Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

Protein micelles from lipoxygenase 3

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.     

The effects of salinity and sodicity upon nodulation and nitrogen fixation in chickpea (Cicer arietinum)

Production of grain legumes is severely reduced in salt-affected soils because their ability to form and maintain nitrogen-fixing nodules is impaired by both salinity and sodicity (alkalinity). Genotypes of chickpea, Cicer arietinum, with high nodulation capacity under stress were identified by field screening in a sodic soil in India and subsequently evaluated quantitatively for nitrogen fixation in a glasshouse study in a saline but neutral soil in the UK. In the field, pH 8.9 was the critical upper limit for most genotypes studied but genotypes with high nodulation outperformed all others at pH 9.0-9.2. The threshold limit of soil salinity for shoot growth was at ECe 3 dS m(-1), except for the high-nodulation selection for which it was ECe 6. Nodulation was reduced in all genotypes at salinities above 3 dS m(-1) but to a lesser extent in the high-nodulation selection, which proved inherently superior under both non-saline and stress conditions. Nitrogen fixation was also much more tolerant of salinity in this selection than in the other genotypes studied. The results show that chickpea genotypes tolerant of salt-affected soil have better nodulation and support higher rates of symbiotic nitrogen fixation than sensitive genotypes.     

Genetic and morphologic differentiation of Bolbophorus confusus and B. levantinus (Digenea: Diplostomatidae), based on rDNA SSU polymorphism and SEM

Metacercariae of Bolbophorus species are serious pathogens of farmed fish. Molecular diagnostic tools, capable of identifying and differentiating these parasites, may assist in the development of rationale control strategies. The rDNA 18S (small sub-unit: SSU) genes of adult B. confusus and B. levantinus obtained from a pelican, Pelecanus onocrotalus, and a night heron, Nycticorax nycticorax, respectively, were amplified, sequenced, and aligned. Based on this alignment, we developed a genetic differentiation assay between B. confusus and B. levantinus. These 2 species were compared genetically with the North American species B. damnificus and Bolbophorus sp. ('Type 2'). The relationship between species is outlined and discussed. In addition to the molecular study, specimens of B. confusus and B. levantinus were compared morphologically, using scanning electron microscopy. Morphologic analysis revealed interspecific differences in details of the holdfast organ and the position of the acetabulum.     

Mechanisms of sodium uptake by roots of higher plants

The negative impact of soil salinity on agricultural yields is significant. For agricultural plants, sensitivity to salinity is commonly (but not exclusively) due to the abundance of Na⁺ in the soil as excess Na⁺ is toxic to plants. We consider reducing Na⁺ uptake to be the key, as well as the most efficient approach, to control Na⁺ accumulation in crop plants and hence to improve their salt resistance. Understanding the mechanism of Na⁺ uptake by the roots of higher plants is crucial for manipulating salt resistance. Hence, the aim of this review is to highlight and discuss recent advances in our understanding of the mechanisms of Na⁺ uptake by plant roots at both physiological and molecular levels. We conclude that continued efforts to investigate the mechanisms of root Na⁺ uptake in higher plants are necessary, especially that of low-affinity Na⁺ uptake, as it is the means by which sodium enters into plants growing in saline soils.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)

H₂S⁺ bacteria responsible for the degradation of sulfur-containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H₂S⁺ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.     

Abdominal obesity in BTBR male mice is associated with peripheral but not hepatic insulin resistance

Insulin resistance is a common feature of obesity. BTBR mice have more fat mass than most other inbred mouse strains. On a chow diet, BTBR mice have elevated insulin levels relative to the C57BL/6J (B6) strain. Male F1 progeny of a B6 x BTBR cross are insulin resistant. Previously, we reported insulin resistance in isolated muscle and in isolated adipocytes in this strain. Whereas the muscle insulin resistance was observed only in male F1 mice, adipocyte insulin resistance was also present in male BTBR mice. We examined in vivo mechanisms of insulin resistance with the hyperinsulinemic euglycemic clamp technique. At 10 wk of age, BTBR and F1 mice had a >30% reduction in whole body glucose disposal primarily due to insulin resistance in heart, soleus muscle, and adipose tissue. The increased adipose tissue mass and decreased muscle mass in BTBR and F1 mice were negatively and positively correlated with whole body glucose disposal, respectively. Genes involved in focal adhesion, actin cytoskeleton, and inflammation were more highly expressed in BTBR and F1 than in B6 adipose tissue. The BTBR and F1 mice have higher levels of testosterone, which may be related to the pathological changes in adipose tissue that lead to systemic insulin resistance. Despite profound peripheral insulin resistance, BTBR and F1 mice retained hepatic insulin sensitivity. These studies reveal a genetic difference in body composition that correlates with large differences in peripheral insulin sensitivity.     

Both the suppressor of cytokine signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-gamma by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-gamma-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, ((53))DTHFRTFRSHSDYRRI((68)) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001-1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr(1007), pJAK2(1001-1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-gamma-induced STAT1-alpha phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-alpha activation. Both Tkip and SOCS1-KIR inhibited IFN-gamma activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001-1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN-gamma activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-gamma activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.     

Improving crop salt tolerance

Salinity is an ever-present threat to crop yields, especially in countries where irrigation is an essential aid to agriculture. Although the tolerance of saline conditions by plants is variable, crop species are generally intolerant of one-third of the concentration of salts found in seawater. Attempts to improve the salt tolerance of crops through conventional breeding programmes have met with very limited success, due to the complexity of the trait: salt tolerance is complex genetically and physiologically. Tolerance often shows the characteristics of a multigenic trait, with quantitative trait loci (QTLs) associated with tolerance identified in barley, citrus, rice, and tomato and with ion transport under saline conditions in barley, citrus and rice. Physiologically salt tolerance is also complex, with halophytes and less tolerant plants showing a wide range of adaptations. Attempts to enhance tolerance have involved conventional breeding programmes, the use of in vitro selection, pooling physiological traits, interspecific hybridization, using halophytes as alternative crops, the use of marker-aided selection, and the use of transgenic plants. It is surprising that, in spite of the complexity of salt tolerance, there are commonly claims in the literature that the transfer of a single or a few genes can increase the tolerance of plants to saline conditions. Evaluation of such claims reveals that, of the 68 papers produced between 1993 and early 2003, only 19 report quantitative estimates of plant growth. Of these, four papers contain quantitative data on the response of transformants and wild-type of six species without and with salinity applied in an appropriate manner. About half of all the papers report data on experiments conducted under conditions where there is little or no transpiration: such experiments may provide insights into components of tolerance, but are not grounds for claims of enhanced tolerance at the whole plant level. Whether enhanced tolerance, where properly established, is due to the chance alteration of a factor that is limiting in a complex chain or an effect on signalling remains to be elucidated. After ten years of research using transgenic plants to alter salt tolerance, the value of this approach has yet to be established in the field.