometrium (n = 11), secretory endometrium (n = 12), simple hyperplasia (n = 13), complex hyperplasia without atypia (n = 9), complex hyperplasia with atypia (n = 17), and adenocarcinoma (n = 21) were stained for HA, CD44s, and CD44v6. HA was detected throughout the normal menstrual cycle but was more intense during the secretory phase. Only during the secretory phase was CD44s expressed in the stromal cells in 11 cases (92%), whereas CD44v6 was detected in glandular epithelium in 9 (75%). CD44s was expressed in the glandular epithelium in 2 (15%) cases of simple hyperplasia, 4 (44%) of complex hyperplasia without atypia, 14 (82%) of complex hyperplasia with atypia, and in 16 (76%) of adenocarcinoma. CD44v6 was expressed in the glandular epithelium in 1 (11%) case of complex hyperplasia without atypia, 17 (100%) cases of complex hyperplasia with atypia, and in 18 (86%) cases of adenocarcinoma, but in none of the cases of simple hyperplasia. The endometrial stromal cells expressed CD44v6 in 1 (8%) case of simple hyperplasia, 6 (67%) of complex hyperplasia without atypia, 8 (47%) of complex hyperplasia with atypia, and in 3 (14%) of adenocarcinoma. We concluded that in the normal menstrual cycle, the timing of peak staining of HA and CD44s in the stroma and the up-regulation of CD44v6 in secretory glands are coincident with the period in which the endometrium is most receptive to embryo implantation. HA is more abundant in the stroma adjacent to the tumor, suggesting that interactions between tumor cells and stromal HA promote tumorigenesis. With progression from hyperplasia and with increasing atypia to adenocarcinoma, levels of stromal HA, glandular CD44v6, and glandular and stromal CD44s all increase. Thus, HA and CD44 are both involved in the development and progression of endometrial cancer.     

Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis

No data on lipopolysaccharide-binding protein (LBP) in newborns with sepsis have been available up to now. We therefore determined levels of LBP and soluble CD14 (sCD14) in plasma of healthy and septic neonates in order to evaluate their potential diagnostic role. The study included prospectively collected patient samples of two recently published studies on cytokine expression in neonatal sepsis. Twenty-nine septic patients were enrolled in the present analysis. Samples—either cord blood or peripheral blood—from patients admitted within the first 24 h of life for suspicion of sepsis and cord blood samples of a control group of 40 healthy mature infants delivered spontaneously were analyzed. For seven patients of the septic group, a second sample collected between 24 and 48 h of life was available. Levels of sCD14 and LBP in plasma were determined by an enzyme immunoassay using recombinant CD14 and LBP as standards. LBP and sCD14 were correlated to cytokine plasma levels. In septic neonates, LBP (median, 36.6 versus 7.8 μg/ml; P < 0.001) and sCD14 (median, 0.42 versus 0.28 μg/ml; P < 0.001) levels were highly elevated when compared to those of healthy neonates and strongly correlated to granulocyte colony-stimulating factor (G-CSF), interleukin-1β (IL-1β), IL-6, and IL-8 levels. LBP levels in septic neonates analyzed between 24 and 48 h of life even increased when compared to samples obtained at or shortly after delivery (median, 36.6 versus 60 μg/ml; P = 0.038). In summary, levels of LBP in plasma of neonates with early-onset sepsis are significantly elevated; the elevated plasma levels seem to persist for more than 24 h, which could provide the clinician with a prolonged time period to identify the newborn with bacterial sepsis.     

Increased Levels of Soluble CD30 in Plasma of Patients with Plasmodium falciparum Malaria

Levels of soluble CD30 (sCD30) in serum were elevated in patients with Plasmodium falciparum malaria but showed decline following treatment. The levels of sCD30 in serum were correlated significantly with the expression of gamma interferon by peripheral T cells. These data suggest that CD30⁺ cells are upregulated during a malaria attack and that they may play a regulating role at the site of inflammation.     

Expression of CD44H and CD44v3 in normal oesophagus, Barrett mucosa and oesophageal carcinoma.

AIMS: To examine CD44H and CD44v3 expression in normal gastric and small bowel mucosa, normal and Barrett oesophagus, and oesophageal epithelial malignancies (squamous cell carcinoma and adenocarcinoma). METHODS: Ninety five specimens, comprised of 40 of normal oesophageal, gastric and small bowel mucosa, 22 of Barrett oesophagus (two with dysplastic changes), 20 of resected adenocarcinomas, and 13 of squamous cell carcinoma, were evaluated. The samples were fixed in formalin and subsequently stained with anti-CD44H and anti-CD44v3 monoclonal antibodies using the avidin-biotin peroxidase technique. RESULTS: In contrast to normal oesophagus, which showed positivity for both CD44 epitopes (CD44H and CD44v3) in the basal third of the epithelium, antral and intestinal subtypes of Barrett oesophagus expressed CD44H only, the distribution being focal in non-dysplastic and diffuse in dysplastic Barrett mucosa. Similarly, normal antral glands and small bowel epithelium were focally immunopositive for CD44H at the base of the crypts. All squamous cell carcinomas were diffusely positive for both isoforms, whereas 75% (15/20) of the adenocarcinomas expressed CD44H and 60% (12/20) expressed CD44v3. CONCLUSIONS: CD44H is expressed in the proliferating areas of both normal squamous epithelium and Barrett mucosa. CD44H expression seems to increase progressively in dysplasia and infiltrating carcinoma, similar to the process described in the stomach. CD44v3 expression, usually not observed in normal or neoplastic gastric mucosa, was present in normal squamous epithelium and oesophageal squamous cell carcinoma. CD44v3 immunoreactivity was also identified in 60% of adenocarcinomas. These findings suggest that CD44v3 may play a role in the development of oesophageal carcinoma of both squamous and glandular types.     

CD44 and hematologic malignancies

The expression of CD44 was upregulated in some hematological malignancies and is associated with metastasis and prognosis. The ligation of CD44 with specific monoclonal antibodies can trigger terminal differentiation of leukemic blasts in some subtypes, so it is probable to develop an anti-CD44 based differentiation therapy in leukemia. The effects of CD44 and its monoclonal antibodies are discussed in this review. Cellular ＆ Molecular Immunology.     

可溶性CD83与免疫抑制

CD83是树突状细胞(DC)上重要的表面标志,同时也是重要的功能分子。对CD83功能的认识是从对可溶性CD83(sCD83)研究开始的。sCD83主要通过膜结合型CD83(mCD83)蛋白水解形成,存在于正常人血清中,具有免疫抑制活性。sCD83通过影响细胞骨架抑制DC的功能。在某些血液病和病毒感染患者血清内浓度升高,并引起免疫抑制,导致血液肿瘤细胞和病毒的免疫逃逸。sCD83能够显著抑制实验性自身免疫病病变的发生,在治疗自身免疫病方面展示了广阔的前景。     

Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated

It has recently been recognized that CD44 comprises a largefamily of alternatively spliced forms.In the thymus, CD44 hasbeen postulated to play an important role in immature T cellmigration and maturation. In this paper, we have studied theexpression of CD44 molecules and two CD44 ligands, hyaluronan(HA) and fibronectin (FN), during human thymic fetal development.We found that mAbs against all CD44 isoforms (A3D8 or A1G3)reacted with both thymic epithelial (TE) cells and thymocytesbeginning at the time of initial colonization of the human thymusby hematopoietic stem cells at 8.2 weeks of fetal gestation.However, mAbs specific for splice variants of CD44 containingmembrane-proximal inserts (11.24, 11.10 and 11.9) reacted onlywith terminally differentiated TE cells in and around Hassall'sbodies beginning at 16–19 weeks of fetal gestation. Studiesof differentiated versus undifferentiated TE cells in vitroconfirmed the selective expression of CD44 variant isoformson terminally differentiated TE cells. Expression of HA andFN was determined by fluorescence microscopy using either biotlnylated-HAbinding protein or an anti-FN mAb. We found that whereas FNwas present throughout the human fetal thymus beginning at 8.2weeks, HA was not present until 16 weeks of gestational age.These data demonstrate the differential expression of standardversus variant CD44 isoforms during thymic ontogeny and implicateCD44 interactions with ligands other than HA as important inthe earlier stages of humanthymus development     

A role for CD44 in T cell development and function during direct competition between CD44+ and CD44- cells

The role of CD44 in T cell biology remains incompletely understood. Although studies using anti-CD44 antibodies have implicated this cell adhesion molecule in a variety of important T cell processes, few T cell defects have been reported in CD44-deficient mice. We have assessed the requirement for CD44 in T cell development and mature T cell function by analyzing mice in which CD44(-/-) and WT cells were produced simultaneously. In mixed (CD44(-/-) + CD44(+/+)) bone marrow chimeras, production of CD44(-/-) T cells was shown to be reduced compared to WT cells due to inefficient intrathymic development. In addition, mature CD44(-/-) CD8(+) T cells generated a substantially lower response than WT T cells after infection of mice with lymphocytic choriomeningitis virus, with the reduction in response apparent in both lymphoid and non-lymphoid tissues. Overall, these results demonstrate a poor capacity of CD44(-/-) T lineage cells to compete with WT cells at multiple levels, implicating CD44 in normal T cell function.     

Expression,Purification and Characterization of C-FADD

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecuiar structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain a FADD variant that containing C-terminal region. In this study, mouse FADD （80-205） containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni^2＋ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical a-helix spectrum in CD （circular dichroism） assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ia-binding activity which was important for its non-apoptotic function.     

Purification, Subunit Characterization and Ultrastructure of Three Soluble Bovine Lectins: Conglutinin, Mannose-Binding Protein and the Pentraxin Serum Amyloid P-Component

Conglutinin and mannose-binding protein (MBP) are members of the C-type lectins which are widely present in mammalian plasma. Serum amyloid P-component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tetramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc-like molecules (diameter 11 nm) in the SAP preparations.