Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates.

We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.     

Slit drainage--progress in drainage treatment after herniotomy]

In a prospective randomized study was conducted on 80 patients undergoing elective hernia repair to assess whether a new slit-suction drainage is less painful than a Redon-suction drainage. Main endpoint of the study was the traction power in order to remove the drain. Other endpoints were pain at the time of removal of the drain and the effectiveness to draw off secretion. To remove redon drain the average traction power was 226 pond and to remove slit drain the average traction power was only 25 pond. The difference was significant (p less than 0.01). At the time of removal patients with slit drain rarely complained about pain (p less than 0.01). On contrary, patients with Redon drain always complained about pain. Clotting were seldom shown in slit drains and were frequently shown in redon drains. Both slit drains and redon drains were effective to drain secretion. As the new slit drain was more comfortable for patients following hernia repair slit drain should always be used as subcutaneous suction drainage.     

High abundances of neurotrophin 3 in atopic dermatitis mast cell

Background  

Neurotrophin 3 (NT-3) is a member of the neurotrophin family, a group of related proteins that are known to regulate neuro-immune interactions in allergic diseases. Their cellular sources and role in the recruitment of mast cell precursors in atopic dermatitis have not been characterized in detail so far.     

Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen

BACKGROUND: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells. Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow's milk. It is not well established, however, how this presence of LPS affects oral tolerance induction. METHODS: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein beta-lactoglobulin (beta-LG) on antigen-specific immune responses. IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against beta-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of beta-LG was evaluated. RESULTS: LPS contamination of beta-LG provoked a beta-LG-specific IgG2a response, as well as an enhanced beta-LG-specific IgG1 response upon intraperitoneal immunization. Oral tolerance induction to beta-LG was induced by aqueous solutions of beta-LG with and without LPS administration. Conversely, oral administration of w/o-emulsified beta-LG prevented oral tolerance to beta-LG only when the beta-LG was contaminated with LPS. CONCLUSIONS: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.     

TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo

The fusion gene of herpes simplex virus thymidine kinase and green fluorescent protein (TK-GFP) was shown to be a versatile tool for examining the features of thymidine kinase/ganciclovir gene therapy in vitro. In this study, we used viral vectors carrying the fusion gene to characterize the aspects of this gene therapy form in rodent tumor models. Growth of subcutaneous 9L rat tumors transduced ex vivo with TK-GFP gene was prevented when ganciclovir (GCV) treatment was initiated immediately after tumor inoculation. Established tumors (>100 mm(3)), however, were untreatable despite the initial 55% proportion of TK-GFP positive cells. This was due to a rapid clearance of TK-GFP positive cells, but not GFP positive cells. Propidium iodide staining revealed that TK-GFP lentivirus vector was able to induce apoptosis/necrosis in 9L cells, as opposed to the respective GFP vector. Furthermore, when a subcutaneous nude mouse tumor model was used, the percentage of TK-GFP positive cells in vivo was maintained similarly as in cultured cells, suggesting contribution of a fully functional immune response to the disappearance of fusion gene positive cells. In vivo gene transfer studies: adenovirus TK-GFP vector injections resulted in about 25% gene transfer efficiency to 9L tumors and showed that their growth could be significantly reduced even when the tumor volumes were already >120 mm(3). Part of the effect was shown to be due to cytotoxicity of the vector. In summary, our results demonstrate the utility of TK-GFP fusion gene-carrying viral vectors in animal studies and show that readily detectable therapeutic genes can help us to understand the complicated nature of in vivo cancer gene therapy experiments.     

Genetic characterization of L-Zagreb mumps vaccine strain

Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide sequence was previously determined (accession numbers , and ). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed. A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s-1970s). L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.     

Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene

We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.     

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer

Recently, we identified increased cathepsin X expression in H. pylori-infected gastric mucosa. Here, we describe further up-regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up-regulation in H. pylori-infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori-infected and non-infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non-contact co-cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5-fold) and protein (1.6-fold) expression than H. pylori-negative patients. Cathepsin X was also up-regulated in gastric cancer (3-12-fold) compared to non-neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)-positive H. pylori-infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up-regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer.