Membrane targeting of RecA during genetic transformation

Recombination in prokaryotes and eukaryotes is mediated by the RecA family of proteins. Although the interactions between RecA and DNA are well studied, the cellular location of these interactions is not known. Using genetic transformation of Streptococcus pneumoniae as a model system, there was increased expression of a protein, colligrin, and RecA, products of the rec locus during genetic transfer. These proteins formed a complex and were found associated with the membranes of genetically competent cells. With immunoelectron microscopy and subcellular fractionation, we showed that the induction of competence led to the translocation of RecA and colligrin to the membrane and to the formation of clusters of RecA in a colligrin-dependent step. Based on the behaviour of colligrin and RecA during genetic exchange and the numerous proteins in prokaryotes and eukaryotes with domains similar to colligrin, we suggest that there may exist a family of proteins, which gathers macromolecules at specific sites in biological membranes.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Within-species correlations in leaf traits of three boreal plant species along a latitudinal gradient

Evergreen boreal plant species express high variability in their leaf traits. It remains controversial whether this within-species variability is constrained to the same leaf trait relationships as has been observed across species. We sampled leaves of three boreal evergreen woody species along a latitudinal gradient (from 57o56′N to 69o55′N). Leaf longevity (LL) of Pinus sylvestris L. and Vaccinium vitis-idaea L. correlated negatively with mean annual air temperature (MAT), whereas the LL of Ledum palustre L. was not affected by MAT. V. vitis-idaea and L. palustre had a negative relationship between leaf mass per area (LMA) and MAT. In P. sylvestris, the LMA–MAT relationship was positive. A negative correlation between LL and LMA was significant only for P. sylvestris. Leaf nitrogen concentration was positively related to leaf phosphorus concentration in all three species. Leaf potassium concentration was related to nitrogen concentration only in L. palustre, and to phosphorus concentration in P. sylvestris and L. palustre. Our results demonstrate that although within the studied species the variation in some of the leaf traits may have the same degree as interspecific variation, there is no such intercorrelation of leaf traits within the studied species as has been observed across species.     

Impact of disease frequency and host density on pollination and transmission of an African anther-smut fungus

The vast majority of flowering plants rely solely on insects for pollination. A number of pathogens have evolved mechanisms to exploit these close associations and use pollinators as vectors of infective propagules. Factors that affect pollinator movements and successful pollination may in turn also influence successful transmission of fungal spores. Here we investigate the effect of host density and the frequency of diseased Oxalis lanata individuals infected by the anther-smut fungus, Thecaphora capensis, on the likelihood of receiving pollen and fungal spores. Specifically, we determined the numbers of spores and pollen grains deposited on stigmatic surfaces of selected flowers under natural and standardized conditions where host density and disease frequency varied. The effect of host flower density and diseased flower frequency on pollen and spore transfer was variable under natural conditions and these factors interacted significantly. However, an increase in host density and disease frequency significantly influenced pollen and spore deposits under standardized conditions. The effect of host density was, however, not linear and an optimal flower density for pollen and fungal spore transmission was found. Similar to other systems of vector-borne disease, the transmission of anther-smut of Oxalis lanata is more frequency-dependent than density-dependent. This study represents a first step towards understanding the disease transmission process of T. capensis on Oxalis and lays the foundation for future comparative studies between this and other systems to develop and test general hypotheses of disease dynamics in vector-borne disease transmission systems.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.     

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.