The activity of ribosome modulation factor during growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.     

Distinct characteristics of Ag+ and Cd2+ binding to CopZ from Bacillus subtilis

The chaperone CopZ together with the P-type ATPase transporter CopA constitute a copper-detoxification system in Bacillus subtilis that is commonly found in bacteria and higher cells. Previous studies of the regulation of the copZA operon showed that expression is significantly upregulated in response to elevated concentrations of environmental silver and cadmium, as well as copper. Here, we have used spectroscopic and bioanalytical methods to investigate in detail the capacity of CopZ to bind these metal ions (as Ag(+) and Cd(2+)). We demonstrate that Ag(+) binding mimics closely that of Cu(+): Ag(+)-mediated dimerisation of the protein occurs, and distinct Ag(+)-bound species are formed at higher Ag(+) loadings. Cd(2+) also binds to CopZ, but exhibits significantly different behaviour. Cd(2+)-mediated dimerisation is only observed at low loadings, such that at 0.5 and one Cd(2+) per CopZ the protein is present mainly in a monomeric form; and multinuclear higher-order forms of Cd(2+)-CopZ are not observed. Competition binding studies reveal that Ag(+) binds with an affinity very similar to that of Cu(+), while Cd(2+) binding is significantly weaker. These data provide support for the proposal that CopZ may be involved in the detoxification of silver and cadmium, in addition to copper.     

Protection and selection for gene therapy in the hematopoietic system

Hematopoietic stem cell gene therapy is potentially curative for a number of inherited and acquired disorders. However, poor gene transfer and expression in repopulating hematopoietic stem cells attenuate this potential. Here we review potential means of conferring a selective advantage to hematopoietic stem cells and their progeny, and discuss the issues that surround the use of selective advantages in vivo.     

Heteroleptic phthalocyaninato-[tetra(15-crown-5)phthalocyaninato] lanthanides(III) double-deckers: Synthesis and cation-induced supramolecular dimerisation

A series of heteroleptic bisphthalocyaninates [(15C5)₄Pc]M(Pc) ((15C5)₄Pc = 2,3,9,10,16,17,24,25-tetrakis(15-crown-5)phthalocyaninate; Pc = unsubstituted phthalocyaninate; M = La, Sm, Dy, Tm) was synthesized. The raise-by-one-story method was applied in the cases of Sm, Dy and Tm complexes, whereas for the La complex we have developed a new synthetic approach. The complex [(15C5)₄Pc]La(Pc) is the first representative of heteroleptic lanthanum diphthalocyaninates. Homoleptic counterparts M[(15C5)₄Pc]₂ and M(Pc)₂, M = La, Sm, Dy, Tm have also been prepared for comparative studies. The UV-Vis spectral properties of all synthesized heteroleptic compounds were investigated and compared to those of the homoleptic unsubstituted and crown-substituted diphthalocyaninates. Cation-induced dimerisation of heteroleptic complexes was studied. The observed spectral effects were explained in terms of excitonic coupling between chromophoric molecules. The unsymmetrical distribution of electronic density over macrocyclic ligands is established.     

The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.     

Synthesis, spectral properties and supramolecular dimerisation of heteroleptic triple-decker phthalocyaninato complexes with one outer crown-substituted ligand

Four complexes - [(15C5)₄Pc]M(Pc)M(Pc), (Pc²⁻ - phthalocyaninato-dianion, [(15C5)₄Pc]²⁻ - 2,3,9,10,16,17,24,25-tetrakis(15-crown-5)phthalocyaninato-dianion, M = Sm, Dy, Tm, Y) were obtained via the reaction of M(Pc)₂, H₂[(15C5)₄Pc] and M(acac)₃. The influence of the stability of starting M(Pc)₂ on the yields of target compounds was investigated. Increasing the stability of M(Pc)₂ leads to higher yields of [(15C5)₄Pc]M(Pc)M(Pc) and lower yields of scrambling products. All complexes were characterized by ¹H NMR, UV-Vis and FT-IR spectroscopy as well as MALDI TOF mass-spectrometry. The analysis of ¹H NMR spectra was performed in terms of lanthanide-induced shifts. Cation-induced dimerisation was studied by means of spectrophotometric titration. Supramolecular dimers {2[(15C5)₄Pc]M(Pc)M(Pc) · 4K⁺} are the largest discrete cofacial supramolecular assemblies built of phthalocyanine building blocks reported up-to-date. The observed increase of the intermolecular excitonic interaction between building blocks with the increase of REE(III) size is tentatively explained in terms of metal-size dependent deformation of phthalocyanine ligands in sandwich complexes.     

Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal

The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.