RNA cleavage and inhibition of protein synthesis by bleomycin

Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.     

Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Experimental models for assaying microvascular endothelial cell pathophysiology in stroke

It is important to understand the molecular mechanisms underlying neuron death following stroke in order to develop effective neuroprotective strategies. Since studies on human stroke are extremely limited due to the difficulty in collecting post-mortem tissue at different time points after the onset of stroke, brain ischaemia research focuses on information derived from in-vitro models of neuronal death through ischaemic injury [1]. This review aims to provide an update on the different in-vitro stroke models with brain microvascular endothelial cells that are currently being used. These models provide a physiologically relevant tool to screen potential neuroprotective drugs in stroke and to study the molecular mechanisms involved in brain ischaemia.     

On-line analysis of volatile chlorinated hydrocarbons in air by gas chromatography–mass spectrometry: Improvements in preconcentration and injection steps

An analytical system composed of a cryofocusing trap injector device coupled to a gas chromatograph with mass spectrometric detection (CTI-GC–MS) specific for the on-line analysis in air of volatile chlorinated hydrocarbons (VCHCs) (dichloromethane; chloroform; 1,1,1-trichloroethane; tetrachloromethane; 1,1,2-trichloroethylene; tetrachloroethylene) was developed. The cryofocusing trap injector was the result of appropriate low cost modifications to an original purge-and-trap device to make it suitable for direct air analysis even in the case of only slightly contaminated air samples, such as those from remote zones. The CTI device can rapidly and easily be rearranged into the purge-and-trap allowing water and air analysis with the same apparatus. Air samples, collected in stainless steel canisters, were introduced directly into the CTI-GC–MS system to realize cryo-concentration (at −120 °C), thermal desorption (at 200 °C) and for the subsequent analysis of volatiles. The operating phases and conditions were customised and optimized. Recovery efficiency was optimized in terms of moisture removal, cold trap temperature and sampling mass flow. The injection of entrapped volatiles was realized through a direct transfer with high chromatographic reliability (capillary column–capillary column). These improvements allowed obtaining limits of detection (LODs) at least one order of magnitude lower than current LODs for the investigated substances. The method was successfully employed on real samples: air from urban and rural areas and air from remote zones such as Antarctica.     

Struggle for space: viral extinction through competition for cells

The design of protocols to suppress the propagation of viral infections is an enduring enterprise, especially hindered by limited knowledge of the mechanisms leading to viral extinction. Here we report on infection extinction due to intraspecific competition to infect susceptible hosts. Beneficial mutations increase the production of viral progeny, while the host cell may develop defenses against infection. For an unlimited number of host cells, a feedback runaway coevolution between host resistance and progeny production occurs. However, physical space limits the advantage that the virus obtains from increasing offspring numbers; thus, infection clearance may result from an increase in host defenses beyond a finite threshold. Our results might be relevant to devise improved control strategies in environments with mobility constraints or different geometrical properties.     

Theoretical investigation of histidine-tryptophan preferential interactions

Several histidine-tryptophan complexes (either stacked or T-shaped), derived from the crystal structures available in the Brookhaven Protein Data Bank, have been examined with molecular mechanics (MM), using the Tripos force field with Gasteiger-Hückel charges, whose trend was found to be analogous to the AMBER or CHARMM ones. The MM results were compared to the ab initio MP2 results, with and without counterpoise (CP) correction, previously obtained using extended basis sets on 5-methylimidazole and indole as model systems. MM seems to underestimate the interaction energy between the two monomers when compared to the uncorrected MP2 results, while the agreement is much better after including the CP correction at the MP2 level in all cases. MM was thus used to qualitatively analyse the dependence of the stacking energy on the ring rotation at a variable distance and ring centroid displacement for these systems, while keeping the rings in parallel planes. An analogous study was carried out for a T-shaped adduct. Received: 24 March 1998 / Accepted: 3 September 1998 / Published online: 1 February 1999     

Influence of structural features on the self‐assembly of short ionic oligopeptides

We investigated the self‐aggregation of 12 short ionic oligopeptides constituted by 4–7 amino acid residues to establish useful structure–property relationships that might be exploited in the biomedical field by using the concept of molecular Lego. We show that the critical aggregation concentration (CAC) of tetrapeptides decreases with increasing hydrophobicity of neutral residues. Additionally, the dependence of the CAC of isomeric oligopeptides on the distribution of amino acid residues confirms the high tendency to self‐organization of molecules with alternating ionic and neutral residues. Indeed, atomic force microscopy (AFM) images recorded on oligopeptide solutions above the CAC show the presence of either fibrillar or spherical aggregates depending on oligopeptide structure and concentration, steric hindrance, solution pH, and time. The potential of the investigated oligopeptides in tissue engineering applications is supported by their in vitro cytocompatibility. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 889–897, 2010