Some effects of nickel toxicity on rye grass

Summary Rye grass (Lolium perenne, cv.S-23) was grown for 4 weeks in a non-calcareous Seaton loam soil with varying amounts of Ni as NiSO₄. The purpose of this investigation was to study the Ni toxicity and the relationship of Ni with other essential elements. Nickel depressed shoot yield at all levels except at the lowest levelviz 30 g Ni/g soil. Nickel concentration of 50 g/g in shoots did not reduce the dry matter production in rye grass although slight chlorosis did appear at this level. The Ni and Fe concentration of the shoots increased and that of Mn and Zn decreased with increasing rates of Ni application. Uptake of Mn and Zn decreased at all level of Ni. But Fe uptake showed a slight increase at the first two levels and a profound depression at the subsequent levels. The pattern of Ni uptake is different, being highest at the middle level and decreasing on both sides which showed that the increase of Ni concentration of shoots is not proportional to the reduction in the yield. The Ni–Fe ratio rather than Ni and Fe concentration in plants has shown better relationship with the toxic effects of Ni. The implications of Ni phytotoxicity are discussed with particular reference to serpentine soils.The work is a part of Ph.D. Thesis submitted to the University of Aberdeen, U.K.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.     

Field‐level effects of preventative management tactics on soybean aphids (Aphis glycines Matsumura) and their predators

A 2‐year field experiment was conducted in northern Illinois to evaluate the effects of host plant resistance and an insecticidal seed treatment (thiamethoxam) on soybean aphids, Aphis glycines Matsumura and their predators. Densities of soybean aphids varied between the 2 years of the experiment. During both years, resistant plants experienced fewer cumulative aphid days than susceptible plants. Populations of soybean aphids on resistant plants rarely exceeded the economic injury level of 250 soybean aphids per plant. The use of thiamethoxam reduced cumulative aphid days in 2007, but not in 2008. Although soybean aphids reached densities that were sufficient to cause yield‐loss for untreated and susceptible plants, no yield‐benefit was associated with using the two management tactics in either year. This latter finding suggests that densities of soybean aphids need to be greater and sustained for a longer period of time than what we observed if the two management tactics are expected to provide a yield‐benefit. Monitoring natural enemies revealed that densities of key aphidophagous predators were relatively unaffected by host plant resistance or thiamethoxam; the effect of these management tactics on densities of predators, as well as the effectiveness of the method used to sample predators, is discussed.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.     

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.     

Polystomatidae (Monogenea) of Southern African Anura: Eupolystoma vanasi n. sp. parasitic in Schismaderma carens (Smith)

Eupolystoma vanasi is described as a new species of the Polystomatidae parasitic in the urinary bladder of Schismaderma carens in Northern Province and KwaZulu-Natal Province, South Africa. This is the third Eupolystoma species described from Africa and the first polystomatid from Schismaderma, an anuran genus that is primitive with respect to the other African bufonids in which Eupolystoma has been recorded. The species is distinguished by body size (this is the largest Eupolystoma known; mean length of adults 6 mm), by genital spine number (4 in comparison with 6-9 in other species), marginal hooklet length (greater than in other African species), and by the small size of the ovary and testis. In a sample of 27 toads, 37% were infected with up to 130 parasites per host (mean intensity 37). Worm burdens of this magnitude are exceptional amongst polystomatids in general but are characteristic of Eupolystoma, where there may be repeated re-infection of adult hosts and, uniquely, a direct, internal cycle of auto-infection.