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Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z- discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly. (c) 2006 Elsevier Inc. All rights reserved
Ultrasensitive biosensors
(1996)
The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.
KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.
The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.
We have identified a gene, ST18 (suppression of tumorigenicity 18, breast carcinoma, zinc-finger protein), within a frequent imbalanced region of chromosome 8q11 as a breast cancer tumor suppressor gene. The ST18 gene encodes a zinc-finger DNA-binding protein with six fingers of the C2HC type (configuration Cys-X5-Cys-X12-His-X4-Cys) and an SMC domain. ST18 has the potential to act as transcriptional regulator. ST18 is expressed in a number of normal tissues including mammary epithelial cells although the level of expression is quite low. In breast cancer cell lines and the majority of primary breast tumors, ST18 mRNA is significantly downregulated. A 160 bp region within the promoter of the ST18 gene is hypermethylated in about 80% of the breast cancer samples and in the majority of breast cancer cell lines. The strong correlation between ST18 promoter hypermethylation and loss of ST18 expression in tumor cells suggests that this epigenetic mechanism is responsible for tumor-specific downregulation. We further show that ectopic ST18 expression in MCF-7 breast cancer cells strongly inhibits colony formation in soft agar and the formation of tumors in a xenograft mouse model
Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.
The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 mu mol/l and 0.0056 s(-1), respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical.
Monoclonal Antibodies
(2006)
The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC and COSTAR cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis. It was shown that coatings with fibronectin (0.2 ;g/ml) lead to a substantial improvement of cell growth by 50-70% and an increase of monoclonal antibody production by 100-120%. Collagen I coatings showed an improvement in cell growth by 30-70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the ;2-chain of the ;2;1-integrin, which is responsible for binding to collagen and laminin. However, the presence of ;1- integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.
Peripheral T-cell (TC) tolerance can be induced by tolerogenic antigen-presenting cell (APC). A prerequisite is the reduction or blockade of B7 of APC. Besides dendritic cell, B cells can be used as APC. Here, we show the generation B cells with reduced B7 expression by lentiviral transduction of endoplasmic reticulum (ER)-directed CTLA4. Vectors coding for the human CTL4-Ig were used for the human B-cell line Raji. Transduction efficiency was over 90% (MOI = 3). For the murine B-cell line A20 and for primary mouse B cells, murine CTLA4 was used. We show that B cells with reduced B7 expression reduce the antigen (Ag) specific TC proliferation in vitro. B cells expressing an ER-directed CTLA4 may reduce Ag-specific immune responses.
CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.
The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse.
Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.
Sperm proteins of the marine sessile mussels of the Mytilus edulis species complex are models to investigate reproductive isolation and speciation. This study aimed at identifying sperm proteins and their corresponding genes. This was aided by the use of monoclonal antibodies that preferentially bind to yet unknown sperm molecules. By identifying their target molecules, this approach identified proteins with relevance to Mytilus sperm function. This procedure identified 16 proteins, for example, enkurin, laminin, porin and heat shock proteins. The potential use of these proteins as genetic markers to study reproductive isolation is exemplified by analysing the enkurin locus. Enkurin evolution is driven by purifying selection, the locus displays high levels of intraspecific variation and species-specific alleles group in distinct phylogenetic clusters. These findings characterize enkurin as informative candidate biomarker for analyses of clinal variation and differential introgression in hybrid zones, for example, to understand determinants of reproductive isolation in Baltic Mytilus populations.
This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies. It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein-monuron conjugate together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron monoclonal antibody) were used as central components of the assay. The fluorescein-monuron conjugate can be bound either by the anti-fluorescein monoclonal antibody or by the anti-diuron/ monuron monoclonal antibody. Due to steric hindrance, binding of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately, a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle for the determination of diuron, which was demonstrated for concentrations of approximately 5 nM.