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Laboratories evaluated whether an interference was causing a false-positive PSA for the Immulite 2000 immunoassay after a time course of increasing prostate-specific antigen (PSA) in a post-prostatectomy patient led to salvage therapy, which had no effect on the elevated PSA. Serial dilutions of PSA for the patient sample (6.1 ng/mL; post-prostatectomy reference range: <0.1 ng/mL [undetectable]) were linear (r > 0.99). However, the PSA measurement was reduced to 0.1 ng/mL after pretreatment of the sample with heterophilic antibody blocking reagent. PSA was undetectable (<0.1 ng/mL) when measured using two alternative immunoassays. These results were consistent with the presence of heterophilic antibody interference for the Immulite 2000 assay. In this case, heterophilic antibody interference with PSA measurement must have originated during the period of post-prostatectomy monitoring, and the apparent progressive increases in PSA may have been due solely to the progressive increase of this heterophilic antibody assay interference. In the absence of clinical correlation, positive PSA monitoring results should always be assessed for heterophilic antibody interference for at least one time point.

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Because proinsulin and insulin have different circulatory half-lives, the ratio of proinsulin to insulin in plasma depends on the dynamics of insulin secretion. This variation can potentially influence comparison of IMX assays and radioimmunoassays (RIAs) for [insulin], given that the antibody used in the IMX assay has negligible cross-reactivity with proinsulin compared to the 40% cross-reactivity with proinsulin of the antibody used in the RIA. Simulation of a simple mass balance model for insulin and proinsulin concentrations during an oral glucose tolerance test predicts that the ratio (R) of RIA to IMX insulin measurements of [insulin] should transiently decrease, pass through a minimum, increase past the initial value, pass through a maximum and eventually return to the initial value. Using time course specimens from patients, this pattern of variation in R was observed in the majority (12/16) of cases studied. The variation in R for time course specimens (CV = 26%) was significantly greater than for other specimens (fasting, random or undesignated; P < 0.05). Thus, when comparing IMX and RIA measurements of [insulin], variation in R for samples from differing states of dynamic insulin secretion contains a component that is attributable to dynamic changes in the ratio of [proinsulin]/[insulin] in plasma.

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Stable isotope dilution gas chromatographic-mass spectrometric (GC-MS) measurement of tricyclic antidepressants (TCA) is a useful alternative to high-performance liquid chromatography (HPLC) methods when interfering substances prevent accurate quantitation by HPLC. For satisfactory GC-MS analysis, secondary amine TCA must be derivatized. Commonly employed trifluoroacetyl and heptafluorobutyryl derivatives are relatively unstable and cause rapid deterioration of capillary GC columns. Therefore we examined 4-carbethoxyhexafluorobutyryl chloride (CHFB-CI) as an alternative derivatizing agent and developed a stable isotope dilution GC-MS method employing ring-labeled [2H4]-desipramine and [2H4]-imipramine internal standards, which permits measurement of desipramine, nortriptyline, imipramine, and amitriptyline in plasma samples containing one or all of these analytes. The GC-MS assay is linear for each analyte from the lower limit of quantitation (25 ng/mL) up to 1500 ng/mL and correlates well with HPLC measurements. The GC-MS analytic coefficient of variation was 9.7 +/- 1.3% for all analytes considered together. Although interferences are observed in the HPLC assay, thioridazine, perphenazine, cyclobenzaprine, and norcyclobenzaprine do not interfere with GC-MS measurements of the TCA examined here. The stability of the CHFB derivative of secondary amine TCA was found to be superior to that of the trifluoroacetyl derivatives of these compounds.

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Measurement of blood concentrations of cystatin C (cysC), a cysteine protease inhibitor present in human plasma, has been suggested for use as an indicator of glomerular filtration rate (GFR) in a manner analogous to the use of plasma creatinine (SCR). In this study, cysC and SCR were measured in plasma from pediatric patients (4-19 years) with renal disease for whom a "gold standard" measurement of GFR via inulin clearance (C(IN)) was available. The data analyses were divided into two age groups: group A (4-12 years, n = 26) and group B (12-19 years, n = 34). For both age groups, the linear correlation coefficient of [cysC](-1) vs C(IN) (mL/min/1.73 m2) (r = 0.765 for group A and r = 0.869 for group B) was less than that of the linear correlation coefficient of [SCR](-1) vs C(IN) (r = 0.841 for group A and r = 0.892 for group B). As a single measurement for detection of abnormal GFR, however, the optimum receiver-operator characteristic point for cysC measurement (for group A at cysC >1.2 mg/L, sensitivity = 80%, specificity = 91%; and for group B at cysC >1.4 mg/L, sensitivity = 87%, specificity = 100%) was numerically superior to that for SCR measurement (for group A at SCR >8.0 mg/L, sensitivity = 67%, specificity = 100%; and for group B at SCR >9.0 mg/L, sensitivity = 91%, specificity = 91%), using a reference value for normal GFR of C(IN) > 90 mL/min/1.73 m2. However, these differences were not statistically significant. CysC measurement appears to be broadly equivalent to SCR measurement for estimation of GFR in pediatric patients.

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The polyol 1,5-anhydroglucitol (AG) present in human plasma is derived largely from ingestion and is excreted unmetabolized. Reduction of plasma [AG] has been noted in diabetics and is due to accelerated excretion of AG during hyperglycemia. Plasma [AG] has therefore been proposed as a marker for glycemic control. A precise understanding of its utility relies on a quantitative understanding of the mass balance for AG. In this study, non-steady-state data from the literature were analyzed to develop a dynamic mass balance model for AG that is based on the two-compartment model proposed by Yamanouchi et al. [T. Yamanouchi, Y. Tachibana, H. Akanuma, S. Minoda, T. Shinohara, H. Moromizato, H. Miyashita, and I. Akaoka. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E268-E273, 1992]. The data are consistent with a model in which exchange between tissue and plasma pools is rapid and in which the tissue compartment mass is two to three times the mass of the plasma compartment. According to model estimates, accelerated excretion of AG due to hyperglycemia can cause marked net depletion of total AG over a time scale of days. Recovery from a depleted state is slow because the total body capacity represents >5 wk of normal intake. Accordingly, AG monitoring should be able to indicate the presence of past glucosuric hyperglycemic episodes during a period of days to weeks, as well as provide information on the extent to which high deviations from the average plasma glucose concentration are operative.

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Arachidonyltrifluoromethyl ketone (ATFMK), an analogue of arachidonic acid (AA), inhibits an 85 kDa cytosolic phospholipase A2 enzyme. Exposure of HIT insulinoma cells to ATFMK induced a delayed, sustained, and irreversible increase in cytosolic [Ca2+] that required extracellular Ca2+ and a concentration-dependent inhibition of depolarization-induced increases in cytosolic [Ca2+] prior to onset of the delayed response to AFTMK. These results suggest a disruptive effect of ATFMK on calcium mobilization which may contribute to its effects on insulin secretion from beta-cells.

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A method is described for detection and quantitation of agmatine [4-(aminobutyl)guanidine] by gas chromatography/negative-ion chemical ionization/mass spectrometry after derivatization with hexafluoroacetylacetone. The lower limit of detection of the derivative was about 25 fmol on-column. For quantitative studies of agmatine content in biological samples, a procedure utilizing an internal standard ([15N4]agmatine prepared from [15N4]arginine) and an extraction step had a lower limit of detection of about 15 pmol for total sample content. Agmatine content was measured in rat tissue samples and normalized to protein content. Kidney and spleen samples exhibited the greatest content of agmatine per unit protein mass but agmatine was also detected in pancreatic islets and brain regions (cerebellum and cerebral cortex). On the basis of these measurements, it is estimated that the pancreatic islet intracellular agmatine concentration may exceed 1 microM. The sensitive and highly specific means of detection and quantitation provided by mass spectrometry may be useful in investigating the physiological role of agmatine in mammalian systems.

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In two intact cell systems in which GTP-binding protein (G) activity is initiated by the presence of agonist-bound receptors (R), it has been demonstrated that the rate of G activation is influenced by the rate of turnover of agonist occupancy among the receptor population. G activity is reduced when a low concentration of agonist-occupied receptors comprised by low fractional occupancy of a large receptor population is replaced by the presence of the same concentration of 100%-occupied receptors. This effect has been proposed to be due to a time interval of interaction between R and G (an encounter) that is long compared to the time of a single collection between R and G and long compared to the lifetime of an agonist-receptor complex. In a recent simulation study of R-G interaction via diffusion, the effect of agonist occupancy turnover was observed but it was assumed that long encounters were not operative. In this study, encounter intervals in simulations of R-G interaction by simple diffusion were measured in order to address that difference. The results demonstrate that relatively long encounters comprised of multiple, separate collisions are an inherent part of R-G interaction as modelled by diffusion. The implications for further implementation of simulation studies of R-G interaction are discussed.

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The cyclic AMP receptor protein (CAP) and lactose repressor bind their regulatory sites in the lactose promoter with moderate cooperativity (omega C101 = 11.8(+/- 3.7)). This cooperativity is significantly reduced by the removal of DNA located upstream of the CAP binding site or by substitution of the dimeric lacI-18 mutant repressor for the wild-type tetrameric protein. These results are consistent with a mechanism of interaction in which CAP bends the DNA and the lac repressor binds simultaneously to its operator site and to promoter-distal sequences. Similar values of omega C101 were obtained with a promoter truncation containing the O3 pseudooperator site and one in which the site is destroyed, suggesting that DNA contacts distal to the O3 site are necessary for cooperative binding.

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The salt concentration dependences of the observed association constants (Kobs) for the binding of wild-type lac repressor tetramer and the dimeric lacI-18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion-binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion-binding affinities requires additional mechanistic or structural information. The simplest ion-binding mechanism consistent with the data is compatible with a current structural model of the repressor-operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild-type repressor binding is slightly greater than that of the lacI-18 mutant. This difference may reflect the availability of ion-binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.

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Transcription in E. coli is often controlled by the binding of specific gene-regulatory proteins. Binding of these proteins to their specific DNA binding sites occurs in the presence of a large excess of "nonspecific" genomic DNA. Binding to a specific DNA site thus depends on the concentration of regulatory protein, on its affinities for specific and competing nonspecific binding sites, and on the free concentrations of those sites. Although it is probable that genomic DNA is largely occluded by protein binding or by condensation in vivo, the actual extent to which the DNA is available to act as a competitor for specific binding (i.e. the effective concentration of nonspecific DNA) is not known. Because many regulatory interactions occur simultaneously in a cell, it is reasonable to expect that they will have evolved to function at equilibrium with a shared concentration of competing nonspecific DNA. This premise was the basis for this study. In vitro binding data were compiled for six regulatory proteins that function in E. coli, and used to calculate theoretical equilibrium binding distributions. The calculated distributions were used to evaluate the regulatory states of promoters according to models based on the equilibrium occupancies of regulatory sites. For four proteins whose DNA-binding affinities are modulated by ligand binding (CAP, lac repressor, trp repressor and araC), regulation was assessed as the extent to which the presence of the modulator could affect the occupancy by protein of the specific sites (e.g. the difference in equilibrium occupancy by CAP of CAP binding sites between conditions of high and low concentrations of CAP's affinity modulator, cAMP). For two proteins whose site affinities are not modulated by ligand binding (lambda repressor and lambda-cro), regulation was assessed by specific site occupancy at equilibrium. These regulation profiles were compared to determine whether a single concentration of nonspecific competing DNA is compatible with effective regulation as defined for all of the systems. For five of the six modeled systems (CAP, trp repressor, araC, lambda repressor and lambda-cro), a free nonspecific DNA concentration on the order of 10(-4) M base pairs is compatible with regulation based on equilibria of the protein-DNA interactions. The lac repressor-operator system is an exception to these results: as has been shown previously, the regulation of operator binding by low molecular weight inducers increases with increasing concentrations of nonspecific DNA (von Hippel et al., 1974 Proc. natn. Acad. Sci. U.S.A. 71, 4808-4812).(ABSTRACT TRUNCATED AT 400 WORDS)

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The equilibrium association constant observed for many DNA/protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that displacement of cations from the DNA is the predominant form of the involvement of ions in the binding reaction. With this assumption, the graph of log K(obs) versus log [MX] is predicted to have a constant slope proportional to the number of ions displaced from the DNA upon protein binding [Record, M. T., Lohman, T. M. & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158]. Experimental data often deviate from linearity, however, at lower salt concentrations. Such deviations can be due to differential cation binding, anion binding or changes in macromolecular hydration, or differential screening effects of the electrolyte on protein and/or DNA charges. Here the theoretical effects on K(obs) of a simple form of ion-protein interaction are examined. A model for binding interactions is used that includes a mass balance of ions bound to both protein and DNA as the protein is transferred from the salt concentration of bulk solvent to the typically higher cation and lower anion concentrations characteristic of the volume adjacent to the DNA. We show that models in which the cation and anion stoichiometries of a protein change as it associates with DNA are consistent with the curvature of plots of log K(obs) versus log [MX]. Such mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.

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The rate of adenylate cyclase activation via agonist-bound receptors in intact cells can be partly dependent on the rate of turnover of occupancy by agonist with respect to individual receptors. For instance, low occupancy of the full complement of receptors by epinephrine in intact S49 cells has been shown to promote a rate of activation that is substantially greater than that for high occupancy of a small number of receptors for which the concentration of epinephrine-bound receptors is the same. According to the encounter coupling model, a partial dependence of the relationship between receptor occupancy and adenylate cyclase activity on the agonist binding frequency can in principle be explained by episodic interactions of finite duration (encounters) between individual pairs of receptor and GTP-binding protein. The mean lifetime of the agonist-receptor complex and the frequency of binding relative to the mean duration of such encounters dictate whether there is variation of the state of the receptor during an encounter and the extent to which the overall rate of GTP-binding protein activation can be dependent on binding frequency. We present here a quantitative analysis of agonist concentration versus cyclase response curves in terms of the encounter coupling model that explicitly includes agonist binding frequency, the encounter frequency, and the encounter duration as parameters. The essential result is that the model is quantitatively consistent with concentration versus response curves for receptor-mediated activation of adenylate cyclase in S49 cells. It is also shown that the model is consistent with data on the differential effects of antagonists to inhibit agonist-stimulated cyclase activation in a manner that is dependent on the antagonist binding frequency.

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A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a protein's helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.

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Experiments measuring epinephrine stimulation of the S49 cell have demonstrated that the rate of adenylate cyclase activation is partly dependent on the rate of turnover of epinephrine occupancy with respect to individual receptors. Specifically, it has been shown that a low occupancy of the full receptor population by epinephrine promotes a rate of adenylate cyclase activation significantly greater than that for a low number of receptors completely occupied by epinephrine with which the concentration of bound receptors is the same. This finding indicated that the interaction of individual receptors with GTP-binding protein (G) occurs on a time scale which is greater than the mean lifetime of the epinephrine-receptor complex; during this period of interaction (an "encounter"), a receptor can change its occupancy state in the presence of a high binding frequency agonist such as epinephrine. Here we present a general analysis, in an extension of the Collision Coupling Model of Tolkovsky and Levitzki (Biochemistry 17: 3795-3810, 1978), of the consequences of encounters (rather than pure collisions) for the relationships of receptor occupancy, receptor-agonist complex lifetime, and receptor-agonist efficiency to G/adenylate cyclase activation. Using this "encounter coupling" model of receptor/G interaction, it is demonstrated from a theoretical standpoint that the net rate of G activation can depend in part on the agonist binding frequency. The predicted dependence is consistent with the data on which the model is based, in which high binding frequency increases the activation rate. A special case of the "encounter coupling model" allows calculation of the frequency of encounters by an analysis of a previous experiment using epinephrine in which the rate of adenylate cyclase activation was measured in response to a small number of fully occupied, highly efficient receptors. Using those results and the model developed here, the encounter frequency was found to be on the order of 100/min in the intact S49 cell. This calculation relied on knowledge of the rate of inactivation of G/adenylate cyclase in intact cells. A method for the measurement of the adenylate cyclase inactivation rate is presented. Using this method, the adenylate cyclase inactivation rate constant was found to be between 0.8 and 3.0/min.

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A given overall level of beta-adrenergic receptor occupancy by agonist can involve either high or low turnover of occupancy with respect to individual receptors, depending on the binding properties of the particular agonist. It was recently demonstrated that, for epinephrine-stimulated adenylate cyclase activation in the S49 cell, a portion of the separation between the beta-adrenergic receptor binding curve and the cyclase response curves is dependent on high occupancy turnover (high binding frequency). By involving a larger number of receptors within a short period of time than are bound at any one instant, the effect of high binding frequency is to increase the rate of GTP-binding protein/adenylate cyclase activation over the rate that is observed when the mobility of the number of receptors occupied at any given instant is the rate-limiting factor. This phenomenon contributes to the normal dose-response curve for epinephrine, according to our analysis, but only in combination with the apparent high efficiency of the receptor in the epinephrine-bound state at cyclase activation. Here we examined the potential combination of the contributions of agonist binding frequency and intrinsic efficiency to the adenylate cyclase activation rate for four other beta-adrenergic agonists (isoproterenol, zinterol, metaproterenol, and dobutamine). This was done by a comparison of the response (1-min cAMP accumulation) between a point on the normal dose versus response curve (control) with the response under conditions in which the concentration of agonist-bound receptors was identical to control but the absolute number of receptors involved in maintaining that concentration was significantly reduced. In the experiments, the majority of the receptors were blocked by the beta-adrenergic antagonist propranolol, which has a relatively long occupancy half-life. The remaining receptors were occupied by agonist such that the concentration of bound receptors was identical to the control condition of low occupancy of the full complement of receptors in the absence of antagonist. Compared with control, the experimental condition was one in which agonist occupancy turnover was inhibited and the potential contribution of agonist binding frequency as a factor contributing to the cyclase activation rate was greatly reduced (producing a point on the receptor mobility-limited dose versus response curve). Isoproterenol and metaproterenol show evidence that their binding frequencies and the efficiency of the receptor when bound to them are of such a combination that the normal dose-response curves for these agonists contain a component that is dependent on the binding frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

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The dissociation constant (Kd) for the binding of epinephrine to beta-adrenergic receptors of the S49 cell is 2 microM, which is the ratio of the rate constants for dissociation (koff) and association (kon), Kd = koff/kon. Although the Kd is known by direct measurement, the individual rate constants kon and koff are unknown since they are both too large to be measured by conventional experimental methods. We present here an analysis in which a minimum value for these constants is calculated. The analysis uses a "transiently private receptor" model for coupling of receptors to G protein/adenylate cyclase that is based on the limits prescribed by the known empirical relationship between the beta-adrenergic receptor occupancy by epinephrine (characterized by Kd) and their coupling to adenylate cyclase (characterized by EC50 = 10 nM) as a function of epinephrine concentration. The model makes only the assumption (based on previous evidence) that a receptor cannot activate more than one cyclase during the course of one cycle of binding and unbinding of an epinephrine molecule. In such a model, and with such a large separation between the Kd and the EC50, the rate of G protein/adenylate cyclase activation by epinephrine-bound receptors can be related to the frequency of receptor binding at low concentrations of epinephrine, from which minimum values for the rate constant for association can be derived. This gives the estimates kon greater than 10(8)/M/min and koff greater than 280/min at 37 degrees. The on-rate constant is comparable to the on-rate constants that have been measured for other beta-adrenergic receptor ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

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The binding of epinephrine to beta-adrenergic receptors is a rapid-on, rapid-off process, such that at any level of receptor occupancy (defined as the fraction of time a receptor is bound or, alternatively, the probability that any particular receptor is bound at any given instant) the entire population of available receptors has periods of occupancy that occur at high frequency. While in the bound state, the receptor acts as a mobile catalyst for the activation of adenylate cyclase. Two processes, then, could conceivably contribute to the access of epinephrine-bound receptors to cyclase and the extent of cyclase activation for a given concentration of epinephrine: 1) the rapid switching of epinephrine among receptors ensures that discontinuous distributed regions of the cell surface experience agonist activity and 2) the mobility of the receptors (and GTP-binding protein) in the cell membrane makes it possible for one receptor to activate numerous GTP-binding protein-adenylate cyclase complexes. In principle, either effect can lead to a wide separation between the binding and response curves (EC50 much less than Kd). It has so far been assumed that mobility is able to account completely for the separation. The extent of the contribution of the process of agonist binding and unbinding to adenylate cyclase activation has not been demonstrated or quantified. Here we examine the distinction between binding frequency and receptor mobility contributions to adenylate cyclase activation in epinephrine-stimulated S49 lymphoma cells for which there is a 200-fold separation between the EC50 and Kd at 37 degrees (EC50 = 10 nM, Kd = 2 microM). Experiments were designed to measure adenylate cyclase activation rates for a constant concentration of epinephrine-bound receptors but with variation of the absolute number of receptors involved in the activation. This was accomplished by blocking a portion of the receptor population with an antagonist (propranolol) that has a long occupancy half-life, while increasing the occupancy of the remaining receptors by compensating increases in epinephrine. With this protocol, a condition is approached in which receptor mobility alone is responsible for activation. This resulted in a 50% decrease in adenylate cyclase activity, compared with a control of 30 nM epinephrine. Thus, for epinephrine concentrations near the EC50, the switching of epinephrine among the receptor population is necessary for greater than 50% of the observed activity; it can be shown in conjunction that receptor mobility nonetheless accounts for the majority of the separation between the EC50 and the Kd.(ABSTRACT TRUNCATED AT 400 WORDS)

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An elderly woman with Streptococcus pneumoniae meningitis relatively resistant (minimum inhibitory concentration, [MIC] = 0.12 micrograms/mL) to penicillin is reported. The occurrence of penicillin-resistant pneumococcal infections is reviewed and management discussed. Because of the importance of recognition of resistant pneumococci, a state-wide clinical laboratory survey was conducted to determine the accuracy of susceptibility testing for this isolate. Of 111 laboratories completing the survey, only 26 performed the 1-microgram oxacillin disk test as recommended by the National Committee for Laboratory Standards (NCCLS). When laboratories were analyzed according to hospital size, the proficiency in performing the proper susceptibility testing was 55% (6 of 11) for hospitals with more than 400 beds versus 3% (2 of 58) for hospitals with fewer than 100 beds (P less than 0.0001 by Fisher's exact test). This contrasts with reported surveys by the College of American Pathologists (CAP), and reasons for this are explored. Guidelines for laboratory testing of S. pneumoniae are reviewed, and additional study of clinical proficiency with attention to laboratory size is recommended.

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