Electrostatic potential of nucleotide-free protein is sufficient for discrimination between adenine and guanine-specific binding sites

Despite sharing many common features, adenine-binding and guanine-binding sites in proteins often show a clear preference for the cognate over the non-cognate ligand. We have analyzed electrostatic potential (ESP) patterns at adenine and guanine-binding sites of a large number of non-redundant proteins where each binding site was first annotated as adenine/guanine-specific or non-specific from a survey of primary literature. We show that more than 90% of ESP variance at the binding sites is accounted for by only two principal component ESP vectors, each aligned to molecular dipoles of adenine and guanine. Projected on these principal component vectors, the adenine/guanine-specific and non-specific binding sites, including adenine-containing dinucleotides, show non-overlapping distributions. Adenine or guanine specificities of the binding sites also show high correlation with the corresponding electrostatic replacement (cognate by non-cognate ligand) energies. High correlation coefficients (0.94 for 35 adenine-binding sites and 1.0 for 20 guanine-binding sites) were obtained when adenine/guanine specificities were predicted using the replacement energies. Our results demonstrate that ligand-free protein ESP is an excellent indicator for discrimination between adenine and guanine-specific binding sites and that ESP of ligand-free protein can be used as a tool to annotate known and putative purine-binding sites in proteins as adenine or guanine-specific.     

Exobasidium vexans infection of Camellia sinensis increased 2,3-cis isomerisation and gallate esterification of proanthocyanidins

Infection of leaves of tea (Camellia sinensis (Kuntze) L, cv TRI 2025) which was susceptible to blister blight (Exobasidium vexans Massee), resulted in a shift of the proanthocyanidin stereochemistry away from 2,3-trans (e.g. catechin and gallocatechin) and towards 2,3-cis (e.g. epicatechin and epigallocatechin). Infection also resulted in increased gallic acid esterification of the initiating subunits of proanthocyanidins. This was shown by both mass spectroscopy and phloroglucinolysis. Proanthocyanidins isolated from healthy tissue had a predominantly 2,3-trans stereochemistry which accounted for 53% and 61% of the total initiating and extension units of proanthocyanidin, respectively. Conversely in infected tissue, proanthocyanidin subunits with a 2,3-trans stereochemistry accounted for 26% and 40% of the total initiating and extension units, respectively. Infection had little impact on the hydroxylation state of the B-rings of proanthocyanidins. The products of acid hydrolysis under oxidative conditions had a slight excess of di-hydroxylated B-rings with cyanidin accounting for 58.3+/-0.05% and 60.4+/-0.2% of the total anthocyanidin recovered following hydrolysis of proanthocyanidin isolated from infected and healthy leaves, respectively. Similar results were obtained by phloroglucinolysis.     

A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control

We have examined a replication terminus (psiL1) located on the left arm of the chromosome of Bacillus subtilis and within the yxcC gene and at or near the left replication checkpoint that is activated under stringent conditions. The psiL1 sequence appears to bind to two dimers of the replication terminator protein (RTP) rather weakly and seems to possess overlapping core and auxiliary sites that have some sequence similarities with normal Ter sites. Surprisingly, the asymmetrical, isolated psiL1 site arrested replication forks in vivo in both orientations and independent of stringent control. In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1 terminus. The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (psiL1) sequence to RTP did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of psiL1. On the basis of the result it would appear that the weak RTP-L1Ter interaction cannot by itself account for fork arrest, thus suggesting a role for DnaB-RTP interaction.     

Oleanane triterpenoid CDDO-Me inhibits Akt activity without affecting PDK1 kinase or PP2A phosphatase activity in cancer cells

Our previous studies have shown that methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane synthetic triterpenoid induces apoptosis in prostate cancer cells by inhibiting the Akt/NF-κB/mTOR signaling cascade; however, the mechanism by which CDDO-Me inhibits Akt/NF-κB/mTOR signaling has remained undetermined. Present studies show that Akt plays a critical role in the response of prostate cancer cells to CDDO-Me. Silencing of Akt sensitized PC-3 cells to CDDO-Me, whereas its overexpression rendered them resistant to CDDO-Me. Evaluation of the effect of CDDO-Me on Akt which lies upstream of NF-κB and mTOR showed that CDDO-Me directly inhibits the Akt kinase activity in cell-free kinase activity assay and in vivo without modulating the activity of PDK1, the upstream kinase that phosphorylates and activates Akt. The inhibition of Akt activity resulted in inhibition of phosphorylation/inactivation of proapoptotic procaspase-9, Bad and Foxo3a. Further, inhibition of p-Akt by CDDO-Me was not attributable to an increase in the activity of protein phosphatase 2A (PP2A) or PH domain/leucine-rich repeat protein phosphatase1 (PHLPP1) both of which dephosphorylate p-Akt. These findings show that Akt is a direct target of CDDO-Me in the Akt/NF-κB/mTOR prosurvival signaling axis.     

All G protein βγ complexes are capable of translocation on receptor activation

Heterotrimeric G proteins transduce signals sensed by transmembrane G protein coupled receptors (GPCRs). A subfamily of G protein βγ subunit types has been shown to selectively translocate from the plasma membrane to internal membranes on receptor activation. Using 4D imaging we show here that Gβγ translocation is not restricted to some subunit types but rather all 12 members of the family of mammalian γ subunits are capable of supporting βγ translocation. Translocation kinetics varies widely depending on the specific γ subunit type, with t(1/2) ranging from 10s to many minutes. Using fluorescence complementation, we show that the β and γ subunits translocate as βγ dimers with kinetics determined by the γ subunit type. The expression patterns of endogenous γ subunit types in HeLa cells, hippocampal neurons and cardiomyocytes are distinctly different. Consistent with these differences, the βγ translocation rates vary widely. βγ translocation rates exhibit the same γ subunit dependent trends regardless of the specific receptor type or cell type showing that the translocation rates are intrinsic to the γ subunit types. βγ complexes with widely different rates of translocation had differential effects on muscarinic stimulation of GIRK channel activity. These results show that G protein βγ translocation is a general response to activation of GPCRs and may play a role in regulating signaling activity.     

Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The K _m and k _cat values of the PG were 2.7 mg/mL and 2.23 × 10³ s^?1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag⁺, Hg²⁺ and KMnO₄, while it was stimulated to some extent by Co²⁺. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.     

Distinct Evolutionary Pressures Underlie Diversity in Simian Immunodeficiency Virus and Human Immunodeficiency Virus Lineages

Simian immunodeficiency virus (SIV) infection of rhesus macaques causes immune depletion and disease closely resembling human AIDS and is well recognized as the most relevant animal model for the human disease. Experimental investigations of viral pathogenesis and vaccine protection primarily involve a limited set of related viruses originating in sooty mangabeys (SIVsmm). The diversity of human immunodeficiency virus type 1 (HIV-1) has evolved in humans in about a century; in contrast, SIV isolates used in the macaque model evolved in sooty mangabeys over millennia. To investigate the possible consequences of such different evolutionary histories for selection pressures and observed diversity in SIVsmm and HIV-1, we isolated, sequenced, and analyzed 20 independent isolates of SIVsmm, including representatives of 7 distinct clades of viruses isolated from natural infection. We found SIVsmm diversity to be lower overall than HIV-1 M group diversity. Reduced positive selection (i.e., less diversifying evolution) was evident in extended regions of SIVsmm proteins, most notably in Gag p27 and Env gp120. In addition, the relative diversities of proteins in the two lineages were distinct: SIVsmm Env and Gag were much less diverse than their HIV-1 counterparts. This may be explained by lower SIV-directed immune activity in mangabeys relative to HIV-1-directed immunity in humans. These findings add an additional layer of complexity to the interpretation and, potentially, to the predictive utility of the SIV/macaque model, and they highlight the unique features of human and simian lentiviral evolution that inform studies of pathogenesis and strategies for AIDS vaccine design.     

Carbon pools of an intact forest in Gabon

Quantitative and qualitative loss of tropical forests prompted international policy agendas to slow down forest loss through reducing emissions from deforestation and forest degradation (REDD)+, ensuring carbon offset payments to developing countries. So far, many African countries lack reliable forest carbon data and monitoring systems as required by REDD+. In this study, we estimate the carbon stocks of a naturally forested landscape unaffected by direct human impact. We used data collected from 34 plots randomly distributed across the Mount Birougou National Park (690 km²) in southern Gabon. We used tree‐level data on species, diameter, height, species‐specific wood density and carbon fraction as well as site‐level data on dead wood, soil and litter carbon to calculate carbon content in aboveground, belowground, dead wood, soil and litter as 146, 28, 14, 186 and 7 Mg ha^?1, respectively. Results may serve as a benchmark to assess ecosystem carbon loss/gain for the Massif du Chaillu in Gabon and the Republic of Congo, provide field data for remote sensing and also may contribute to establish national monitoring systems.     

Reproduction numbers for infections with free-living pathogens growing in the environment

The basic reproduction number ?(0) for a compartmental disease model is often calculated by the next generation matrix (NGM) approach. When the interactions within and between disease compartments are interpreted differently, the NGM approach may lead to different ?(0) expressions. This is demonstrated by considering a susceptible-infectious-recovered-susceptible model with free-living pathogen (FLP) growing in the environment. Although the environment could play different roles in the disease transmission process, leading to different ?(0) expressions, there is a unique type reproduction number when control strategies are applied to the host population. All ?(0) expressions agree on the threshold value 1 and preserve their order of magnitude. However, using data for salmonellosis and cholera, it is shown that the estimated ?(0) values are substantially different. This study highlights the utility and limitations of reproduction numbers to accurately quantify the effects of control strategies for infections with FLPs growing in the environment.     

The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern–Triggered Immunity in Rice

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.