The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications

Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitination apparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3 enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry of its active site cysteine residue since this residue must form a thioester bond with the carboxyl terminus of ubiquitin. Despite the plethora of structural information that is available, there has been a notable dearth of information regarding the chemical basis of E2 function. Toward filling this large void in our understanding of E2 function, we have examined the pK(a) of the active site cysteine using a combination of experimental and theoretical approaches. We find, remarkably, that the pK(a) of the active site cysteine residue is elevated by approximately 2 pH units above that of a free cysteine. We have identified residues that contribute to the increase in this pK(a). On the basis of experimental values obtained with three different E2 proteins, we believe this to be a general and important characteristic of E2 protein chemistry. Sequence comparison suggests that the electrostatic environment is maintained not through strict residue conservation but through different combinations of residues near the active site. We propose that the elevated pK(a) is a regulatory mechanism that prevents the highly exposed cysteine residue in free E2 from reacting promiscuously with electron deficient chemical moieties in the cell.     

A dynamic model of thoracic differentiation for the control of turning in the stick insect

Leg movements of stick insects (Carausius morosus) making turns towards visual targets are examined in detail, and a dynamic model of this behaviour is proposed. Initial results suggest that front legs shape most of the body trajectory, while the middle and hind legs just follow external forces (Rosano H, Webb B, in The control of turning in real and simulated stick insects, vol. 4095, pp 145–156, 2006). However, some limitations of this explanation and dissimilarities in the turning behaviour of the insect and the model were found. A second set of behavioural experiments was made by blocking front tarsi to further investigate the active role of the other legs for the control of turning. The results indicate that it is necessary to have different roles for each pair of legs to replicate insect behaviour. We demonstrate that the rear legs actively rotate the body while the middle legs move sideways tangentially to the hind inner leg. Furthermore, we show that on average the middle inner and hind outer leg contribute to turning while the middle outer leg and hind inner leg oppose body rotation. These behavioural results are incorporated into a 3D dynamic robot simulation. We show that the simulation can now replicate more precisely the turns made by the stick insect. This work was supported by CONACYT México and the European Commission under project FP6-2003-IST2-004690 SPARK.     

Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure–mutagenicity relationships for arylamine metabolites

The structure–activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure–mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.     

LIMaS: the JAVA-based application and database for microarray experiment tracking

Microarrays allow monitoring of gene expression for tens of thousands of genes in parallel and are being used routinely to generate huge amounts of valuable data. Handling and analysis of such data are becoming major bottlenecks in the utilization of the technology. To enable the researcher to interpret the results postanalysis, we have developed a laboratory information management system for microarrays (LIMaS) with an n-tier Java front-end and relational database to record and manage large-scale expression data preanalysis. This system enables the laboratory to replace the paper trail with an efficient and fully customizable interface giving it the ability to adapt to any working practice, e.g., handling many resources used to form many products (chaining of resources). The ability to define sets of activities, resources, and workflows makes LIMaS MIAME-supportive.LIMaS is available for download at (http://www.mgu.har.mrc.ac.uk/microarray.)     

Bacterial RNA isolation with one hour centrifugation in a table-top ultracentrifuge.

A procedure for the rapid preparation of cesium-chloride purified RNA from E. coli and the cyanobacterium Synechococcus sp. PCC7942 is described. Cells are lysed in modified sucrose, Triton X-100, EDTA, Tris buffer with phenol/chloroform. The cleared lysate is extracted further with phenol/chloroform and RNA is peleted by centrifugation through a 5.7 M CsCl cushion. High quality RNA can be prepared in three hours using this procedure.     

Macro shocks and micro outcomes: child nutrition during Indonesia’s crisis

A survey of households in rural Java is used to assess the nutritional impact of Indonesia's drought and financial crisis of 1997/1998. A time-age-cohort decomposition reveals significant nutritional impacts. However, child weight-for-age (WAZ) remained constant throughout the crisis, despite rapid increases in food prices and the consequent household consumption shock. The evidence is consistent with the hypothesis that within households, mothers buffered children's caloric intake, resulting in increased maternal wasting. However, reductions in the consumption of high-quality foods further resulted in increased prevalence of anemia for both mothers and children. The combined effects were particularly severe for cohorts conceived and weaned during the crisis.     

Patterns of Protozoan Infections: Spatiotemporal Associations with Cattle Density

Waste from cattle production contains protozoa, such as Cryptosporidium spp. and Giardia, which can be transmitted to humans. People residing in areas of high cattle density may be at increased risk for protozoan infections. The objective of this study was to assess spatial and temporal associations between cattle density and hospitalizations for protozoan infections in the U.S. elderly. Data on protozoan infections were abstracted from Centers for Medicare and Medicaid Services datasets for a 14-year period (1991–2004). Cattle inventory data were abstracted from the 2002 U.S. Census of Agriculture. Counties were classified into one of five exposure categories based on both cattle density and human density. Our analyses considered differences in rates, trends, and variations in seasonal patterns based on exposure categories. Cryptosporidiosis demonstrated a trend of increasing annual rates related to increased potential exposure to cattle. Both cryptosporidiosis and giardiasis demonstrated significant seasonal patterns peaking during the fourth week of October in areas of high cattle/low population density and the second week of September in counties with low cattle/low human density, respectively. Counties with low human population density (regardless of cattle density) had the highest rate of all protozoan infections, peaking in the summer. These results demonstrate the elderly population is at increased risk of protozoan infections in areas of high cattle density, particularly cryptosporidiosis. The seasonal patterns and higher annual rates seen in rural areas suggest time-variant environmental exposures, which may be affected with geographical and temporal targeting of agricultural policies and interventions to improve public health.