Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).

Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.     

Revealing the dynamics of the 20 S proteasome phosphoproteome: a combined CID and electron transfer dissociation approach

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO(2)), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential beta5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (beta1 caspase-like, beta2 trypsin-like, and beta5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.     

Cumulative Risk of Bovine Mastitis Treatments in Denmark,Finland, Norway and Sweden

Data from the national dairy cow recording systems during 1997 were used to calculate lactation-specific cumulative risk of mastitis treatments and cumulative risk of removal from the herds in Denmark, Finland Norway and Sweden. Sweden had the lowest risk of recorded mastitis treatments during 305 days of lactation and Norway had the highest risk. The incidence risk of recorded mastitis treatments during 305 days of lactation in Denmark, Finland, Norway and Sweden was 0.177, 0.139, 0.215 and 0.127 for first parity cows and 0.228, 0.215, 0.358 and 0.204 for parities higher than three, respectively. The risk of a first parity cow being treated for mastitis was almost 3 times higher at calving in Norway than in Sweden. The period with the highest risk for mastitis treatments was from 2 days before calving until 14 days after calving and the highest risk for removal was from calving to 10 days after calving in all countries.The study clearly demonstrated differences in bovine mastitis treatment patterns among the Nordic countries. The most important findings were the differences in treatment risks during different lactations within each country, as well as differences in strategies with respect to the time during lactation mastitis was treated.     

Physiological susceptibility of the predator Macrolophus basicornis (Hemiptera: Miridae) to pesticides used to control of Tuta absoluta (Lepidoptera: Gelechiidae)

In order to aid the integration of biological and chemical controls for the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), this study evaluated the relative toxicity of five insecticides to the leaf miner predator Macrolophus basicornis (Stal) (Hemiptera: Miridae). The insecticides evaluated were teflubenzuron, abamectin, chlorantraniliprole, chlorfenapyr, and cartap hydrochloride, all of which are recommended for control of T. absoluta in Brazil. Nymphs and adults of M. basicornis were exposed to tomato leaves treated with the insecticides, under laboratory and greenhouse conditions. The overall mortality caused by the products in both situations was recorded, and the survival of congeneric groups was analysed using the Weibull model. The persistence of the insecticides was also evaluated and they were categorised into toxicity classes proposed by the International Organisation for Biological Control (IOBC) based on predator mortality and persistence. Abamectin and chlorfenapyr were toxic to M. basicornis nymphs and adults in all bioassays. Cartap hydrochloride was slightly harmful to adults in laboratory assays, but harmful to nymphs, and moderately harmful under greenhouse conditions. Chlorantraniliprole and teflubenzuron were harmless in most assays, except when nymphs were exposed in the laboratory, where they were moderately and slightly harmful, respectively. Chlorantraniliprole and teflubenzuron should be preferred insecticides for use in tomato leaf miner IPM programmes that aim to conserve M. basicornis populations.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes

Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.     

Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.     

Phase II protocol for the evaluation of new treatments in patients with advanced gastric carcinoma: results of ECOG 5282

The study was a Phase II randomized study to evaluate the efficacy of new agents for the treatment of advanced gastric carcinoma. Patients were randomized to receive single agent chemotherapy with mitoxantrone, etoposide, aclacinomycin-A or spirogermanium. The patients were stratified by prior use of chemotherapy, prior doxorubicin use and ECOG performance status. Patients with a history of cardiac disease or prior doxorubicin exceeding a dose of 400 mg/m² were restrictively randomized to sopirogermanium or etoposide only. One hundred and fourteen patients were registered for the study. Among 98 evaluable patients there were only two partial responses (both in the etoposide arm), and one complete response in the mitoxantrone arm. The median survival on the study was 3.3 months. One hundred and six patients were analyzable for toxicity. There were four treatment-related deaths and four life-threatening toxicities. Because of low response rates and relatively high toxicities the studied compounds were not deemed worth further investigation for advanced gastric cancer.