Evolution of a multi‐step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer‐binding protein triggers acid‐activated gene expression

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid‐tolerant strain Ensifer medicae WSM419, the acid‐activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi‐step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer‐binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid‐induced. RACE mapping revealed that lpiA/acvB were co‐transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN‐binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co‐located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid‐activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.     

Osmoregulatory capacity,health status and growth as functions of moult stages from various weight classes in marron (Cherax cainii) and yabbies (Cherax destructor)

The study investigated the relationship between five moult stages and the osmoregulatory capacities (OC), moisture content of hepatopancreas (HM%), hepatosomatic indices (HI_wet, HI_dry) and growth of 6 weight classes of 235 marron (Cherax cainii) and 235 yabbies (Cherax destructor) under laboratory conditions. In each moult stage, OC increased linearly with the increase in wet body weight. Intermoult stage C showed the highest OC significantly lower than premoult stages D₀, D₁, D₂ and postmoult stage AB in every weight class in both crayfish. HM% decreased from AB to D₂, while both HI_wet and HI_dry reduced from C to D₂. Percentage dry matter of whole body carcass was highest in stage C and lowest during the AB stage with a significant difference between the two species. Larger than 15-g marron and yabbies differ with each other in OC, HI_wet, and HI_dry, moult intervals and growth rates.     

Comprehensive physiological analyses and reactive oxygen species profiling in drought tolerant rice genotypes under salinity stress

Rice being a staple cereal is extremely susceptible towards abiotic stresses. Drought and salinity are two vital factors limiting rice cultivation in Eastern Indo-Gangetic Plains (EIGP). Present study has intended to evaluate the consequences of salinity stress on selected drought tolerant rice genotypes at the most susceptible seedling stage with an aim to identify the potential multi-stress (drought and salt) tolerant rice genotype of this region. Genotypic variation was obvious in all traits related to drought and salt susceptibility. IR84895-B-127-CRA-5-1-1, one of the rice genotypes studied, exhibited exceptional drought and salinity tolerance. IR83373-B-B-25-3-B-B-25-3 also displayed enhanced drought and salt tolerance following IR84895-B-127-CRA-5-1-1. Variations were perceptible in different factors involving photosynthetic performance, proline content, lipid peroxidation, K⁺/Na⁺ ratio. Accumulation of reactive oxygen species (ROS) disintegrated cellular and sub-cellular membrane leading to decreased photosynthetic activities. Therefore, accumulation and detoxification of reactive oxygen species was also considered as a major determinant of salt tolerance. IR84895-B-127-CRA-5-1-1 showed improved ROS detoxification mediated by antioxidant enzymes. IR84895-B-127-CRA-5-1-1 seedlings also displayed significant recovery after removal of salt stress. The results established a direct association of ROS scavenging with improved physiological activities and salt tolerance. The study also recommended IR84895-B-127-CRA-5-1-1 for improved crop performance in both drought and saline environments of EIGP. These contrasting rice genotypes may assist in understanding the multiple stress associated factors in concurrent drought and salt tolerant rice genotypes.     

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition.

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.     

Aromatic inhibitors of dehydroquinate synthase: synthesis, evaluation and implications for gallic acid biosynthesis.

The role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of whether Co(+2) or Zn(+2) was the active site metal ion.     

GroEL/S Overexpression Helps to Purge Deleterious Mutations and Reduce Genetic Diversity during Adaptive Protein Evolution

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way.     

Epistasis Creates Invariant Sites and Modulates the Rate of Molecular Evolution

Invariant sites are a common feature of amino acid sequence evolution. The presence of invariant sites is frequently attributed to the need to preserve function through site-specific conservation of amino acid residues. Amino acid substitution models without a provision for invariant sites often fit the data significantly worse than those that allow for an excess of invariant sites beyond those predicted by models that only incorporate rate variation among sites (e.g., a Gamma distribution). An alternative is epistasis between sites to preserve residue interactions that can create invariant sites. Through computer-simulated sequence evolution, we evaluated the relative effects of site-specific preferences and site-site couplings in the generation of invariant sites and the modulation of the rate of molecular evolution. In an analysis of ten major families of protein domains with diverse sequence and functional properties, we find that the negative selection imposed by epistasis creates many more invariant sites than site-specific residue preferences alone. Further, epistasis plays an increasingly larger role in creating invariant sites over longer evolutionary periods. Epistasis also dictates rates of domain evolution over time by exerting significant additional purifying selection to preserve site couplings. These patterns illuminate the mechanistic role of epistasis in the processes underlying observed site invariance and evolutionary rates.     

Structure-function relationships in human salivary <Emphasis Type="Italic">α</Emphasis>-amylase: role of aromatic residues in a secondary binding site

Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrolysis of starch; (ii) binding to hydroxyapatite; and (iii) binding to bacteria (e.g. viridans streptococci). Oral bacteria utilize the starch hydrolyzing activity of HSAmy to derive their nutrients from dietary starch. Localized acid production by bacteria, through the metabolism of maltose generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries formation. HSAmy is a component of the acquired enamel pellicle and is used by Streptococcus gordonii to colonize the oral cavity. Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one of the secondary saccharide-binding sites harboring the aromatic residues W316 and W388, may play an important role in bacterial binding. To test this hypothesis, the aromatic residues W316 and W388 were mutated to alanine. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding and bacterial binding. Our results clearly showed that (i) the mutants W316A and W388A were not impaired in starch binding or bacterial binding; (ii) mutation of aromatic residues at these sites does not alter the overall conformation of the molecule; and (iii) the hydrolytic activity of the enzyme is unaffected against starch as substrates but reduced significantly against oligosaccharides.     

Myostatin inhibits myoblast differentiation by down-regulating MyoD expression

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.     

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies

In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.