Estimation of growth hormone secretion rate: impact of kinetic assumptions intrinsic to the analytical approach

We compared four common mathematical techniques to determine daily endogenous growth hormone (GH) secretion rates from diurnal plasma GH concentration profiles in 24 women (16 upper- or lower-body obese and 8 normal-weight individuals). Two forms of deconvolution analysis and two techniques based on a priori determined GH clearance estimates were employed. Deconvolution analyses revealed significant differences in the 24-h GH secretion rate between normal-weight and upper-body obese women, whereas the other two techniques did not. Moreover, deconvolution analyses predicted that the reduction in mean plasma GH concentrations in upper-body obese women was accounted for by impaired GH secretion, whereas the other methods suggested that obesity increases GH metabolic clearance. Thus we infer that disparate conclusions concerning GH secretion can be drawn from the same primary data set. The different inferences likely reflect dissimilar kinetic assumptions and the particular limitations intrinsic to each analytical approach. Accordingly, we urge caution in the facile comparison of calculated GH secretion data in humans, especially when kinetic and secretion measurements are performed under different conditions. The most appropriate way to determine the GH secretion rate in humans must be balanced by the exact intent of the experiment and the acceptability of different assumptions in that context.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Bayesian deconvolution analysis of pulsatile hormone concentration profiles

Many hormones are secreted into the circulatory system in a pulsatile manner and are cleared exponentially. The most common method of analyzing these systems is to deconvolve the hormone concentration into a secretion function and a clearance function. Accurate estimation of the model parameters depends on the number and location of the secretion pulses. To date, deconvolution analysis assumes the number and approximate location of these pulses are known a priori. In this article, we present a novel Bayesian approach to deconvolution that jointly models the number of pulses along with all other model parameters. Our method stochastically searches for the secretion pulses. This is accomplished by viewing the set of parameters that define the pulses as a point process. Pulses are determined by a birth-death process which is embedded in Markov chain Monte Carlo algorithm. This idea originated with Stephens (2000, Annals of Statistics 28, 40-74) in the context of finite mixture model density estimation, where the number of mixture components is unknown. There are several advantages that our model enjoys over the traditional frequentist approaches. These advantages are highlighted with four datasets consisting of serum concentration levels of luteinizing hormone obtained from ovariectomized ewes.     

Rhythmic and Nonrhythmic Modes of Anterior Pituitary Gland Secretion

Because of confounding effects of subject-specific and hormone-specific metabolic clearance, the nature of anterior pituitary secretory events in vivo is difficult to ascertain. We review an approach to this problem, in which deconvolu-tion analysis is used to dissect the underlying secretory behavior of an endocrine gland quantitatively from available serial plasma hormone concentration measurements assuming one- or two-compartment elimination kinetics. This analytical tool allows one to ask the following physiological questions: (a) does the anterior pituitary gland secrete exclusively in randomly dispersed bursts, and/or does a tonic (constitutive) mode of interburst hormone secretion exist? and (b) what secretory mechanisms generate the circadian or nyctohemeral rhythms in blood concentrations of pituitary hormones? Waveform-independent deconvolution analysis of 24-h serum hormone concentration profiles of immunoreactive growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and β-endorphin in normal men sampled every 10 min showed that (a) anterior pituitary gland secretion in vivo occurs in an exclusively burstlike mode for all hormones except TSH and prolactin (for the latter two, a mixed burst and basal mode pertains); (b) significant nyctohemeral regulation of secretory burst frequency alone is not demonstrable for any hormone; (c) prominent 24-h variations in secretory-burst amplitude alone are delineated for ACTH and LH; (d) TSH, GH, and β-endorphin are both frequency and amplitude controlled; (e) prolactin manifests 24-h rhythms in both secretory-burst amplitude and nadir secretory rates; (f) no significant diurnal variations occur in FSH secretory parameters; and (g) a fixed hormone half-life yields good fits of the 24-h serum hormone concentration series, which indicates that there is no need to introduce diurnal variations in hormone half-lives. In summary, the normal human anterior pituitary gland appears to release its various (glyco)protein hormones via intermittent secretory episodes that are apparently unassociated with significant basal hormone secretion, except in the case of TSH and prolactin. Hormone-specific amplitude and/or frequency control of secretory burst activity over 24 h provides the mechanistic basis for the classically recognized nyctohemeral rhythms in plasma concentrations of adenohypophyseal hormones in the human.     

Rhythmic and Nonrhythmic Modes of Anterior Pituitary Gland Secretion

Because of confounding effects of subject-specific and hormone-specific metabolic clearance, the nature of anterior pituitary secretory events in vivo is difficult to ascertain. We review an approach to this problem, in which deconvolu-tion analysis is used to dissect the underlying secretory behavior of an endocrine gland quantitatively from available serial plasma hormone concentration measurements assuming one- or two-compartment elimination kinetics. This analytical tool allows one to ask the following physiological questions: (a) does the anterior pituitary gland secrete exclusively in randomly dispersed bursts, and/or does a tonic (constitutive) mode of interburst hormone secretion exist? and (b) what secretory mechanisms generate the circadian or nyctohemeral rhythms in blood concentrations of pituitary hormones? Waveform-independent deconvolution analysis of 24-h serum hormone concentration profiles of immunoreactive growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and β-endorphin in normal men sampled every 10 min showed that (a) anterior pituitary gland secretion in vivo occurs in an exclusively burstlike mode for all hormones except TSH and prolactin (for the latter two, a mixed burst and basal mode pertains); (b) significant nyctohemeral regulation of secretory burst frequency alone is not demonstrable for any hormone; (c) prominent 24-h variations in secretory-burst amplitude alone are delineated for ACTH and LH; (d) TSH, GH, and β-endorphin are both frequency and amplitude controlled; (e) prolactin manifests 24-h rhythms in both secretory-burst amplitude and nadir secretory rates; (f) no significant diurnal variations occur in FSH secretory parameters; and (g) a fixed hormone half-life yields good fits of the 24-h serum hormone concentration series, which indicates that there is no need to introduce diurnal variations in hormone half-lives. In summary, the normal human anterior pituitary gland appears to release its various (glyco)protein hormones via intermittent secretory episodes that are apparently unassociated with significant basal hormone secretion, except in the case of TSH and prolactin. Hormone-specific amplitude and/or frequency control of secretory burst activity over 24 h provides the mechanistic basis for the classically recognized nyctohemeral rhythms in plasma concentrations of adenohypophyseal hormones in the human.     

Spectral analysis of the bc1 complex components in situ: Beyond the traditional difference approach

The cytochrome (cyt) bc₁ complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc₁ complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc₁ complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc₁ turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b_L. From LS analysis of the chromophoric components (RC, c_tot, b_H and b_L), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc₁ complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.     

In situ kinetics of cytochromes c1 and c2

In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

Fast, automated implementation of temporally precise blind deconvolution of multiphasic excitatory postsynaptic currents

Records of excitatory postsynaptic currents (EPSCs) are often complex, with overlapping signals that display a large range of amplitudes. Statistical analysis of the kinetics and amplitudes of such complex EPSCs is nonetheless essential to the understanding of transmitter release. We therefore developed a maximum-likelihood blind deconvolution algorithm to detect exocytotic events in complex EPSC records. The algorithm is capable of characterizing the kinetics of the prototypical EPSC as well as delineating individual release events at higher temporal resolution than other extant methods. The approach also accommodates data with low signal-to-noise ratios and those with substantial overlaps between events. We demonstrated the algorithm's efficacy on paired whole-cell electrode recordings and synthetic data of high complexity. Using the algorithm to align EPSCs, we characterized their kinetics in a parameter-free way. Combining this approach with maximum-entropy deconvolution, we were able to identify independent release events in complex records at a temporal resolution of less than 250 μs. We determined that the increase in total postsynaptic current associated with depolarization of the presynaptic cell stems primarily from an increase in the rate of EPSCs rather than an increase in their amplitude. Finally, we found that fluctuations owing to postsynaptic receptor kinetics and experimental noise, as well as the model dependence of the deconvolution process, explain our inability to observe quantized peaks in histograms of EPSC amplitudes from physiological recordings.     

Spectral and kinetic resolution of the bc1 complex components in situ: a simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc(1) complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The "traditional" set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c(tot) (cyt c(1)+cyt c(2)), cyt b(L), cyt b(H), and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c(1) and c(2) is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c(1), c(2), b(L), and b(H)) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc(1) complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc(1) complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Comparison of four computerized models to estimate 24-hour growth hormone secretion in girls with Turner's syndrome.

Daily pituitary growth hormone (GH) secretion can be estimated from a 24-hour GH profile by various methods. We have used four methods to assess GH secretion in 36 girls with Turner's syndrome: the method described by Thompson et al., the Pulsar algorithm combined with the method of Hellman et al. and two deconvolution techniques. The number of detected peaks varied considerably among the methods. The mean (+/- SD) total daily secretion per square meter body surface was 0.53 (0.19) U/m2.day by deconvolution, in contrast to 0.31 (0.17) with the Hellman method and 1.06 (0.37) according to Thompson. The differences are explained by different assumptions about the metabolic clearance rate and various methodological aspects. Assuming a degradation rate of 50%, the growth hormone substitution dosage would be 1-2 IU/m2.day in GH-deficient children. The usual dosage in girls with Turner's syndrome is expected to lead to serum GH levels approximately 4 times higher than in the untreated state.     

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.     

Robust dynamic experiments for the precise estimation of respiration and fermentation parameters of fruit and vegetables

Dynamic models based on non-linear differential equations are increasingly being used in many biological applications. Highly informative dynamic experiments are valuable for the identification of these dynamic models. The storage of fresh fruit and vegetables is one such application where dynamic experimentation is gaining momentum. In this paper, we construct optimal O₂ and CO₂ gas input profiles to estimate the respiration and fermentation kinetics of pear fruit. The optimal input profiles, however, depend on the true values of the respiration and fermentation parameters. Locally optimal design of input profiles, which uses a single initial guess for the parameters, is the traditional method to deal with this issue. This method, however, is very sensitive to the initial values selected for the model parameters. Therefore, we present a robust experimental design approach that can handle uncertainty on the model parameters.     

Estimation of the size and shape of GH secretory bursts in healthy women using a physiological estradiol clamp and variable-waveform deconvolution model

Because estrogen production and age are strong covariates, distinguishing their individual impact on hypothalamo-pituitary regulation of growth hormone (GH) output is difficult. In addition, at fixed elimination kinetics, systemic GH concentration patterns are controlled by three major signal types [GH-releasing hormone (GHRH), GH-releasing peptide (GHRP, ghrelin), and somatostatin (SS)] and by four dynamic mechanisms [the number, mass (size), and shape (waveform) of secretory bursts and basal (time invariant) GH secretion]. The present study introduces an investigative strategy comprising 1) imposition of an experimental estradiol clamp in pre- (PRE) and postmenopausal (POST) women; 2) stimulation of fasting GH secretion by each of GHRH, GHRP-2 (a ghrelin analog), and l-arginine (to putatively limit SSergic restraint); and 3) implementation of a flexible-waveform deconvolution model to estimate basal GH secretion simultaneously with the size and shape of secretory bursts, conditional on pulse number. The combined approach unveiled the following salient percent POST/PRE contrasts: 1) only 27% as much GH secreted in bursts during fasting (P < 0.001); 2) markedly attenuated burstlike GH secretion in response to bolus GHRP-2 (29%), bolus GHRH (30%), l-arginine (37%), constant GHRP-2 (38%), and constant GHRH (42%) (age contrasts, 0.0016 相似文献   

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.     

Validation of Bayesian analysis of compartmental kinetic models in medical imaging

IntroductionKinetic compartmental analysis is frequently used to compute physiologically relevant quantitative values from time series of images. In this paper, a new approach based on Bayesian analysis to obtain information about these parameters is presented and validated.Materials and methodsThe closed-form of the posterior distribution of kinetic parameters is derived with a hierarchical prior to model the standard deviation of normally distributed noise. Markov chain Monte Carlo methods are used for numerical estimation of the posterior distribution. Computer simulations of the kinetics of F18-fluorodeoxyglucose (FDG) are used to demonstrate drawing statistical inferences about kinetic parameters and to validate the theory and implementation. Additionally, point estimates of kinetic parameters and covariance of those estimates are determined using the classical non-linear least squares approach.Results and discussionPosteriors obtained using methods proposed in this work are accurate as no significant deviation from the expected shape of the posterior was found (one-sided P > 0.08). It is demonstrated that the results obtained by the standard non-linear least-square methods fail to provide accurate estimation of uncertainty for the same data set (P < 0.0001).ConclusionsThe results of this work validate new methods for a computer simulations of FDG kinetics. Results show that in situations where the classical approach fails in accurate estimation of uncertainty, Bayesian estimation provides an accurate information about the uncertainties in the parameters. Although a particular example of FDG kinetics was used in the paper, the methods can be extended for different pharmaceuticals and imaging modalities.     

Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4.

Insulin stimulates adipose cells both to secrete proteins and to translocate the GLUT4 glucose transporter from an intracellular compartment to the plasma membrane. We demonstrate that whereas insulin stimulation of 3T3-L1 adipocytes has no effect on secretion of the alpha3 chain of type VI collagen, secretion of the protein hormone adipocyte complement related protein of 30 kD (ACRP30) is markedly enhanced. Like GLUT4, regulated exocytosis of ACRP30 appears to require phosphatidylinositol-3-kinase activity, since insulin-stimulated ACRP30 secretion is blocked by pharmacologic inhibitors of this enzyme. Thus, 3T3-L1 adipocytes possess a regulated secretory compartment containing ACRP30. Whether GLUT4 recycles to such a compartment has been controversial. We present deconvolution immunofluorescence microscopy data demonstrating that the subcellular distributions of ACRP30 and GLUT4 are distinct and nonoverlapping; in contrast, those of GLUT4 and the transferrin receptor overlap. Together with supporting evidence that GLUT4 does not recycle to a secretory compartment via the trans-Golgi network, we conclude that there are at least two compartments that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes: one for ACRP30 secretion and one for GLUT4 translocation.     

Towards a quantitative definition of plant hormone sensitivity

Abstract A definition of plant hormone sensitivity is proposed which is based on the initial rates of responses at different hormone concentrations. By analogy with enzyme kinetics, it is concluded that simple concentration-response curves can be described by a function containing three ‘sensitivity parameters’. Objective methods for determining the values of these parameters and for comparing curves are described. These methods are used to describe the responses of Commelina communis L. stomata to abscisic acid. If certain assumptions are valid, the sensitivity parameters can be assigned physicochemical meanings. To validate the assumptions, certain experimental criteria must be fulfilled and these are discussed.     

PNA-Based Dynamic Combinatorial Libraries (PDCL) and screening of lectins

Selections from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection from >6,000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better in affinity than the corresponding glycan alone (41 µM).