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At least 991 records · Page 55Linked to original sources
PubMed2001

Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies.

We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.

Amino Acid Sequence↗
PubMed1990

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.

B-Lymphocytes↗
PubMed2004

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.

Animals↗
PubMed1986

Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect.

Highly purified pro-urokinase (pro-UK) or single-chain urokinase-type plasminogen activator (scu-PA) was treated with diisopropylfluorophosphate (1 mmol/L) to eliminate traces of two-chain UK activity. This preparation was found to retain a low activity against a chromogenic substrate (S2444), equivalent to 0.1% to 0.5% of the activity of its plasmin-activated derivative. Evidence is presented that the intrinsic activity of pro-UK (scu-PA) was sufficient to activate plasminogen on a fibrin plate or in buffer and was far more reactive against Lys-plasminogen than against Glu-plasminogen. The relative resistance of Glu-plasminogen to activation was overcome by the addition of lysine (25 mmol/L) to the reaction mixture. By contrast, in plasma, pro-UK (scu-PA) was stable and nonreactive for greater than 72 hours when incubated (37 degrees C). Pro-UK (scu-PA) did not form sodium dodecyl sulfate-stable inhibitor complexes, whereas complexation occurred rapidly with UK. Only at high concentrations of pro-UK (scu-PA) (greater than or equal to 250 IU/mL) did plasminogen activation in plasma occur. The relative inertness of pro-UK (scu-PA) in plasma, in contrast to its low-grade enzymatic activity in buffer, was attributed to the effect of inhibitors. The addition of EDTA or the removal of divalent cations by dialysis was associated with a lower threshold for nonspecific plasminogen activation by pro-UK (scu-PA) in plasma. Replacement of Ca++ but not other cations restored baseline conditions. In the presence of a clot, fibrin-selective plasminogen activation and clot lysis were triggered. Lysis was accompanied by less than 10% conversion of pro-UK (scu-PA) to two-chain UK, suggesting that the intrinsic activity of pro-UK (scu-PA) itself may have been responsible for fibrinolysis, although a contribution by the small amount of UK generated could not be excluded. Similarly, pro-UK (scu-PA) supported clot lysis for several days in the same plasma before the effect dissipated as a result of degradation to UK. When Glu-plasminogen in plasma was replaced by Lys-plasminogen, or when lysine was added to normal plasma, nonselective plasminogen activation and fibrinogenolysis occurred. It was concluded that under the experimental conditions, the fibrin specificity of pro-UK (scu-PA) can be explained by its selective activation of fibrin-bound plasminogen and is due to the latter's Lys-plasminogen-like conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed1994

Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.

Animals↗
PubMed1994

Fibroblasts determine the fate of Fc epsilon RI+ cell populations in vitro by selectively supporting the viability of mast cells while internalizing and degrading basophils.

To determine the fate of Fc epsilon RI+ cells on fibroblasts in vitro, human bone marrow derived CD34+ cells were cultured in the presence of recombinant human interleukin 3 and recombinant human hematopoietic stem cell factor for 3 weeks, and Fc epsilon RI+ cells were purified by immunomagnetic selection. This enriched Fc epsilon RI+ cell population consisted of 92-94% basophils and 3-5% mast cells as determined by morphologic, immunohistochemical, and ultrastructural criteria. The Fc epsilon RI+ cells were then cocultured with 3T3 fibroblasts. Basophils decreased markedly by 1 week and were absent from cocultures by 2-3 weeks, while the mast cell numbers on the fibroblast monolayers remained constant. Ultrastructural examination of cocultures at 2 days demonstrated phagocytosis of basophils by fibroblasts. By 1 week, phagocytosed basophil membranes and granules gave fibroblasts the superficial appearance of mast cells by toluidine blue staining. Mast cells surviving in cocultures could be distinguished from granule-containing fibroblasts by IgE surface labeling and by ultrastructural demonstration of tryptase-positive granules. Thus, while mast cells remain viable in coculture with 3T3 fibroblasts, basophils do not survive and are internalized and degraded by the fibroblast monolayer.

Basophils↗
PubMed1995

Biochemical status of renal epithelial Na+ channels determines apparent channel conductance, ion selectivity, and amiloride sensitivity.

Purified bovine renal papillary Na+ channels, when reconstituted into planar lipid bilayers, reside in three conductance states: a 40-pS main state, and two subconductive states (12-13 pS and 24-26 pS). The activity of these channels is regulated by phosphorylation and by G-proteins. Protein kinase A (PKA)-induced phosphorylation increased channel activity by increasing the open state time constants from 160 +/- 30 (main conductance), and 15 +/- 5 ms (both lower conductances), respectively, to 365 +/- 30 ms for all of them. PKA phosphorylation also altered the closed time of the channel from 250 +/- 30 ms to 200 +/- 35 ms, thus shifting the channel into a lower-conductance, long open time mode. PKA phosphorylation increased the PNa:PK of the channel from 7:1 to 20:1, and shifted the amiloride inhibition curve to the right (apparent K(i)amil from 0.7 to 20 microM). Pertussis toxin-induced ADP-ribosylation of either phosphorylated of either phosphorylated or nonphosphorylated channels decreased the PNa:PK to 2:1 and 4:1, respectively, and altered K(i)amil to 8 and 2 microM for phosphorylated and nonphosphorylated channels, respectively. GTP-gamma-S treatment of either phosphorylated or nonphosphorylated channels resulted in an increase of PNa:PK to 30:1 and 10:1, respectively, and produced a leftward shift in the amiloride dose-response curve, altering K(i)amil to 0.5 and 0.1 microM, respectively. These results suggest that amiloride-sensitive renal Na+ channel biophysical characteristics are not static, but depend upon the biochemical state of the channel protein and/or its associated G-protein.

Amiloride↗
PubMed1977

Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

Ammonium Chloride↗
PubMed1980

Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro.

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.

Animals↗
PubMed1990

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17 beta.

A sensitive test system has been developed for estimation of estradiol-17 beta (E2) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E2 is estimated by displacement of biocytinyl-E2, that was produced by ligation of estradiol-17 beta, D-glucuronic acid and biocytin. Bound biocytinyl-E2 is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to approximately 120 fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E2 were found in bovine plasma: male or female calves less than 2.7 pg/ml, cycling cow 0.5-7 pg/ml, cow during the last month of pregnancy 9-310 pg/ml, mature bull 5-30 pg/ml. However, up to 1110 pg E2/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sensitive estimation of estradiol-17 beta in plasma from all type of cattle and for control of improper use of E2 after commitment of a threshold level.

Animals↗
PubMed1999

Cell-specific glycoforms of sialoadhesin and CD45 are counter-receptors for the cysteine-rich domain of the mannose receptor.

We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.

Animals↗
PubMed1969

Radioimmunoassay of beta lipoprotein-protein of rat serum.

A double antibody radioimmunoassay for rat serum beta lipoprotein-protein (beta Lp-protein) is described. The protein was purified by ultracentrifugation, selective heparin-manganous precipitation, and gel filtration on Sephadex G-200. Antiserum was prepared in rabbits by biweekly immunization and absorbed with nonbeta lipoprotein containing rat serum. Iodination with (125)I and purification by gel filtration provided a radiolabeled protein which was > 98% displaced by purified beta lipoprotein in the immunoassay. The radioimmunoassay was sensitive to beta Lp-protein concentrations from 0.1 to 1.5 mug. Specificity of the immunoassay for beta Lp-protein was established by comparison of the displacement curves obtained with serum very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and density (d) > 1.21 fractions and with the beta and alpha migrating lipoproteins eluted from paper electrophoretograms. Suitability of the assay for measuring beta Lp-protein in serum was established by demonstrating 100% recovery of beta lipoprotein added to whole serum and by the absence of immunoreactive beta Lp-protein in serum of orotic acid-treated rats. Examination of sera from six other vertebrates species revealed partial cross-reactivity. Normal rat serum was found to contain 0.25+/-0.01 mg/ml of beta Lp-protein and hepatic production by an isolated perfused rat liver system was determined as 0.145 mg/hr.

Animals↗
PubMed1982

The isolation and characterization of a mouse myeloma protein with anti-dextran activity.

A myeloma protein in ascitic fluid from BALB/c mice bearing W3129 plasma cell tumors was isolated by affinity chromatography. This protein exhibits anti-dextran activity and has been obtained in highly purified form by selective adsorption on isomaltosyl-Sepharose and elution with isomaltose solution. The isomaltosyl-Sepharose was synthesized from maltose, p-aminophenyl glucoside and cyanogen bromide-activated Sepharose by a new procedure utilizing glucosyltransferase and chemical coupling reactions. Results of gel electrophoresis, isoelectrofocusing and agar diffusion experiments showed that the purified myeloma protein consisted of 6 isomeric proteins with each isomer possessing anti-dextran activity. Data from hapten inhibition studies were interpreted to show that the W3129 myeloma protein combines with terminal isomaltosyl units of branched dextrans and oligosaccharides.

Animals↗
PubMed2002

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.

Apoproteins↗
PubMed1999

Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it.

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The alpha or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(LB400)beta(P6), and ht-alpha(B-356)beta(P6). ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E. coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

Biphenyl Compounds↗
PubMed2002

Identifying in vivo targets of cyclin-dependent kinase inhibitors by affinity chromatography.

Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription, differentiation and many functions in the nervous system. The frequent deregulation of CDKs in cancers and in numerous other pathologies justifies the active search for chemical inhibitors capable of reversibly and selectively inhibiting this class of enzymes. Intensive screening of collections of natural and synthetic compounds has led to the identification of several families of ATP competitive CDK inhibitors. As the therapeutic potential of the most promising compounds is currently being evaluated in preclinical and clinical trials, their mechanism of action is still unclear. In particular, the real spectrum of their intracellular targets remains largely unknown. Determination of the selectivity of the compounds and identification of their intracellular targets constitute a prerequisite to understand their cellular effects and to improve their efficiency on a rational basis. The classical method for the determination of a compound's selectivity consists in testing the compound in a panel of purified kinases. However, the selectivity study is then restricted to the panel's enzymes. As a consequence, many, if not most other potential targets are not evaluated. As an alternative way to investigate the range of true targets of CDK inhibitors, we propose an affinity chromatography approach based on immobilized inhibitors. Briefly, the inhibitor is covalently bound to a resin and cellular extracts are batch loaded on this inhibitor matrix. After extensive washing, the bound proteins are resolved by SDS-PAGE and identified by microsequencing. In addition to confirming the interaction of CDK inhibitors with CDKs, this method has led to the identification of additional, sometimes unexpected, targets. We here illustrate the potential of this technique through a few examples.

Animals↗