Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.     

Structural studies on phospho-CDK2/cyclin A bound to nitrate,a transition state analogue: implications for the protein kinase mechanism

Eukaryotic protein kinases catalyze the phosphoryl transfer of the gamma-phosphate of ATP to the serine, threonine, or tyrosine residue of protein substrates. The catalytic mechanism of phospho-CDK2/cyclin A (pCDK2/cyclin A) has been probed with structural and kinetic studies using the trigonal NO(3)(-) ion, which can be viewed as a mimic of the metaphosphate transition state. The crystal structure of pCDK2/cyclin A in complex with Mg(2+)ADP, nitrate, and a heptapeptide substrate has been determined at 2.7 A. The nitrate ion is located between the beta-phosphate of ADP and the hydroxyl group of the serine residue of the substrate. In one molecule of the asymmetric unit, the nitrate is close to the beta-phosphate of ADP (distance from the nitrate nitrogen to the nearest beta-phosphate oxygen of 2.5 A), while in the other subunit, the nitrate is closer to the substrate serine (distance of 2.1 A). Kinetic studies demonstrate that nitrate is not an effective inhibitor of protein kinases, consistent with the structural results that show the nitrate ion makes few stabilizing interactions with CDK2 at the catalytic site. The binding of orthovanadate was also investigated as a mimic of a pentavalent phosphorane intermediate of an associative mechanism for phosphoryl transfer. No vanadate was observed bound in a 3.4 A resolution structure of pCDK2/cyclin A in the presence of Mg(2+)ADP, and vanadate did not inhibit the kinase reaction. The results support the notion that the protein kinase reaction proceeds through a mostly dissociative mechanism with a trigonal planar metaphosphate intermediate rather than an associative mechanism that involves a pentavalent phosphorane intermediate.     

Automated Bayesian model development for frequency detection in biological time series

Background

The ability to perform quantitative studies using isotope tracers and metabolic flux analysis (MFA) is critical for detecting pathway bottlenecks and elucidating network regulation in biological systems, especially those that have been engineered to alter their native metabolic capacities. Mathematically, MFA models are traditionally formulated using separate state variables for reaction fluxes and isotopomer abundances. Analysis of isotope labeling experiments using this set of variables results in a non-convex optimization problem that suffers from both implementation complexity and convergence problems.

Results

This article addresses the mathematical and computational formulation of ¹³C MFA models using a new set of variables referred to as fluxomers. These composite variables combine both fluxes and isotopomer abundances, which results in a simply-posed formulation and an improved error model that is insensitive to isotopomer measurement normalization. A powerful fluxomer iterative algorithm (FIA) is developed and applied to solve the MFA optimization problem. For moderate-sized networks, the algorithm is shown to outperform the commonly used 13CFLUX cumomer-based algorithm and the more recently introduced OpenFLUX software that relies upon an elementary metabolite unit (EMU) network decomposition, both in terms of convergence time and output variability.

Conclusions

Substantial improvements in convergence time and statistical quality of results can be achieved by applying fluxomer variables and the FIA algorithm to compute best-fit solutions to MFA models. We expect that the fluxomer formulation will provide a more suitable basis for future algorithms that analyze very large scale networks and design optimal isotope labeling experiments.     

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.     

Identification and characterization of 17 microsatellite primers for the tick,Ixodes ricinus,using enriched genomic libraries

Ixodes ricinus is the main vector for important infectious diseases in both humans and in animals. Microsatellite loci were isolated from a dinucleotide‐enriched library made from I. ricinus sampled in Norway. Seventeen polymorphic microsatellites were further characterized among 24 individuals sampled from an island in the Oslofjord region. The number of observed alleles ranged from two to 17 and the observed heterozygosities between 0.10 and 0.83. Analysis of family materials gives evidence of non‐Mendelian inheritance of several of the characterized loci, among which most could be explained by presence of null alleles.     

First Record of Teratopactus nodicollis (Coleoptera: Curculionidae) in Dry Bean (Phaseolus vulgaris)

Observations on the bioecology and damage of Teratopactus nodicollis Boheman on Phaseolus vulgaris were carried out on field samples by assessing the number of larvae and root damage in 40?ha of a dry bean field from the Federal District, Brazil (16°4??28.41???W; 47°30??21.13???S). Larvae caused the greatest damage at the stage of germination, emergence, and primary leaves, producing 50?% stand reduction. Most larvae pupated in August and September, and adult emergence occurred in middle October. Some larvae were infected with the fungus Metarhizium spp., a biological agent that would be naturally controlling this insect.