Anti-complement activity of polynucleotides.

A biologic assay system, based on complement (C′) inhibition, is described to unravel structural differences among polynucleotides. The C′ system appears particularly suitable to distinguish (1) homo- from copoly-ribonucleotides, (2) deoxyribo- from 2′-OH and other 2′-modified polynucleotides, and (3) single homopolynucleotides from double- or triple-stranded complexes.From these studies a number of polynucleotides emerged with potent anti-C′ activities, worthy of further investigation. The most active polymers were (G)_n (polyguanylic acid), (dCc1)_n [poly(2′-chloro-2′deoxycytidylic acid)] and (dUz)_n [poly(2′-azido-2′-deoxyuridylic acid)].     

Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.     

Multiple attack in the mechanism of endoglucanase action]

The review deals with the phenomenon of repetitive catalytic acts occurring in one enzyme-substrate encounter of certain endo-glucanases with water soluble polysaccharides. Here are discussed experimental results and theoretical models applied in analysis of data.     

Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. II. Nucleophilic attack

Dynamic systems, modelling the elementary act of nucleophilic attack on the carbonyl carbon atom of amide (N-methyl-acetamide) and ester (methylacetate) substrates by some compounds, simulating the nucleophilic group of various types proteases active site were calculated and analysed by the CNDO/2 method, namely: 1) methoxyanion and the methanol (serine proteases), 2) water molecule in the presence of formate anion (carboxylic proteases) and 3) mercaptide anion (CH3S-) (thiolic proteases). The formation of productive enzyme-substrate complex was shown not only to orient reactive groups of the enzyme and substrate, but also to activate it by induction of a certain degree of cleavable pyramidalization, as a result of the partial resonance stabilization energy loss.     

Localization of the metabolic processes affected by calcium during corticotropin action.

To define the role of calcium during corticotropin-induced steroidogenesis, adrenal sections were incubated under conditions of varying degrees of calcium depletion. Corticosterone production, [14C]leucine incorporation into protein, and tissue cyclic AMP levels were measured concomitantly. Omitting calcium from the incubation media inhibited all three processes to variable extents, thus limiting conclusions regarding which process is most dependent on calcium. While calcium was required during the early phase of corticotropin action, it was not required during later phases: rapid induction of calcium deficiency did not diminish the heightened rate of steroidogenesis previously induced by corticotropin in the presence of calcium. Thus, while calcium is required for induction of steroidogenesis factor(s), the operation of the latter is not dependent upon calcium in the extracellular fluid.     

Proton-coupled electron-transfer mechanism for the radical scavenging activity of cardiovascular drug dipyridamole

Dipyridamole (DIP) is a well-known pharmaceutical drug used as a coronary vasodilator and anti-platelet agent in clinics for treating several cardiovascular diseases. Primarily, the therapeutic effects of the drug are attributed to its antioxidant potency. In this research, we aim to declare the unknown antioxidant mechanism of DIP as well as its potent chain-breaking antioxidant activity in polar aqueous medium inside the cells, using different experimental methods and theoretical quantum calculations. Data demonstrated the higher antioxidant capacity of DIP against ROS and free radicals in polar cell's interior. DIP is capable of generating long living and noninvasive DIP(?) radicals in oxidant condition that leads to an effective "chain-breaking antioxidant" activity. Quantum computational data indicated that DIP antioxidant has more favorable ionization potential than trolox which means DIP has higher antioxidant activity. Also, data showed that the direct hydrogen-transfer is not a favorable process to construct DIP(?) because of high barrier energy, though electron-transfer process can more easily to produce DIP(?+) with the lowest barrier energy. Altogether, the electron donating potency of DIP to reduce ferric ion, having the low anodic oxidation peak potential, producing long lived stable DIP(?) radicals and protecting myoblast cells from oxidation, proposed the excellent "chain-breaking antioxidant" potency via electron-transfer mechanism of this vasodilator DIP drug in polar aqueous medium.     

Blood levels of opioid peptides and alpha interferon under the action of dipyridamole

The levels of met-enkephalin (ME), beta-endorphin (BE), and alpha-interferon (a-IFN) have been determined in the human blood 1 day after dipyridamole administration. Dipyridamole led to an increase in serum a-IFN concentration up to 3 times, and to simultaneous rise of the lymphocytes ability to produce a-IFN. The content of BE did not depend on dipyridamole treatment, but ME level achieved 110 +/- 4.8 pg/ml (compare to 79.5 +/- 7.6 pg/ml in control). Positive interrelation has been found out between individual ME concentrations and lymphocyte abilities to a-IFN production with the coefficient of correlation equal to 0.69. The effect of dipyridamole on ME level is suggested to develop via a-IFN interaction with the opioid systems.     

Milk caseins as useful vehicle for delivery of dipyridamole drug

The interaction of bovine milk α- and β-caseins as an efficient drug carrier system with Dipyridamole (DIP) was investigated using spectroscopy and molecular docking studies at different temperatures (20–37 °C). FTIR, CD, and fluorescence spectroscopy methods demonstrated that α- and β-caseins interact with DIP molecule mainly via hydrophobic and hydrophilic interactions and change in secondary structure of α- and β-caseins. DIP showed a higher quenching efficiency and binding constant of α-casein than β-casein. There was only one binding site for DIP and it was located on the surface of the protein molecule. The thermodynamic parameters of calculation showed that the binding process occurs spontaneously and demonstrated that α- and β-caseins provide very good binding and entrapment to DIP via hydrogen bonds, Van der Waals forces, and hydrophobic interactions. Fluorescence resonance energy transfer, synchronous fluorescence spectroscopy, and docking study showed that DIP binds to the Trp residues of α- and β-casein molecules with short distances. Docking study showed that DIP molecule made several hydrogen bonds and van der Waals interactions with α- and β-caseins. The study of cell culture and micellar solubility of DIP demonstrated α- and β-caseins relatively the same helping in delivery of DIP. Milk α- and β-caseins are considered as a useful vehicle for the solublization and stabilization of DIP in aqueous solution at natural pH.     

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.     

Anticonvulsant drug mechanisms of action

The effects of clinically used anticonvulsant drugs on high-frequency sustained repetitive firing (SRF) of action potentials and on postsynaptic responses to iontophoretically applied gamma-aminobutyric acid (GABA) have been compared to establish a classification of anticonvulsant drugs based on cellular mechanisms of action. By using concentrations in the range of therapeutic cerebrospinal fluid values in humans, drugs have been separated into three categories: Phenytoin, carbamazepine, and valproic acid limited SRF, but did not alter GABA responses. Phenobarbital, clonazepam, and diazepam augmented GABA responses and limited SRF only at concentrations above the therapeutic range in ambulatory patients but that are achieved in the acute treatment of status epilepticus. Ethosuximide failed to affect SRF or GABA responses even at supratherapeutic concentrations. Ability of an anticonvulsant to limit SRF correlated well with efficacy against generalized tonic-clonic seizures clinically and against maximal electroshock seizures in experimental animals. Augmentation of GABA responses and lack of limitation of SRF correlated with efficacy against generalized absence seizures in humans and against pentylenetetrazol-induced seizures in animals. However, ethosuximide must act against generalized absence seizures and against pentylenetetrazol-induced seizures by a third, as yet unknown, mechanism. Other actions occurring at supratherapeutic concentrations correlated with clinical toxicity.