Plasma determination of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using d,l-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide. Standard curves were linear from 0.5 to 50 and from 2 to 100 μg/ml of plasma, using 1.5 and 5 μg of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 μg/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 μg/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

Accumulation of epidermal growth factor within cells does not depend on receptor recycling

Quiescent cells seemingly have a constant number of surface epidermal growth factor receptors. However, exposure of cells to agents which interfere with normal protein turnover suggests that these receptors are internalized and degraded with an apparent half-life of ～6 hours. We show that the time course of maximal accumulation of ligand-receptor complexes is not altered under conditions where degradation of the ligand is inhibited, indicating that no degradation occurs during its first hour of exposure to cells. We also conclusively demonstrate that epidermal growth factor receptors are not recycled during the initial uptake of the ligand, and that a component of pinocytosis of this growth factor is dependent on $\text{de}\text{novo}$ protein synthesis.     

The mobile receptor hypothesis and “cooperativity” of hormone binding. Application to insulin

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone.A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from “negative cooperativity”. (2) Receptor occupancy and biological response may be coupled in a non-linear fashion.By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of “cooperatively.” These considerations may also be pertinent to intepretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder

Summary The possibility that sodium from the serosal bathing medium back-diffuses into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO₂ production by the tissue and enhance the stoichiometric ratio of sodium ions transported across the bladder per molecule of sodium transport dependent CO₂ produced simultaneously by the bladder (J _Na/J _{CO 2}). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO₂ production by the bladder [(a) rate of basal, nontransport related, CO₂ production (J _CO2 ^b ) equals 1.54±0.52 with serosal sodium and 1.54±0.37 without serosal sodium; (b)J _CO2 ^b equals 2.18±0.21 with serosal sodium and 2.09±0.21 without serosal sodium; (c) 1.14±0.26 without ouabain and 1.13±0.25 with ouabain; unite ofJ _CO2 ^b are nmoles mg d.w.^–1 min^–1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the toad bladder.     

Quantitative aspects of hormone-receptor interactions of high affinity: Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones

It is demonstrated that because of limitations in the magnitude of the specific activity of radiolabeled hormone derivatives, direct binding studies of hormone-receptor interactions of high affinity (10^?9–10^?11 M, depending on whether ³H- or ¹²³I-labeled hormones are used) will be subject to artifactual distortions due to the need to utilize high concentrations of the receptor. If the concentration of the receptor is not ten times lower than the true affinity constant, the apparent dissociation constant obtained from direct concentration binding curves will vary as a linear function of the receptor concentration. In addition, at high receptor concentrations saturability becomes difficult to demonstrate experimentally and the binding data yield apparently non-hyperbolic, sigmoidal curves which can be mistakenly interpreted to depict cooperative interactions. Similar artifacts related to receptor concentration are predicted for measurements of the hormone concentration dependence of biological processes (e.g. activation of adenylate cyclase, transport processes, etc.). Methods for detecting these effects, and correctly measuring affinities for labeled and unlabeled hormones under these conditions, are described. The implications for measuring the binding properties of hormone-receptor interactions are discussed, especially in reference to studies of the comparative analysis of receptor function in altered metabolic states and to studies relating the biological and binding properties of hormones.     

Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.     

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.