Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice

The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.     

A diminished role for hydrogen bonds in antifreeze protein binding to ice

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.     

Measurement and analysis of unbound drug concentrations

The plasma protein binding of drugs has been shown to have significant effects on numerous aspects of clinical pharmacokinetics and pharmacodynamics. In many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the unbound fraction of drug in plasma which is available for distribution, elimination, and pharmacodynamic action. Thus, accurate determination of unbound plasma drug concentrations is essential in the therapeutic monitoring of drugs. Many methodologies are available for determining the extent of plasma protein binding of drugs, however, in the clinical evaluation of drug therapy, equilibrium dialysis and ultrafiltration are the most routinely utilised methods. Both of these methods have been proven to be experimentally sound and to yield adequate protein binding data. Furthermore, the characterisation of the interactions between drug and protein molecules is essential for the assessment of the pharmacokinetic implications of drug-protein binding. Protein binding parameters which characterise the affinity of the drug-protein association, the number of classes of binding sites, the number of binding sites per class or protein and the binding capacity are useful for predicting unbound drug concentrations. Simple graphical methods have often been used to obtain protein binding parameters, but these methods have limitations and are not useful for drugs with more than 1 class of binding site. Therefore, the fitting of protein binding models which characterise the drug-protein binding interaction for experimental data is the preferred method of calculating binding parameters. Using the appropriate model, values for binding parameters are typically estimated by using nonlinear least-squares regression analysis.     

Kinetic and immunohistochemical characteristics of mitogen-induced cutaneous hypersensitivity in chickens selected for antibody responsiveness

Mitogen-induced cutaneous hypersensitivity was evaluated in chickens selected for high and low antibody responses to SRBC, and in a random bred control line. Wing web swelling responses were found after subcutaneous administration of phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide (LPS), respectively, in all three lines. All mitogens induced significant acute 4 h wing web swelling responses, followed by a significant (classical) late 24 h wing web swelling response. The 4 h responses were significantly lower in the L line, whereas a tendency for lower responses at 24 h in the L line was found as well. Immunohistochemical evaluation of the early and late wing web swelling responses revealed extravascular localisation of leukocytes at 24 h after sensitization with mitogens, which consisted of CD4+ cells, CD8+ cells, TCR-1+ cells, and heterophils, but no B cells, whereas the 4 h swelling response was primarily characterized by oedema. Cutaneous hypersensitivity either initiated by T-cell mitogens as well as B-cell mitogens may depend for an important part on the rapid induction of local homing of lymphocytes towards the sensitizing agent, which may be mediated by an acute local expression of molecules with chemo-attractive capacities. Interpretation of cellular immunity responses in vivo such as delayed-type hypersensitivity should therefore incorporate oedema-initiating characteristics of sensitizing agents. The relationship between the magnitude of cutaneous hypersensitivity to mitogens and selection for antibody responsiveness is discussed.