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Biomedical subjects

R F Ebert

Publications and source records attributed to R F Ebert.

At least 19 recordsLinked to original sources
PubMed1998

Highly sensitive thromboplastins do not improve INR precision.

For determination of the international normalized ratio (INR), it has been suggested that "highly sensitive" thromboplastin reagents (International Sensitivity Index [ISI] < or = 1.2) provide the most consistent performance and minimize interlaboratory variability. We compared the INR values obtained from 69 specimens drawn from patients receiving long-term oral anticoagulant therapy, using four thromboplastin preparations (manufacturer-assigned ISI range of 0.96-1.10) and two automated photo-optical analyzers. Multivariate analysis of the INR response matrix (552 INR values) indicated that the eight reagent-coagulometer combinations did not produce equivalent INR values. Similar analysis indicated that INR values were not normalized when uncorrected prothrombin ratios or INR values, calculated after assignment of "local ISI values" to each thromboplastin reagent, were compared. The INR differences also seemed to be clinically significant because 17% to 29% of paired thromboplastin values were discordant when all INR values were assigned to one of four therapeutic categories used in oral anticoagulant therapy (< 2.0; 2.0-3.0; 3.0-4.5; or > 4.5). These differences in INR values obtained with two photo-optical coagulometers and four highly sensitive thromboplastin reagents suggest that the existing INR system has not achieved the goal of standardized prothrombin time values and does not support the recommendation to use only highly sensitive reagents for the regulation of oral anticoagulant therapy.

Anticoagulants↗
PubMed1996

Keyhole limpet hemocyanin: structural and functional characterization of two different subunits and multimers.

Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

Amino Acid Sequence↗
PubMed1994

Immunotherapy with keyhole limpet hemocyanin: efficacy and safety in the MB-49 intravesical murine bladder tumor model.

The antitumor activity and potential toxicity of a clinical-grade keyhole limpet hemocyanin preparation (KLH-Immune Activator; KLH-IA) were determined in the MB-49 intravesical murine bladder tumor model. Mice were immunized subcutaneously with KLH-IA two weeks prior to intravesical implantation of MB-49 tumor cells. Treatment consisted of intravesical KLH-IA (10 or 100 micrograms.) 1, 4, 7, 14 and 21 days after implantation. Control animals either were not immunized prior to tumor implantation and KLH-IA treatment, or were immunized with KLH-IA and treated with the vehicle. By 4 weeks after implantation tumor outgrowth in the treated groups was significantly decreased (p < 0.01, Fisher's Exact) relative to the control groups. Prior subcutaneous immunization was required to elicit antitumor activity of KLH-IA; thus, the mechanism of action is immune-mediated and not due to spurious interference with tumor implantation by intravesical instillations. Animals treated with a dissociated form of KLH exhibited decreased tumor outgrowth approaching, but not attaining, significance (p < 0.09, Fisher's Exact). A separate toxicity study in which KLH-IA was given subcutaneously (4 mg./kg.), intraperitoneally (40 mg./kg.), or intravesically (40 mg./kg.) disclosed no significant gross or histopathologic abnormalities except for mild-to-moderate papillary hyperplasia in all catheterized animals. These results establish the efficacy and safety of KLH-IA in mice and suggest that clinical trials for intravesical treatment of superficial bladder cancer may be warranted.

Adjuvants, Immunologic↗
PubMed1994

A high-sensitivity thromboplastin reagent prepared from cultured human cells.

High-sensitivity thromboplastin reagents suitable for use in the prothrombin time (PT) assay are typically prepared from human brain and placenta, tissues that are in limited supply and subject to viral contamination. Cloning and expression of recombinant human tissue factor (TF) has enabled production of a new generation of thromboplastin reagents whose performance and utility are under active investigation. The purpose of this study was to determine the feasibility of producing a sensitive human thromboplastin reagent from a non-recombinant source: cultured human cells. Several human cell lines with apparently high constitutive TF synthesis were identified, and a viable thromboplastin reagent (Humaplastin) was produced from a human lung cell line via a non-conventional process that did not require reconstitution or rehydration of TF in cell membranes. When calibrated against BCT/253, a human brain international reference thromboplastin, Humaplastin exhibited a mean normal prothrombin time of 12.6 +/- 0.7 s (mean +/- SD: n = 20) and an International Sensitivity Index of 1.09 +/- 0.019. The performance of this reagent was well correlated (r = 0.983) with Thromborel S, a commercially available human placental thromboplastin reagent. Orthogonal least squares regression of the log PT values from the placental thromboplastin reagent versus Humaplastin and two recombinant TF-based thromboplastin reagents suggested that the latter three reagents are somewhat more sensitive than the placental thromboplastin reagent, although such differences should not be expected to have a significant impact on clinical utility. It is concluded that cultured human lung cells represent a suitable source of tissue thromboplastin for production of a high-sensitivity non-recombinant thromboplastin reagent.

Adenocarcinoma↗
PubMed1993

Immunotherapy of murine bladder cancer with keyhole limpet hemocyanin (KLH).

Keyhole limpet hemocyanin (KLH) is a potent immunogen that is being evaluated as an immunotherapeutic alternative to BCG in the treatment of bladder cancer. In the mouse bladder tumor model (MBT2) intralesional KLH significantly reduced tumor incidence, growth rate, and mortality and exhibited antitumor activity similar to that achievable with BCG. Endotoxin contamination of KLH was not responsible for the antitumor activity, although endotoxin alone was shown to have anti-tumor activity in this animal model. Keyhole limpet hemocyanin is both safe and effective in the MBT2 model, and is an immunomodulator to consider for clinical trials.

Adjuvants, Immunologic↗
PubMed1993

A comparative evaluation of ELISAs for D-dimer and related fibrin(ogen) degradation products.

This study was designed to (i) characterize two new monoclonal antibodies that react with neoepitopes on fibrin(ogen) fragments D-dimer and D1 and (ii) compare the specificity of these antibodies with that of two commercially available 'D-dimer' ELISAs [Dimertest EIA (American Diagnostica, Inc.) and Asserachrom D-Di (Diagnostica Stago)] and also with an ELISA for fibrinolytic fragments containing the E domain complexed with a D (or D-dimer) moiety [Fibrinostika FbDP (Organon Teknika)]. All assays were in a capture (sandwich) ELISA format. Fibrin(ogen) degradation products were isolated in high yield by a novel method involving hydrophobic interaction chromatography. The results disclosed considerable differences in sensitivity and specificity for purified fibrinolytic fragments among all five ELISAs. Although the diagnostic utility of monitoring plasma fibrinolytic fragments is not questioned, our results provide a rational basis for understanding why mAb-based ELISAs for these analytes are not well correlated. If, as our data suggest, the D-dimer ELISAs are not actually measuring the same analytes, then interpretation of studies that determine D-dimer concentrations with different methods will be problematic and standardization of D-dimer measurements across assays will be impossible.

Antibodies, Monoclonal↗
PubMed1989

Fibrinogen Baltimore IV: congenital dysfibrinogenemia with a gamma 275 (Arg----Cys) substitution.

The major functional characteristics of fibrinogen Baltimore IV include delayed fibrin monomer polymerization which is not corrected by the addition of calcium, inhibition of normal plasma coagulation upon mixing, and a biphasic polymerization profile in which the abnormal fibrin monomers appear to polymerize at about 2% of the normal rate (Ebert & Bell, Thromb. Res. 38:121-128, 1985). We now report the detection of an abnormal peptide in a comparative peptide map from a cyanogen bromide digest of fibrinogen Baltimore IV. The sequence of this peptide corresponds to the fibrinogen sequence gamma 265-310, except that Arg275 is replaced by Cys. Amino acid analyses of carboxymethylated peptides from control vs. fibrinogen Baltimore IV confirmed the sequencing data. Both the propositus and his affected daughter were heterozygous for the trait.

Amino Acid Sequence↗
PubMed1988

Fibrinogen Baltimore III: congenital dysfibrinogenemia with a shortened gamma-subunit.

An abnormal fibrinogen has been found in an asymptomatic Negro female. Clinical laboratory findings were normal, except for a prolonged thrombin time which was corrected by addition of calcium. Fibrinopeptide release by thrombin and crosslinking by factor XIII also occurred normally, but fibrin monomer polymerization was delayed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis disclosed that 50% of the gamma-subunits migrated with an apparent Mr of 45,500, approximately 1,500 Da smaller than normal. The evidence suggests that an internal sequence of 10-15 residues is missing from the gamma-subunit of the abnormal fibrinogen.

Adult↗
PubMed1988

Isolation and comparative peptide mapping of fibrinogen subunits by reversed-phase high-performance liquid chromatography.

Comparative peptide mapping represents one approach to identification of structural defects in variant human fibrinogens. In view of the large size of this protein, we chose to generate peptide maps of fibrinogen subunits. A reversed-phase high-performance liquid chromatographic method was developed to isolate the subunits: fibrinogen was reduced with dithioerythritol and alkylated with iodoacetamide. Subunits were isolated on a Vydac TP, C4 column (25 x 1.0 cm). Eluent A was 0.1% aqueous trifluoroacetic acid (TFA); eluent B was 0.1% TFA in acetonitrile. Initial conditions were 65% A, 35% B, at 2 ml/min. The reduced-alkylated subunits were lyophilized, redissolved in 0.1% TFA plus 4-8 M guanidine-HCl, and chromatographed using a linear gradient (1%/min) to 50% B. This procedure provides homogeneous subunits in yields exceeding 90%, and is therefore superior to conventional cation-exchange chromatography. For comparative peptide mapping, the same stationary and mobile phases were used, except that the initial conditions were 90% A/10% B, and a linear gradient to 60% B (1%/min) was used. Alternatively, peptide maps were generated with a 10 x 0.46 cm Spherisorb ODS-2 column and very shallow gradients. The mapping procedure resolves 45-60 peptides with excellent reproducibility, and has been applied to the identification of an apparent polymorphism in fibrinogen Baltimore II, and the structural defect in fibrinogen Baltimore IV.

Chromatography, High Pressure Liquid↗
PubMed1988

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity.

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.

Animals↗
PubMed1987

Intracellular growth factors in polycythemia vera and other myeloproliferative disorders.

In polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis, hematopoietic cell proliferation is increased in the absence of a recognizable stimulus, suggesting the autonomous production of growth factors in these disorders. Sonicates of peripheral blood mononuclear cells (PBMNC) from patients with polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis contained soluble factors that stimulated the proliferation of quiescent-confluent 3T3 cells. PBMNC sonicates from normal individuals; from patients with secondary erythrocytosis, chronic myelogenous leukemia, B-cell chronic lymphocytic leukemia, and acute myelogenous leukemia; and from K-562 and HL-60 cells did not stimulate proliferation. Polycythemia vera PBMNC sonicates also induced anchorage-independent colony formation in soft agar by normal rat kidney fibroblasts. Both the mitogenic and transforming activities of the polycythemia vera PBMNC sonicates resided in the T-lymphocyte-depleted mononuclear fraction of the PBMNC and were not secreted. By gel filtration, reversed-phase HPLC and NaDodSO4/PAGE, the mitogenic and transforming activities in the polycythemia vera PBMNC were localized to three proteins with molecular masses of 13-, 17-, and 65-kDa. The 13-kDa protein was only mitogenic, and the 17-kDa protein was only transforming, whereas the 65-kDa protein had both mitogenic and transforming activity. These proteins may be involved in the autonomous hematopoiesis that characterizes polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis.

Animals↗
PubMed1986

Fibrinogen Seattle II: congenital dysfibrinogenemia with an Arg (A alpha 16)----his substitution.

Incubation of fibrinogen Seattle II with thrombin (17 mu/ml) resulted in the release of two forms of fibrinopeptide A (FpA) which were resolved by high-performance liquid chromatography. Amino acid analysis disclosed that the abnormal FpA contained histidine in place of arginine. At lower, approximately physiologic thrombin concentrations only half the normal amount of FpA was released, and fibrinopeptide B (FpB) release was delayed. The effect of this substitution on the time course of fibrinopeptide release is consistent with conclusions drawn from other studies on the kinetics of fibrinopeptide release, viz., that prior removal of FpA is not required before FpB hydrolysis by thrombin, and that optimal rates of FpB release occur after formation of fibrin I polymer.

Afibrinogenemia↗
PubMed1986

Amino acid analysis by HPLC: optimized conditions for chromatography of phenylthiocarbamyl derivatives.

A method for analysis of phenylthiocarbamyl-amino acids by HPLC is described. Solvent A contains 0.05 M sodium acetate plus triethylamine (2.75 ml/liter) adjusted to pH 6.40 with phosphoric acid. Solvent B consists of 50% solvent A, 40% acetonitrile, and 10% methanol. The stationary phase is a 3-micron Spherisorb ODS-2 column (4.6 X 100 mm). A new sample can be injected every 27 min. Variation of the triethylamine concentration from 1 to 4 ml/liter is shown to affect column selectivity in a predictable manner, and this provides a rational basis for optimizing chromatographic conditions. Resolution of certain hydrophilic amino acids (Arg, Thr, Ala, and Pro), and mobile phase stability are improved over previously described procedures.

Amino Acids↗
PubMed1986

Purification and characterization of two isoforms of Acanthamoeba profilin.

Acanthamoeba profilin purified according to E. Reichstein and E.D. Korn (1979, J. Biol. Chem. 254:6174-6179) consists of two isoforms (profilin-I and-II) with approximately the same molecular weight and reactivity to a monoclonal antibody but different isoelectric points and different mobilities on carboxymethyl-agarose chromatography and reversed-phase high-performance liquid chromatography. The isoelectric points of profilin-I is approximately 5.5 and that of profilin-II is greater than or equal to 9.0. Tryptic peptides from the two proteins are substantially different, which suggests that there are major differences in their sequences. At similar concentrations, both profilins prolong the lag phase at the outset of spontaneous polymerization and inhibit the extent of polymerization. Both forms also inhibit elongation weakly at the barbed end and strongly at the pointed end of actin filaments.

Actins↗
PubMed1985

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence.

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.

Amino Acid Sequence↗
PubMed1985

Protective effect of divalent cations in the plasmin degradation of fibrinogen.

Calcium limits the plasmic proteolysis of fibrinogen fragment D by binding to a specific site on the carboxy-terminal segment of the D gamma chain. Employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize plasmic fragments, Sr2+, Ba2+, and Mn2+ were found to have an equivalent capacity to limit the degradation of fibrinogen fragment D (Mr 94,000). Mg2+, Fe2+, Co2+, and Zn2+ did not comparably limit the digestion of fragment D. Equilibrium dialysis demonstrated that Ba2+ competitively inhibited Ca2+ binding to fibrinogen, suggesting that the ions occupied the Ca2+ binding site of fibrinogen and thereby limited the plasmic digestion of fragment D. The results suggest that Ca2+, Sr2+, Ba2+, and Mn2+ limit plasmin digestion of fragment D by interacting with a Ca2+ binding site in the D domain of the fibrinogen molecule.

Binding Sites↗