Enzymes in Heloderma horridum Venom

A mixture of venom and saliva from the lizard Heloderma horridum was analyzed for esterase, phosphomonoesterase, phosphodiesterase, 5\u27nucleotidase, and protease activities. Hydrolysis of N-benzoyl-L-arginine ethyl ester occurred at a pH optimum between pH 8.6 and 9.1 with a maximum activity of 452 units per mg per min. Hydrolysis of ptoluenesulfonyl-L-arginine methyl ester occurred at a pH optimum between pH 8.1 and 8.5 with a maximum of only 36 units per mg per min. One mg of the venom mixture liberated 9.3 μM of p nitrophenol from p-nitrophenyl phosphate per minute at an optimum pH between 8.2 and 8.3. Over a wide range of pH, only low phosphodiesterase and 5\u27nucleotidase activities were observed. A trace of caesinolytic activity occurred at pH 9.0

Evaluation and Ranking of Geothermal Resources for Electrical Generation or Electrical Offset in Idaho, Montana, Oregon and Washington. Executive Summary

In 1983, the Bonneville Power Administration contracted for an evaluation and ranking of all geothermal resource sites in the states of Idaho, Montana, Oregon, and Washington which have a potential for electrical generation and/or electrical offset through direct utilization of the resource. The objective of this program was to consolidate and evaluate all geologic, environmental, legal, and institutional information in existing records and files, and to apply a uniform methodology to the evaluation and ranking of all known geothermal sites. This data base would enhance the making of credible forecasts of the supply of geothermal energy which could be available in the region over a 20 year planning horizon. The four states, working together under a cooperative agreement, identified a total of 1,265 potential geothermal sites. The 1,265 sites were screened to eliminate those with little or no chance of providing either electrical generation and/or electrical offset. Two hundred and forty-five of the original 1,265 sites were determined to warrant further study. The Four-State team proceeded to develop a methodology which would rank the sites based upon an estimate of development potential and cost. Development potential was estimated through the use of weighted variables selected to approximate the attributes which a geothermal firm might consider in its selection of a site for exploration and possible development. Resource; engineering; and legal, institutional, and environmental factors were considered. Cost estimates for electrical generation and direct utilization sites were made using the computer programs CENTPLANT, WELLHEAD, and HEATPLAN. Finally, the sites were ranked utilizing a technique which allowed for the integration of development and cost information. On the basis of the developability index, 78 high temperature sites and 120 direct utilization sites were identified as having ''good'' or ''average'' potential for development and should be studied in detail. On the basis of cost, at least 29 of the high temperature sites appear to be technically capable of supporting a minimum total of at least 1,000 MW of electrical generation which could be competitive with the busbar cost of conventional thermal generating technologies. Sixty direct utilization sites have a minimum total energy potential of 900+ MW and can be expected to provide substantial amounts of electrical offset at or below present conventional energy prices. The combined development and economic rankings can be used to assist in determining sites with superior characteristics of both types. Five direct utilization sites and eight high temperature sites were identified with both high development and economic potential. An additional 27 sites were shown to have superior economic characteristics, but development problems. The procedure seems validated by the fact that two of the highest ranking direct utilization sites are ones that have already been developed--Boise, Idaho and Klamath Falls, Oregon. Most of the higher ranking high temperature sites have received serious examination in the past as likely power production candidates

Phosphoproteomic Analyses Reveal Signaling Pathways That Facilitate Lytic Gammaherpesvirus Replication

<div><p>Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication.</p></div

Global phosphoprotein network of MHV68-infected cells.

<p>Interaction-network analyses were performed for all proteins identified in infected data sets using STRING to define a global phosphoprotein network. Standard STRING-defined confidence values of 0.40 were used as cut-offs for putative interactions. All connected nodes were imported into Cytoscape, and specific identifiers were assigned to each node according to MaxQuant values. Solid red denotes detection only in infected cells. Solid blue (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003583#ppat-1003583-g004" target="_blank">Fig. 4</a>) denotes detection only in control cells. Red borders denote increased abundance during infection. Blue borders denote reduced abundance during infection. Black borders indicate no infection-related change in protein intensity. Gene names for each protein are shown. Kinases are depicted as square nodes. Node size corresponds to betweenness centrality, a measure of a particular node's capacity to connect disparate protein clusters within the network. Edge weight corresponds to STRING-defined confidence values, where a thicker line indicates a higher confidence prediction. Nodes highlighted with green, orange, or purple circles represent common nodes in both control (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003583#ppat-1003583-g004" target="_blank">Fig. 4</a>) and infected networks and are provided to facilitate orientation. Subnetworks represent the 6 highest scoring functional protein clusters identified within each global network using MCODE.</p

Overview of integrated approach combining phosphoproteomics, bioinformatics, and biological tests to define signaling events that regulate MHV68 lytic replication.

<p>Overview of integrated approach combining phosphoproteomics, bioinformatics, and biological tests to define signaling events that regulate MHV68 lytic replication.</p

MHV68 D-type cyclin ortholog enhances CDK-related phosphorylation in infected cells.

<p>(A) 3T3 fibroblasts were mock infected or infected with WT or transposon mutant (tn) MHV68 viruses at MOI = 5 PFU/cell. (B) Cells were mock infected or infected with v-cyclin-null (ORF72-null) or genetically repaired WT control (ORF72-MR) MHV68 recombinant viruses. Cells were harvested 18 h post-infection, and proteins were resolved by SDS-PAGE. Immunoblot analyses were performed using antibodies directed against the indicated phosphorylated residues or proteins.</p

Viral phosphopeptides identified through phosphoproteomic analyses.

<p>Viral phosphopeptides identified through phosphoproteomic analyses.</p

Biochemical validation of mass-spectrometry identified infection-related phosphorylation events.

<p>(A) 3T3 fibroblasts were mock-infected or infected with MHV68 at MOI = 5 PFU/cell. Cells were harvested 18 h post-infection, proteins were resolved by SDS-PAGE, and immunoblot analyses were performed using antibodies to detect the indicated phosphorylated or total proteins. (B) 3T3 fibroblasts were mock-infected or infected with MHV68 at MOI = 5 PFU/cell. Cells were harvested 18 h post-infection, and phosphoproteins were immunoprecipitated using antibodies directed against p-Tyr, p-Ser, or p-Thr. Whole cell lysates (1/40^th) or immunoprecipitates were resolved by SDS-PAGE. Immunoblot analyses were performed to detect ORF21 protein or MHV68 lytic antigens. (C) Cells were mock infected in the presence or absence of the antiviral drug cidofovir (Cid.), or infected with WT MHV68, UV-inactivated (UVI) MHV68, ORF50-null MHV68, or WT MHV68 in the presence of cidofovir. Cells were harvested, and immunoblot analyses were performed as in A. (D and E) Mice were intraperitoneally mock-inoculated or inoculated with 10⁶ PFU of H2B-YFP-expressing MHV68. Animals were sacrificed 4 days post-infection, and bulk splenocytes were isolated and processed for flow cytometry to detect H2B-YFP and S73-phosphorylated c-Jun. A representative histogram (D) depicts phospho-S73 c-Jun detection levels in mock-infected (blue), H2B-YFP− cells from infected animals (gray), and MHV68 infected H2B-YFP+ cells (red). The H2B-YFP gating strategy and a comparison to productively-infected fibroblasts is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003583#ppat.1003583.s003" target="_blank">Fig. S3</a>. Data graphed in (E) represent compiled geometric mean fluorescence intensities (GMI) from 3 mock-infected or 4 infected mice.</p