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Biomedical subjects

D N Dietzler

Publications and source records attributed to D N Dietzler.

At least 19 recordsLinked to original sources
PubMed1989

Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.

We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.

Animals↗
PubMed1988

Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine.

Exposure of Escherichia coli to 0.8 M NaCl caused a rapid and large decrease in colony-forming activity. When such osmotically upshocked cells were exposed to betaine, colony-forming activity was restored. Betaine was able to restore colony-forming activity even when chloramphenicol inhibited protein synthesis. Thus, restoration was not the result of cell turnover. The cells were not killed by exposure to 0.8 M NaCl, because during exposure they accumulated ATP intracellularly. Betaine treatment caused this cellular ATP to decrease to a lower level. This work may provide the foundation for a simple plating procedure to quantitatively detect nonculturable E. coli in ocean beach recreational waters.

Adenosine Triphosphate↗
PubMed1988

Quantification of lactate dehydrogenase-1 in serum with use of an M-subunit-specific monoclonal antibody.

We have developed a rapid, one-step assay for measuring lactate dehydrogenase-1 (LD-1) activity in serum after extraction of LD-2, LD-3, LD-4, and LD-5 isoenzymes by an immobilized M-subunit-specific monoclonal antibody (D.8.1). In the assay, 100 microL of serum is mixed with 50 microL of a suspension of 0.8-micron-diameter latex particles coated with 30 micrograms of the monoclonal antibody D.8.1, then incubated at room temperature for 5 min. The latex particles, to which LD-2 through LD-5 are bound, are pelleted by centrifugation for 2 min at 12,000 X g, and the LD-1 activity is measured kinetically in the supernatant fluid. We optimized the assay for antibody immobilization, antibody concentration, and time and temperature of incubation. Serum bilirubin concentrations up to 0.33 g/L (0.56 mmol/L) did not interfere in the assay. Hemolysis interfered solely through LD-1 released from erythrocytes. The within-assay CV for low-concentration quality-control material (LD-1 33 U/L) was 3.5% (n = 9) and for high-concentration material (LD-1 185 U/L) was 1.9% (n = 8); the between-assay CVs for the two materials were 6.1% (n = 9) and 2.5% (n = 10), respectively. The LD-1 activity measured in 98 samples by our assay compared well with a two-step polyclonal antibody-based assay (Isomune-LD, Roche Diagnostic Systems; r = 0.998) and with an electrophoretic method (Paragon, Beckman Instruments; r = 0.956).

Antibodies, Monoclonal↗
PubMed1988

Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody.

This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.

Adsorption↗
PubMed1986

Quantitation of serum lactate dehydrogenase-5 with monoclonal antibodies.

Spleen cells from BALB/cJ mice which had been immunized with human lactate dehydrogenase-1 (LDH-1) were fused with SP2/0-Ag14. Two hybridomas were produced which recognized the antigen. Competitive RIA revealed that one antibody ('Smit-LDH') recognized the H subunit of LDH while the other ('Hem-LDH') recognized both H and M subunits of LDH. With the use of these antibodies we developed an assay for LDH-5 activity in which serum is incubated for 30 min at room temperature with the two antibodies ('Smit-LDH' and 'Hem-LDH' in the ratio 64:1.3, micrograms/ml) immobilized on latex beads to extract LDH-1 through LDH-4. After centrifugation, the LDH activity of the supernatant is measured and represents LDH-5 activity. Latex beads coated with bovine serum albumin were used as control. The LDH-5 activity as determined by our assay correlated well (r = 0.98) with the values obtained by an electrophoresis method. There was no interference due to LDH-1 through LDH-3 up to 3,000 U/l and LDH-4 up to 350 U/l. Serum samples with total LDH activity above 1,000 U/l were appropriately diluted in order to avoid interference by LDH-4. Use of these monoclonal antibodies allows precise, rapid and direct measurement of LDH-5 activity in serum.

Animals↗
PubMed1986

Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme.

Fusion of splenocytes from A/J mice immunized by creatine kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/0-Ag14 myeloma cell line generated hybridomas producing monoclonal antibodies specific to CK-MB. One of these monoclonal antibodies ("Conan-MB") was used to develop a direct assay for CK-MB activity. In the assay, serum is incubated for 30 min at room temperature with "Conan-MB" immobilized on latex beads. The beads are then washed, and CK-MB activity bound to the antibody is measured after incubation with CK enzyme reagent for 30 min at 37 degrees C. Results with the assay correlated well (r = 0.997) with those for CK-MB concentration as measured by a two-site immunoassay. Neither CK-MM, CK-BB, mitochondrial CK, nor a hemolysate of erythrocytes interfered. Use of this unique monoclonal antibody allows rapid, precise, and direct determination of CK-MB activity.

Animals↗
PubMed1985

Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli.

In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding-protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose). Detailed study of glucose transport showed that this inhibition of transport was not secondary to the inhibition of growth by osmotic stress, but rather that the inhibition of transport of a source of carbon and energy was sufficient to cause the complete inhibition of growth observed during severe osmotic upshock. Transport and growth inhibition did not result from cell death; upshocked cells were viable and metabolically active.

Biological Transport, Active↗
PubMed1985

Restoration of cell volume and the reversal of carbohydrate transport and growth inhibition of osmotically upshocked Escherichia coli.

Resumption of growth in osmotically upshocked Escherichia coli was effected only by an external stimulus (betaine treatment) in severe upshock, but was spontaneous in less severe upshock. In either case, growth resumption was preceded by a reversal of glucose transport inhibition, and that reversal was preceded by a recovery of cell volume. We hypothesize that deformation of the membrane by osmotic stress results in conversion of a membrane component of the transport system to a less functional conformation, which results in the inhibition of transport and the consequent inhibition of growth. Relief of the deformation would then allow recovery to a more functional conformation, reversal of transport inhibition, and then resumption of growth.

Betaine↗
PubMed1985

Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies.

Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.

Animals↗
PubMed1985

Independence of cyclic AMP and relA gene stimulation of glycogen synthesis in intact Escherichia coli cells.

Previous studies from our laboratory established that in Escherichia coli, glycogen synthesis is regulated by both the relA gene, which mediates the stringent response, and by cyclic AMP. However, those studies raised the question of whether this dual regulatory system functions in an independent or a dependent manner. We show here that this regulation is independent, i.e., each regulatory process can express its action in the absence of the other. Triggering the stringent response by amino acid starvation increased glycogen synthesis even in mutants lacking the ability to synthesize cyclic AMP or lacking cyclic AMP receptor protein; and cyclic AMP addition stimulated glycogen synthesis in relA mutant strains. We also show that physiological concentrations of GTP inhibit ADP-glucose synthetase (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), the rate-limiting enzyme of bacterial glycogen synthesis, in vitro. Because the stringent response is known to cause an abrupt decrease in the cellular level of GTP, modulation of ADP-glucose synthetase activity by this nucleotide could account for a substantial portion of the step-up in the cellular rate of glycogen synthesis observed when the stringent response is triggered.

Adenylyl Cyclases↗
PubMed1984

Purification of five creatine kinase-MM variants from human heart and skeletal muscle.

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.

Creatine Kinase↗
PubMed1984

Identification of GTP as a physiologically relevant inhibitor of Escherichia coli ADP-glucose synthetase.

We show that physiological concentrations of GTP can significantly inhibit wild-type Escherichia coli ADP-glucose synthetase (the rate-limiting enzyme of bacterial glycogen synthesis) and that mutant-strain enzymes known to show less inhibition by physiological AMP levels also show less inhibition by physiological levels of GTP. This decreased inhibition by both AMP and GTP can almost totally account for the higher cellular rates of glycogen synthesis observed in the mutant strains. In addition, in metabolic conditions where we have shown that cellular glycogen synthesis increases, cellular GTP levels are known to decrease. Thus, we conclude that GTP inhibition is physiologically relevant.

Adenosine Monophosphate↗
PubMed1984

Evidence that cyclic AMP stimulates bacterial glycogen synthesis by relieving AMP inhibition of and by increasing the cellular level of ADP-glucose synthetase.

Using Escherichia coli mutants that possess an ADP-glucose synthetase (EC 2.7.7.27, the rate-limiting enzyme of bacterial glycogen synthesis) that differs in its inhibition by physiological levels of AMP, evidence was obtained that cyclic AMP stimulates cellular glycogen synthesis during nitrogen starvation by relieving AMP inhibition of this enzyme (without altering the cellular AMP level). Deinhibition for AMP of an enzyme controlled by the adenylate energy charge allows selective release from this control despite the maintenance of a constant cellular energy charge value. It was also shown that an additional increase in rate, not accounted for by AMP deinhibition, was due to an increase in the cellular level of ADP-glucose synthetase.

Adenosine Monophosphate↗
PubMed1983

Regulation of bacterial glycogen synthesis. Stimulation of glycogen synthesis by endogenous and exogenous cyclic adenosine 3':5'-monophosphate in Escherichia coli and the requirement for a functional CRP gene.

In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis.

Cyclic AMP↗
PubMed1983

Logit-log calibration curves for EMIT assays.

The logit-log and four-parameter logistic procedures when appropriate for calculation of enzyme-multiplied immunoassay (EMIT) data have the advantage that they can be applied regardless of the kinetic analyzer or reaction conditions. To use these procedures correctly one must determine the change in absorbance at an infinite drug concentration (delta A infinity). The marked variation of delta A infinity with equipment and reaction conditions and the difficulty in determining this value have hindered broad use of these otherwise universally applicable procedures. We have evaluated two simple methods for determining delta A infinity, both based on its equivalence to delta A in the absence of specific antibody: (1) cross-kit reaction using antibody/substrate and enzyme-drug reagents from kits for different drugs, and (2) substitution of an antibody-free substrate reagent with composition based on direct analysis. The cross-kit procedure was tested with EMIT assays for phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, and theophylline. In some cases an unexpected type of cross-reaction occurred, giving an erroneously low value for delta A infinity. The antibody-free substrate reagent always permitted accurate determination of delta A infinity.

Anticonvulsants↗