Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protien kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.     

Priming effects of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 on the development of adenomyosis in the mouse uterus

The possibility of therapeutic application of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 for preventing the development of uterine adenomyosis was investigated in mice. First priming effects of CP8816 on 17beta-estradiol (E2)-induced cell division in uterine tissues were examined. As a result, pretreatment with CP8816 or progesterone significantly suppressed the elevation of the mitotic activity in the luminal epithelial cells of mice treated with E2 later. Priming with CP8816 had little effect on the stromal cells, but progesterone priming caused an increase of stromal mitotic activity in mice treated with E2 later. To evaluate the inhibitory effect of these compounds on the development of adenomyosis induced experimentally by pituitary grafting, 7-week-old female mice were isografted with a single anterior pituitary in the uterus and divided into four groups. Two groups of mice were given daily subcutaneous injections of 1 mg of CP8816 or the vehicle alone for 6 weeks from the day after the grafting. Remaining two groups of mice were given oral administration of 1 mg of CP8863 or the vehicle only for 5 weeks starting one week after the grafting. The incidence of adenomyosis was significantly lower in the groups of mice treated with CP8816 and CP8863 than in the respective control groups. The mechanism by which CP compounds inhibited the development of adenomyosis might be related to their priming effects, i.e., their inhibitory effect on epithelial cell division and lack of effect on stromal cell division after subsequent exposure to E2.     

Modulation of Ligand-gated Ion Channels by Antidepressants and Antipsychotics

It is generally accepted that antidepressants and antipsychotics mediate their therapeutic effects via specific interaction with processes related to synaptic neurotransmission in the central nervous system. Besides their well-known classical mechanisms of action, antidepressants and antipsychotics show widely unknown effects, which might also contribute to the pharmacological profile of these agents. There is growing evidence that an interaction of these drugs with allosteric modulatory sites of ligand-gated ion channels (LGICs) might represent a yet unknown principle of action. Such interactions of psychopharmacological drugs with LGICs might play an important role both for the therapeutic efficacy and the side effect profile of these agents. In this review, we focus on the direct interaction of antidepressants and antipsychotics with LGICs, which may provide a basis for the development of novel psychopharmacological drugs.     

Quinazolinones as γ-secretase modulators

Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.     

Benzotriazinone and benzopyrimidinone derivatives as potent positive allosteric AMPA receptor modulators

AMPA receptors (AMPARs) have been demonstrated to be an important therapeutic CNS target. A series of substituted benzotriazinone and benzopyrimidinone derivatives were prepared with the aim to improve in vivo activity over the previously reported bis-benzoxazinone based AMPAKINE series from our laboratory. These compounds were shown to be potent, positive allosteric AMPAR modulators that have better in vivo activity and improved metabolic stability over the analogous benzoxazinone derivatives.     

Pyridazine-derived γ-secretase modulators

SAR of a novel series of pyridazine-derived γ-secretase modulators is described. Compound 25 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42 and Aβ40, and maintain the levels of total Aβ. Furthermore, 25 demonstrated excellent pharmacokinetic parameters as well as good CNS penetration in the rat.     

The Effect of the Gamma Modulator on Na/K Pump Activity of Intact <Emphasis Type="Italic">Mammalian Cells</Emphasis>

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, γa and γb, decrease the apparent affinity of the pump for Na⁺ and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive ⁸⁶Rb(K⁺) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K_0.5(Na) at high K⁺ concentration and a decrease in K′_ATP. Comparison of clones of γ-transfected and mock-transfected cells (with similar V_max values) indicated that γ causes a modest ≈30% increase in the steady-state concentration of intracellular Na⁺. Furthermore, for both γa and γb, values of intracellular Na⁺ were similar to those predicted from the kinetic constants, K_0.5(Na) and V_max. Finally, there was a γ-mediated increase in apparent affinity for extracellular K⁺, which had not been detected in assays of permeabilized membranes.

Towards toxicity detection using a lab-on-chip based on the integration of MOEMS and whole-cell sensors

A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo-detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02 mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.     

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.

In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.