DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionising radiation in fusion experiments?

Recent reports have revealed that the asymmetric nature of the nuclear genome of somatic hybrids, produced following the irradiation of one of the parents with X- or gamma rays, is generally much less than had been anticipated. As a consequence, we have begun to investigate whether UV radiation might be used as an alternative or indeed a supplement to the presently-used ionising radiation techniques in such experiments. Cell culture studies have revealed that UV radiation induces the desired physiological effects in sugar beet ( Beta vulgaris ) protoplasts, namely, a prevention of cell division without immediate cytotoxicity. Preliminary studies using denaturing and pulsed field gel electrophoresis have shown that UV can also induce substantial physical fragmentation of DNA. When using the same techniques, less breakdown was observed following gamma radiation. All results were highly reproducible. Such results augur well for the potential use of UV in asymmetric somatic cell fusion experiments.     

Synthesis and structural characterisation of four saccharinate - Metal complexes with 2,2′:6,2″-terpyridine (terpy) as a co-ligand

Four saccharinate complexes of divalent transition metals with 2,2′:6,2″-terpyridine (terpy) as a co-ligand have been synthesised, and characterised by elemental analysis and single crystal X-ray diffraction at low temperature. The complexes [M(terpy)(sac)(H₂O)₂] sac · H₂O (1, M = Mn; 2, M = Co; 3, M = Ni) are isostructural, crystallising in space group Pbca. The metal ions have approximately octahedral coordination, with the two coordinated water molecules occupying cis-positions. These water molecules are hydrogen-bonded to the oxygen atom in the free water molecule. The copper(II) ion in the anhydrous complex [Cu(terpy)(sac)₂] 4 is five-coordinate; the compound crystallises in the space group P2(1)/c.     

Identification of amino acid residues within the N-terminal domain of EspA that play a role in EspA filament biogenesis and function

Enteropathogenic Escherichia coli employs a filamentous type III secretion system, made by homopolymerization of the translocator protein EspA. In this study, we have shown that the N-terminal region of EspA has a role in EspA's protein stability, interaction with the CesAB chaperone, and filament biogenesis and function.     

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an ‘ordered’ mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.     

Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC(50) = 4.6 +/- 0.4 ng/ml) and crustaceans (Artemia nauplii LD(50) = 10 +/- 2 mug/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-A resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.     

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.     

Examining Cuphea as a potential host for western corn rootworm (Coleoptera: Chrysomelidae): larval development

In previous crop rotation research, adult emergence traps placed in plots planted to Cuphea PSR-23 (a selected cross of Cuphea viscosissma Jacq. and Cuphea lanceolata Ait.) caught high numbers of adult western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), suggesting that larvae may have completed development on this broadleaf plant. Because of this observation, a series of greenhouse and field experiments were conducted to test the hypothesis that Cuphea could serve as a host for larval development. Greenhouse-grown plants infested with neonates of a colonized nondiapausing strain of the beetle showed no survival of larvae on Cuphea, although larvae did survive on the positive control (corn, Zea mays L.) and negative control [sorghum, Sorghum bicolor (L.) Moench] plants. Soil samples collected 20 June, 7 July, and 29 July 2005 from field plots planted to Cuphea did not contain rootworm larvae compared with means of 1.28, 0.22, and 0.00 rootworms kg(-1) soil, respectively, for samples collected from plots planted to corn. Emergence traps captured a peak of eight beetles trap(-1) day(-1) from corn plots on 8 July compared with a peak of 0.5 beetle trap(-1) day(-1) on 4 August from Cuphea plots. Even though a few adult beetles were again captured in the emergence traps placed in the Cuphea plots, it is not thought to be the result of successful larval development on Cuphea roots. All the direct evidence reported here supports the conventional belief that rootworm larvae do not survive on broadleaf plants, including Cuphea.     

Catalytically requisite conformational dynamics in the mRNA-capping enzyme probed by targeted molecular dynamics

The addition of a N7-methyl guanosine cap to the 5' end of nascent mRNA is carried out by the mRNA-capping enzyme, a two-domain protein that is a member of the nucleotidyltransferase superfamily. The mRNA-capping enzyme is composed of a catalytic nucleotidyltransferase domain and a noncatalytic oligonucleotide/oligosaccharide binding (OB) domain. Large-scale domain motion triggered by substrate binding mediates catalytically requisite conformational rearrangement of the GTP substrate prior to the chemical step. In this study, we employ targeted molecular dynamics (TMD) on the PBCV-1 capping enzyme to probe the global domain dynamics and internal dynamics of conserved residues during the conformational transformation from the open to the closed state. Analysis of the resulting trajectories along with structural and sequence homology to other members of the superfamily allows us to suggest a conserved mechanism of conformational rearrangements spanning all mRNA-capping enzymes and all ATP-dependent DNA ligases. Our results suggest that the OB domain moves quasi-statically toward the nucleotidyltransferase domain, pivoting about a short linker region. The approach of the OB domain brings a conserved RxDK sequence, an element of conserved motif VI, within proximity of the triphosphate of GTP, destabilizing the unreactive conformation and thereby allowing thermal fluctuations to partition the substrate toward the catalytically competent state.