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CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cellcell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the Lisoform (GST-Cyto-L) bound poorly to F-actin in a cosedimentation assay, the S-isoform fusion protein (GSTCyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K D ‫؍‬ 7.0 M). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 ؋ 10 ؊8 and 1.0 ؋ 10 ؊7 M for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 ؋ 10 ؊8 and 1.3 ؋ 10 ؊7 M for GSTCyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K D values of 1.3 ؋ 10 ؊5 and 1.8 ؋ 10 ؊5 M, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin. CEACAM1 1 (biliary glycoprotein, CD66a) is a member of the carcinoembryonic antigen (CEA) family, which in turn belongs to the Ig superfamily (1-4). Alternative splicing of the transcripts of a single gene results in expression of at least four CEACAM1 isoforms (5, 6), all of which contain a transmembrane region, followed by a 74-amino acid long (CEACAM1-L) or a 14-amino acid short (CEACAM1-S) cytoplasmic domain. CEACAM1 is a highly glycosylated type 1 transmembrane protein expressed on the surface of epithelial, endothelial, and granulocytic cells (7). The human as well as the rat and mouse isoforms of CEACAM1 have been characterized as homotypic cell adhesion molecules (8, 9). Murine CEACAM1 also functions as a receptor for murine hepatitis virus (10), whereas human CEACAM1 can bind bacterial membrane proteins from Escherichia coli, Salmonella typhimurium, or Neisseria gonorrhoeae (11,12). CEACAM1 expression is down-regulated in human colon (13) and prostate (14) cancer and in 30% of breast cancers (15). Transfection of rat CEACAM1-L into a human tumorig...

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.Keywords: bispecific single-chain antibody; recombinant antibodies; protein targeting; tobacco mosaic virus; transgenic plants.Antibodies are essential tools in medicine, biology and biochemistry. Their high affinity and specificity make them invaluable for diagnostic and therapeutic applications in medicine and human healthcare. To broaden the potential use of antibodies in medical and biological applications, bispecific antibody molecules, which have two independent binding sites for two epitopes are often desirable. Bispecific antibody molecules are unique therapeutic agents with their ability to crosslink two different antigens, which can be exploited in cancer therapy for the recruitment of cytotoxic T cells to a tumor cell [1,2].Various strategies have been used to generate bispecific antibodies. The first bivalent bispecific full-size antibodies or F(ab H ) 2 fragments were produced by in-vitro chemical crosslinking of two different antibodies [3±5]. In addition, bispecific F(ab H ) 2 fragments have been created by heterodimerization of Escherichia coli-expressed Fab fragments through cysteine residues [6] or leucine zippers [7]. Bispecific antibodies have been produced in vivo using the hybrid hybridoma (quadroma) technology [8], but a limitation of this procedure is the low yield of bispecific antibody of the desired dual specificity. Protein engineering has permitted the design of even smaller bispecific fragments based on single-chain Fv fragments (scFv) fragments, such as the scFv heterodimer diabody, which is formed in vivo by noncovalent association of two single-chain fusion products [9]. Alternatively, the two different binding specificities can be combined in a single polypeptide using a flexible linker peptide to form...

Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, e.g., scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p < 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, by which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of α5β1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.