Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

Amphiphilic porphyrin film on glass as a simple and selective solid-state chemosensor for aqueous Hg2+

Deposition of amphiphilic porphyrin derivatives occurs spontaneously on silanised glass surfaces, in a controlled fashion. The resulting porphyrin films show appreciable fluorescence emission. This emission can be effectively quenched by immersion of the slides into a diluted solution of Hg(2+) (microM concentration). The initial intensity can be restored by washings with a solution of N,N,N',N'-tetrakis(2-pyridilmethyl)ethylenediammine with no loss of efficiency. A remarkable selectivity is featured toward the detection of Hg(2+) over Cu(2+), Cd(2+), Pb(2+) and Zn(2+) counterparts. This protocol can be extended to a flow-through apparatus. The presented results are of importance for the achievement of a solid-state chemosensor for mercuric ions, at micromolar concentration, in water.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes

Abstract: The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the ¹²⁵I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, ¹²⁵I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying ¹²⁵I-labeled S-100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.     

Praziquantel derivatives I: Modification of the aromatic ring

Several analogues of the potent anthelmintic praziquantel were prepared with variation in the aromatic ring. The biological activity of these analogues was evaluated and compared against known analogues. Amination of the ring was tolerated while other variations were not. These results have important implications for drug development for schistosomiasis.     

High-performance liquid chromatography (HPLC) determination of inosine,a potential biomarker for initial cardiac ischaemia,using isolated mouse hearts

Abstract

Each year in the USA approximately 7–8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2–5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs–Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C₁₈ column at a flow rate of 0.6 ml min^?1 with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml^?1 using a 100-µl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.     

Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation