Development and validation of co-dominant gene based markers for <Emphasis Type="Italic">Pi9</Emphasis>, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.     

Production of extracellular hydrolase enzymes by fungi from King George Island

Fungi are known to produce a range of extracellular enzymes and other secondary metabolites. Investment in extracellular enzyme production may be an important element of the survival strategy of these fungi in maritime Antarctic soils. This study focuses on fungi that were isolated from ornithogenic, undisturbed and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica, during the austral summer in February 2007. We (1) describe fungal diversity based on molecular approaches, (2) describe the thermal characteristics of the fungal isolates, and (3) screen extracellular hydrolase enzyme production (amylase and cellulase) by the isolates. Soil samples were cultured using the Warcup soil plating technique and incubated at 4 and 25 °C to allow basic thermal classification. In total, 101 isolates were obtained. All the isolates were screened at culture temperatures of 4 and 25 °C in order to detect activity of extracellular hydrolase enzymes. At 25 °C, ornithogenic penguin rookery soils recorded the lowest diversity of fungi, with little difference in diversity apparent between the other soils examined. At 4 °C, an undisturbed site recorded the lowest and a human-impacted site the highest diversity of fungi. The majority of the fungi identified in this study were in the mesophilic thermal class. Six strains possessed significant activity for amylase and 13 for cellulase at 25 °C. At 4 °C, four strains showed significant amylase and 22 significant cellulase activity. The data presented increase our understanding of microbial responses to environmental temperature.     

Muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase in human skeletal muscle

Physiological stress associated with muscle damage results in systemic insulin resistance. However, the mechanisms responsible for the insulin resistance are not known; therefore, the present study was conducted to elucidate the molecular mechanisms associated with insulin resistance after muscle damage. Muscle biopsies were obtained before (base) and at 1 h during a hyperinsulinemic-euglycemic clamp (40 mU x kg(-1) x min(-1)) in eight young (age 24+/-1 yr) healthy sedentary (maximal O(2) consumption, 49.7+/-2.4 ml x kg(-1) x min(-1)) males before and 24 h after eccentric exercise (ECC)-induced muscle damage. To determine the role of cytokines in ECC-induced insulin resistance, venous blood samples were obtained before (control) and 24 h after ECC to evaluate ex vivo endotoxin-induced mononuclear cell secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Glucose disposal was 19% lower after ECC (P<0.05). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was 45% lower after ECC (P<0.05). Insulin-stimulated phosphatidylinositol (PI) 3-kinase, Akt (protein kinase B) serine phosphorylation, and Akt activity were reduced 34, 65, and 20%, respectively, after ECC (P < 0.05). TNF-alpha, but not IL-6 or IL-1beta production, increased 2.4-fold 24 h after ECC (P<0.05). TNF-alpha production was positively correlated with reduced insulin action on PI 3-kinase (r = 0.77, P = 0.04). In summary, the physiological stress associated with muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase, presumably leading to decreased insulin-mediated glucose uptake. Although more research is needed on the potential role for TNF-alpha inhibition of insulin action, elevated TNF-alpha production after muscle damage may impair insulin signal transduction.     

Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases

The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three cysteine proteinases as follows: papain, human cathepsin B, and cathepsin L. The stoichiometry of enzyme inhibition reveals that both Thyr-1 domains of saxiphilin inhibit papain (apparent K(i) = 1. 72 nm), but only one of these domains inhibits cathepsin B (K(i) = 1. 67 nm) and cathepsin L (K(i) = 0.02 nm). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by cross-linking with glutaraldehyde. The rate of association of saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two H(+)-titratable groups. These results further demonstrate that various Thyr-1 domains are selective inhibitors of cysteine proteinases with utility in the study of protein interactions and degradation.     

Saccharomyces cerevisiae BUB2 prevents mitotic exit in response to both spindle and kinetochore damage

The spindle assembly checkpoint-mediated mitotic arrest depends on proteins that signal the presence of one or more unattached kinetochores and prevents the onset of anaphase in the presence of kinetochore or spindle damage. In the presence of either damage, bub2 cells initiate a preanaphase delay but do not maintain it. Inappropriate sister chromatid separation in nocodazole-treated bub2 cells is prevented when mitotic exit is blocked using a conditional tem1(c) mutant, indicating that the preanaphase failure in bub2 cells is a consequence of events downstream of TEM1 in the mitotic exit pathway. Using a conditional bub2(tsd) mutant, we demonstrate that the continuous presence of Bub2 protein is required for maintaining spindle damage-induced arrest. BUB2 is not required to maintain a DNA damage checkpoint arrest, revealing a specificity for spindle assembly checkpoint function. In a yeast two-hybrid assay and in vitro, Bub2 protein interacts with the septin protein Cdc3, which is essential for cytokinesis. These data support the view that the spindle assembly checkpoint encompasses regulation of distinct mitotic steps, including a MAD2-directed block to anaphase initiation and a BUB2-directed block to TEM1-dependent exit.     

Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability

Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates.     

Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.     

Spectral kinetics ratiometry: a simple approach for real-time monitoring of fluorophore distributions in living cells.

BACKGROUND: Spectral Imaging Microscopy is gaining attention in biological research. Most of the commercial systems in vogue employ linear spectral un-mixing algorithms and/or spectral profile matching algorithms to extract the component spectral information from the measured specimen spectra. The need to accurately deconvolve multiple spectra with minimal cross-contamination is always accompanied by an increase in system complexity and cost. METHODS: We describe here a variant of the spectral waveform cross-correlation analysis (SWCCA) method where the master reference spectral library is constructed by composite spectra with varying ratios of component spectra, unlike the conventional spectral library where pure spectra form the components. We demonstrate that this spectral kinetics ratiometric approach gives realistic estimates of fluorophore distribution in living cells with a better spectral correlation as compared with pure component spectral libraries. RESULTS: Biological applications demonstrated in this article include acceptor photobleaching FRET, caspase activity during cell death and mitochondrial membrane polarization kinetics during substrate metabolism. CONCLUSIONS: Beyond the representative applications presented in this article, we think the proposed approach can be valuable in dynamic studies of a variety of other cellular processes such as pH oscillations, photobleaching and quenching kinetics. Besides giving better spectral correlation and real-time monitoring of biophysical processes in living cells, this method can serve as an economical solution for high-throughput spectral classification requirements.     

Is the titer of adipokinetic peptides in Leptinotarsa decemlineata fed on genetically modified potatoes increased by oxidative stress?

The level of adipokinetic hormones (AKHs) (Peram-CAH-I and II) in the corpora cardiaca and the hemolymph of Leptinotarsa decemlineata enormously increases in the adults fed on genetically modified potatoes containing either GNA lectin or Cry 3Aa toxin concomitant with increased oxidative stress in gut tissues. A similar enhancement of the AKH titer is achieved when the adults are injected with paraquat that evokes oxidative stress. On the other hand, an injection of exogenous AKH reduces oxidative stress biomarkers in the hemolymph by reducing protein carbonyls and enhancing reduced glutathione levels. These facts indicate that there is a feedback regulation between an oxidative stressor action and the level of AKH in the insect body, and that AKHs might be involved in the activation of an antioxidant protection mechanism. These results are to our knowledge, the first evidence for the involvement of AKHs in oxidative stress mitigation, in addition to a plethora of other roles.     

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.