Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.
(33/1806)
A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology. (+info)
Methods to avoid adverse effect of circulating antigen on biodistribution of 125I-labeled antiTac dsFv: preinjection of intact antibody versus clearance of antigen with adivin-biotin system.
(34/1806)
The presence of circulating antigen may adversely affect the biodistribution of a radiolabeled antibody. The alpha subunit of the interleukin-2 receptor (IL-2Ralpha) is a cell-surface receptor that is overexpressed in various hematologic malignancies and in benign disorders. This receptor is cleaved from the cell surface and can be found in high concentrations in serum. Radiolabeled antiTac antibodies are being evaluated to target this receptor. Previous studies have shown that circulating soluble IL-2Ralpha (slL-2Ralpha) adversely affected the biodistribution of radiolabeled antiTac disulfide-stabilized (ds)Fv. In this study, we compared blocking and clearing sIL-2Ralpha to see which better minimized its interference with the biodistribution of radiolabeled antiTac dsFv. METHODS: Two models of sIL-2Ralpha were used: one consisted of mice given intravenous sIL-2Ralpha and the other consisted of mice bearing SP2/Tac tumor xenografts (IL-2Ralpha positive), which shed sIL-2Ralpha. We biotinylated humanized antiTac monoclonal antibody (bt-HuTac) and radiolabeled it with 125I. We then compared its biodistribution with that of humanized antiTac monoclonal antibody IgG (HuTac). We examined the biodistribution of an injected dose of 125I-labeled antiTac dsFv after a preinjection of HuTac to block the sIL-2Ralpha epitope and after a preinjection of bt-HuTac, followed by an avidin chase. RESULT: The 125I-labeled bt-HuTac cleared from the serum at a rate similar to that of HuTac. The avidin chase effectively cleared >92% of circulating 125I-labeled bt-HuTac within 20 min and was also effective in clearing sIL-2Ralpha. In comparison, HuTac prolonged the retention of 125I-labeled sIL-2Ralpha in the circulation, and the avidin chase decreased 125I-labeled sIL-2Ralpha to <18% of control. Although the two-step antigen-clearing system effectively cleared the antigen from the circulation and improved the biodistribution of 125I-labeled dsFv, the HuTac preinjection method had a similar but longer lasting beneficial effect on 125I-labeled dsFv biodistribution. CONCLUSION: Preinjection of either HuTac or bt-HuTac with avidin chase improved the biodistribution of subsequently administered 125I-labeled antiTac dsFv by preventing the dsFv from binding to the sIL-2Ralpha, but the HuTac blocking method is simpler and longer lasting. (+info)
Enhancement of tumor-to-nontumor localization ratios by hepatocyte-directed blood clearance of antibodies labeled with certain residualizing radiolabels.
(35/1806)
To increase tumor-to-nontumor localization ratios of injected radiolabeled antibodies (Abs), several interrelated methods were used. METHODS: The model systems used were two human carcinoma xenografts grown in nude mice, targeted by antibodies RS11 (antiepithelial glycoprotein-2) or MN-14 (anticarcinoembryonic antigen). The Abs were conjugated with biotin and 111In-benzyl diethylenetriamine pentaacetic acid, and, at various times after injection, were cleared by intraperitoneal injection of galactosylated streptavidin, which delivers the complexes to hepatocytes. The radiolabel used was selected because it is retained within tumors after catabolism of the Ab by the tumor cell but is quite rapidly excreted from hepatocytes into bile. RESULTS: With blood clearance induced at 24 h, and dissection 5 h later, high tumor-to-nontumor ratios were attained. Depending on the model used, tumor-to-blood ratios were 16:1 to 31:1, and tumor-to-nontumor ratios for the kidney, lungs and bone were also high and greatly increased by the clearance regimen. Despite clearance into the liver, tumor-to-liver ratios remained >1, due to fairly rapid biliary excretion of the label. The absolute antibody uptake by the tumors was also high, because 24 h was allowed for the Ab to penetrate and bind to cells within the subcutaneous tumors. CONCLUSION: The method described produced high tumor-to-nontumor ratios at 1 d after injection and may be advantageous for tumor imaging with antibodies. Radiation dosimetry calculations indicate that there is only a slight advantage with this approach for radioimmunotherapy. (+info)
Truncation of the C terminus of the rat brain Na(+)-Ca(2+) exchanger RBE-1 (NCX1.4) impairs surface expression of the protein.
(36/1806)
The C terminus of the rat brain Na(+)-Ca(2+) exchanger (RBE-1; NCX1. 4) (amino acids 875-903) is modeled to contain the last transmembrane alpha helix (amino acids 875-894) and an intracellular extramembraneous tail of 9 amino acids (895-903). Truncation of the last 9 C-terminal amino acids, Glu-895 to stop, did not significantly impair functional expression in HeLa or HEK 293 cells. Truncation, however, of 10 amino acids (Leu-894 to stop; mutant C10) reduced Na(+) gradient-dependent Ca(2+) uptake to 35-39% relative to the wild type parent exchanger, and further truncation of 13 or more amino acids resulted in expression of trace amounts of transport activity. Western analysis indicated that Na(+)-Ca(2+) exchanger protein was produced whether transfection was carried out with functional or non-functional mutants. Immunofluorescence studies of HEK 293 cells expressing N-Flag epitope-tagged wild type and mutant Na(+)-Ca(2+) exchangers revealed that transport activity in whole cells correlated with surface expression. All cells expressing the wild type exchanger or C9 exhibited surface expression of the protein. Only 39% of the cells expressing C10 exhibited surface expression, and none was detected in cells transfected with non-functional mutants C13 and C29. Since functional and non-functional mutants were glycosylated, the C terminus is not mandatory to translocation into the endoplasmic reticulum (ER). Endoglycosidase H digestion of [(35)S]methionine-labeled protein derived from wild type Na(+)-Ca(2+) exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the "cellular quality control machinery" can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER-->Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression. (+info)
Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene.
(37/1806)
Conidia (spores) of Colletotrichum lindemuthianum, a fungal plant pathogen causing bean anthracnose, adhere to the aerial parts of host plants to initiate the infection process. These spores possess a fibrillar 'spore coat' as well as a cell wall. In a previous study a mAb, UB20, was raised that recognized glycoproteins on the spore surface. In this study UB20 was used to localize and characterize these glycoproteins and to investigate their possible role in adhesion. Glycoproteins recognized by UB20 were concentrated on the outer surface of the spore coat and, to a lesser extent, at the plasma membrane/cell wall interface. Extraction of spores with hot water or 0.2% SDS resulted in removal of the spore coat. Western blotting with UB20 showed that a relatively small number of glycoproteins were extracted by these procedures, including a major component at 110 kDa. Biotinylation of carbohydrate moieties, together with cell fractionation, confirmed that these glycoproteins were exposed at the surface of the spores. In adhesion assays, > 90% of ungerminated conidia attached to polystyrene Petri dishes within 30 min. UB20 IgG at low concentrations inhibited attachment in an antigen-specific manner. This suggests that the glycoproteins recognized by this mAb may function in the initial rapid attachment of conidia to hydrophobic substrata. Polystyrene microspheres bound selectively to the 110 kDa glycoprotein in Western blots, providing further evidence that this component could mediate interactions with hydrophobic substrata. (+info)
Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.
(38/1806)
We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor. (+info)
Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.
(39/1806)
Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3. (+info)
Display cloning: functional identification of natural product receptors using cDNA-phage display.
(40/1806)
BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence. (+info)