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Biomedical subjects

B Zeeberg

Publications and source records attributed to B Zeeberg.

At least 19 recordsLinked to original sources
PubMed2005

Comparison of strand displacement and ligase chain amplification for detection of Chlamydia trachomatis infection in urogenital specimens.

Two amplification tests for the diagnosis of Chlamydia trachomatis infection, namely the ligase chain reaction (LCx) and the strand displacement assay (ProbeTec), were compared using samples from 1183 patients at sexually transmitted disease clinics. The overall prevalence of positive results was 8.0%, with agreement between the two assays of 98.8%. For endocervical, urethral and male urine samples, agreement was 99.3%, 99.4% and 97.7%, respectively. For ten discrepant samples, alternative amplification assays suggested that the LCx and ProbeTec assays gave erroneous results in six and four cases, respectively. Inhibition of amplification was observed with three (0.25%) urine specimens.

Cervix Uteri↗
PubMed1997

Identifying chimerism in proteins using hidden Markov models of codon usage.

Protein chimerism is a phenomenon involving the combination of multiple ancestral sequences into a single, multi-domain protein through evolution. We propose a novel method for detecting chimeric proteins by analyzing their nucleotide sequence. The method tests for differences in the distributions of synonymous (isoaccepting) codons in different regions of the protein. The test involves the comparison of the ability of varying size hidden Markov models (HMMs) of codon usage to fit the natural sequence, relative to a set of randomized controls. We demonstrate the method on the families of yeast nuclear and mitochondrial amino-acyl tRNA synthetases. The method is potentially useful for the automated screening of entire genomes or large databases.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Evaluation of 1-azabicyclo[2.2.2]oct-3-yl alpha-fluoroalkyl-alpha-hydroxy-alpha-phenylacetates as potential ligands for the study of muscarinic receptor density by positron emission tomography.

Both 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoroeth-2-yl)-alpha-hydroxy-alpha-phenylacetate (FQNE, 5) and 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoropent-5-yl)-alpha-hydroxy-alpha-phenylacetate (FQNPe, 6) were prepared and evaluated as potential candidates for the determination of muscarinic cholinergic receptor (mAChR) density by positron emission tomography (PET). The results of in vitro binding assays demonstrated that although both 5 and 6 had high binding affinities for m1 and m2 mAChR subtypes, 6 displayed a higher affinity (nM, m1; KD, 0.45, m2; KD, 3.53) as compared to 5 (nM, m1; KD, 12.5, m2; KD, 62.8). It was observed that pretreatment of female Fisher rats with either 5 or 6 prior to the i.v. administration of Z-(-)(-)-[131I]-IQNP, a high-affinity muscarinic ligand, significantly blocked the uptake of radioactivity in the brain and heart measured 3 h postinjection of the radiolabeled ligand. These new fluoro QNB analogues represent important target ligands for evaluation as potential receptor imaging agents in conjunction with PET.

Animals↗
PubMed1995

Resolution and in vitro and initial in vivo evaluation of isomers of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate: a high-affinity ligand for the muscarinic receptor.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 1), is a highly selective ligand for the muscarinic acetylcholinergic receptor (mAChR). There are eight stereoisomers in the racemic mixture. The optical isomers of alpha-hydroxy-alpha-phenyl-alpha-(1-propyn-3-yl)acetic acid were resolved as the alpha-methylbenzylamine salts, and the optical isomers of 3-quinuclidinol were resolved as the tartrate salts. The E and Z isomers were prepared by varying the reaction conditions for the stannylation of the triple bond followed by purification utilizing flash column chromatography. In vitro binding assay of the four stereoisomers containing the (R)-(-)-3-quinuclidinyl ester demonstrated that each isomer of 1 bound to mAChR with high affinity. In addition, (E)-(-)-(-)-IQNP demonstrated the highest receptor subtype specificity between the m1 molecular subtype (KD, nM, 0.383 +/- 0.102) and the m2 molecular subtype (29.6 +/- 9.70). In vivo biodistribution studies demonstrated that iodine-125-labeled (E)-(-)-(+)-1 cleared rapidly from the brain and heart. In contrast, iodine-125-labeled (E)-(-)-(-)-, (Z)-(-)-(-)-, and (Z)-(-)-(+)-1 have high uptake and retention in mAChR rich areas of the brain. It was also observed that (E)-(-)-(-)-IQNP demonstrated an apparent subtype selectivity in vivo with retention in M1 (m1, m4) mAChR areas of the rain. In addition, (Z)-(-)-(-)-IQNP also demonstrated significant uptake in tissues containing the M2 (m2) mAChR subtype. These results demonstrate that the iodine-123-labeled analogues of the (E)-(-)-(-)- and (Z)-(-)-(-)-IQNP isomers are attractive candidates for single-photon emission-computed tomographic imaging of cerebral and cardiac mAChR receptor densities.

Animals↗
PubMed1994

Muscarinic receptor selectivities of 3-Quinuclidinyl 8-xanthenecarboxylate (QNX) in rat brain.

We have determined the binding of (R)-3-Quinuclidinyl 8-xanthenecarboxylate to muscarinic acetylcholine receptor preparations from rat cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The competition curves determined with [3H]quinuclidinyl benzilate as the radioligand are well described by a two site model with a difference in affinity between the two sites of 12-fold. The proportions of high affinity site vary from 100% in the caudate/putamen to 0% in the pons/medulla. The selectivities are different from those measured by pirenzepine and are consistent with QNX exhibiting similar affinity for the M1, M3, and M4 receptors with lower affinity for the M2 receptor. This assignment was confirmed by determining the affinities of QNX for the cloned receptor subtypes.

Animals↗
PubMed1992

Optimal survival of Helicobacter pylori under various transport conditions.

The ability of clinical isolates and type strains of Helicobacter pylori to survive in Stuart transport medium, isotonic saline solution, and urea-containing isotonic saline was evaluated. The influences of temperature (4, 10, 15, 20, and 30 degrees C) and holding time (6 to 48 h) and the effect of exposure to air on survival were also studied. The recovery rate of H. pylori was highest from Stuart transport medium in comparison with the recoveries from the other transport media tested. We found that at holding temperatures above 15 degrees C the organisms became noncultivable within 6 h or less, while they survived for 2 days or longer at 10 degrees C. The presence of urea at a concentration of 2% (wt/vol) in isotonic saline resulted in the loss of viability of the organisms tested.

Air↗
PubMed1982

Concerning the efficiency of the treadmilling phenomenon with microtubules.

Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.

Animals↗
PubMed1982

Dynamic properties of microtubules at steady state in the presence of taxol.

The dynamic properties of steady-state microtubules in the presence of the antitumor drug taxol and GTP, but in the absence of microtubule-associated proteins have been studied. The molecular rate constants for the loss or gain of subunits at steady state was found to be dramatically decreased as compared with that for microtubules formed in the presence of GTP and microtubule-associated proteins but in the absence of taxol [Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899]. In light of this change it was surprising to find that the degrees of directionality for subunit flux into the microtubule at steady state are nearly identical within 1.5% of each other) in the two systems. One mechanism to account for this would be for taxol to cause a nearly identical decrease in the rate constants for subunit dissociation at both ends of the microtubule, with no effect on the rate constants for subunit addition. Similar results have previously been found in studies with an endogenous effector of the microtubule steady state, a protein kinase [Jameson, J. L. and Caplow, M. (1981) Proc. Natl Acad. Sci. USA, 78, 3413-3417]. In this case it was found that phosphorylation of microtubule-associated proteins altered the molecular rate constants for tubulin subunit addition and dissociation, but had no effect on the degree of directionality for subunit flux. It will be of interest to determine whether other exogenous or endogenous effectors also act in a manner such as to leave the directionality unaltered.

Alkaloids↗
PubMed1981

An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules.

We have developed a coupled enzyme system composed of hexokinase, glucose, and nucleoside diphosphate kinase which is able to rapidly convert GTP at the exchangeable nucleotide binding site of tubulin to GDP. Using this method, we have studied the dynamic properties of microtubules and tubulin subunits in the presence of GDP. Conversion of GTP to GDP causes microtubules to change to a new, somewhat lower steady state level; dilution studies show that subunits disassemble from microtubules at steady state in the presence of GDP at a rate comparable to that in the presence of GTP; in reactions of existing microtubules with tubulin-GDP subunits it was found that tubulin-GDP subunits do not participate in net microtubule elongation. From these observations we conclude that in the presence of tubulin-GDP the microtubule steady state has an unusual property; in spite of the fact that the microtubule is continuously undergoing rapid subunit loss, but not subunit addition, a nearly constant steady state level of microtubule mass is maintained. This must mean that tubulin-GDP subunits, although unable to participate in a net addition, can participate in additions each of which compensates for the dissociation of a single subunit from the microtubule. This is equivalent to the existence of an isoenergetic exchange of a tubulin-GDP in solution for a subunit which had been lost from the end of a microtubule.

Adenosine Triphosphate↗
PubMed1981

Incorporation of radioactive tubulin into microtubules at steady state. Experimental and theoretical analysis of the effect of podophyllotoxin.

Addition of a substoichiometric amount of podophyllotoxin to microtubules which are at steady state decreases the rate of incorporation of radioactive subunits into the microtubules. The magnitude of this effect depends on the fraction of the total number of each of the microtubule ends which is capped by tubulin-podophyllotoxin subunits; these fractions depend on the concentration of podophyllotoxin used. A quantitative relationship is presented which correlates the extent of tubulin-podophyllotoxin capping of the microtubule ends with alterations in the rate for steady state subunit incorporation. An analysis based upon this relationship reveals that tubulin-podophyllotoxin caps both ends of the microtubule. Previously proposed mechanisms which involve tubulin-podophyllotoxin capping at only one end of the microtubule are analyzed.

Animals↗
PubMed1981

Toxic effect of sampling swabs and transportation test tubes on the formation of intracytoplasmic inclusions of Chlamydia trachomatis in McCoy cell cultures.

The ability of Chlamydia trachomatis, immunotype E, to produce intracytoplasmic inclusions in cycloheximide-treated McCoy cells after being exposed to different types of sampling swabs in experimentally infected transport medium was studied. A larger number of inclusions was obtained with cotton-tipped aluminium and rayon-tipped plastic swabs than with calcium-alginate-tipped aluminium and cotton-tipped wooden swabs (P less than 0.0001). Transport medium stored in glass tubes caused a cytopathic effect when inoculated on to McCoy cell cultures; no such effect occurred with plastic tubes. When cotton-tipped aluminium instead of calcium-alginate-tipped aluminium swabs were used to collect 50 make urethral specimens significantly more were chlamydia-positive (P less than 0.025). This was also true when cotton-tipped aluminium swabs were used instead of alginate-tipped swabs in a study of 123 cervical specimens (P less than 0.01). When the calcium-alginate-tipped aluminium and cotton-tipped wooden swabs were shaken in the transport medium after sampling from the male urethra and the cervix, instead of being left in the medium during transport to the laboratory, more specimens were chlamydia-positive and a greater number of chlamydial inclusions were found per culture-positive sample; these results were, however, not statistically significant (P greater than 0.05).

Cell Line↗
PubMed1980

Exchange of tubulin dimer into rings in microtubule assembly--disassembly.

We have prepared native radioactive tubulin dimer from two species: [35S]tubulin dimer, by in vivo labeling of rat brain, and porcine [3H]ethyltubulin, as previously described [Zeeberg, B., Cheek, J., & Caplow, M. (1980) Anal. Biochem. 104, 321--327]. After microtubule assembly with radioactive tubulin dimer and nonradioactive dimer and rings, the tubulin in the rings and the dimer obtained upon disassembly have approximately equal specific activities. Therefore, during the reaction sequence dimer + rings leads to 37 degrees C microtubules leads to 0 degrees C dimer + rings the tubulin initially in rings becomes indistinguishable from tubulin initially in dimer. Under nonpolymerizing conditions (0 degrees C) radioactive tubulin dimer and radioactive guanine nucleotide are incorporated into rings at approximately equal rates. This indicates that there is a pathway for nucleotide incorporation into rings under nonpolymerizing conditions which involves the incorporation of dimer-bound nucleotide. We also report results on the lack of the mirror image equilibrium during the disassembly process, using porcine [3H]-ethyltubulin dimer, rat [35S]tubulin dimer, and a [3H]-GDP.porcine tubulin dimer complex. In all three cases there is no significant disassembly-dependent incorporatioin of radioactivity into rings when microtubules are disassembled in the presence of radioactive dimer. These results demonstrate that, for rat and porcine tubulin, rings are formed during microtubule disassembly by direct cleavage of intact rings, without a tubulin dimer intermediate.

Animals↗
PubMed1980

Incorporation of radioactive tubulin into microtubules at steady state. Experimental and theoretical analyses of diffusional and directional flux.

Subunit flux into porcine brain microtubules at steady state has been studied using both radioactive guanine nucleotide and a radioactive tubulin dimer, [3H]ethyltubulin, which is obtained by a reductive ethylation (Zeeberg, B., Cheek, J., and Caplow, M., (1980) Anal. Biochem. 104, 321--327). We have also determined the molecular rate constants for steady state dimer loss from and addition to the microtubule. This is the first study where the rate for flux of radioactive subunits at steady state has been correlated with the measured molecular rate constants. Also, we derive a theoretical analysis of the observed flux of subunits into a polymer at steady state in pulse and chase experiments; this is used to relate the flux and molecular rate constant to a steady state model. Analysis of our results shows that, at steady state, there is only a small difference in the number of assembly and disassembly events at a given microtubule end (as compared with the total number of events at both ends) in a unit of time; predominant opposite end assembly-disassembly (Margolis, R. L., and Wilson, L. (1978) Cell 13, 1--8) is, therefore, ruled out. The almost complete absence of opposite end assembly-disassembly in the steady state provides a means for very efficient regulation of the microtubule network in vivo, since a small modulation in the relative magnitudes of the molecular rate constants can increase the subunit flux dramatically.

Animals↗
PubMed1980

Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Animals↗
PubMed1980

New approach for studying macromolecular-ligand binding. Determination of the dissociation constant for macromolecule-bound ligand by gel filtration.

A graph is presented which permits facile determination of the dissociation constant constant for a macromolecule . ligand complex from gel filtration results; it can be used without a detailed understanding of the remainder of the text. When a solution of macromolecules and radioactive ligand is subjected to gel filtrations, the amount of ligand associated with the eluted macromolecule is easily measured. This quantity is then used for obtaining the corresponding dissociation constant from the graph. Values given in the graph had been determined from an equation, which is presented in the text, by means of a program which is run on a programmable calculator (Texas Instruments SR-52). It is also demonstrated how the validity of dissociation constants so obtained can be checked independently by means of a second calculation. A modification of these procedures permits determination of the dissociation constant for those situations where there is moderate dilution of the eluted sample relative to that applied to the gel filtration column, and the graph incorporates results from such calculations. The successful application of the present approach to the determination of the dissociation constants for a tubulin . [3H]GDP complex is described. Also, the potential importance of the present approach relative to other techniques for dissociation constant determination is discussed.

Chromatography, Gel↗