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.     

X-ray structure determination and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus refined at 1.85 A resolution.

The X-ray structure determination, refinement and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus is described. Under identical conditions crystals were obtained in the orthorhombic space group P2(1)2(1)2(1) (form I) and the rhombohedral space group R32 (form II). For both space groups the structures of the protease were solved by molecular replacement and refined at 1.85 A resolution. The final R-factors are 17.9% and 17.1% for form I and form II, respectively. The root-mean-square deviation between the two forms is 0.48 A and 0.86 A for main-chain and side-chain atoms, respectively. Due to differences in crystal lattice contacts and packing, the structures of the two crystal forms differ in intermolecular interaction affecting the local conformation of three flexible polypeptide sequences (Ser50-Glu55, Ser99-Gly102, Gly258-Ser259) at the surface of the protein. While the two overall structures are very similar, the differences are significantly larger than the errors inherent in the structure determination. As expected, the differences in the temperature factors in form I and II are correlated with the solvent accessibility of the corresponding amino acid residues. In form II, two symmetry-related substrate binding sites face each other, forming a tight intermolecular interaction. Some residues contributing to this intermolecular interaction are also found to be involved in the formation of the complex between subtilisin Carlsberg and the proteinaceous inhibitor eglin C. This demonstrates that the two symmetry-related molecules interact with each other at the same molecular surface area that is used for binding of substrates and inhibitors.     

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.     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

GTP plus water mimic ATP in the active site of protein kinase CK2

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs that target CK2 or other kinases with this property.     

Efficient methods for filtering and ranking fragments for the prediction of structurally variable regions in proteins

The prediction of protein 3D structures close to insertions and deletions or, more generally, loop prediction, is still one of the major challenges in homology modeling projects. In this article, we developed ranking criteria and selection filters to improve knowledge-based loop predictions. These criteria were developed and optimized for a test data set containing 678 insertions and deletions. The examples are, in principle, predictable from the used loop database with an RMSD < 1 A and represent realistic modeling situations. Four noncorrelated criteria for the selection of fragments are evaluated. A fast prefilter compares the distance between the anchor groups in the template protein with the stems of the fragments. The RMSD of the anchor groups is used for fitting and ranking of the selected loop candidates. After fitting, repulsive close contacts of loop candidates with the template protein are used for filtering, and fragments with backbone torsion angles, which are unfavorable according to a knowledge-based potential, are eliminated. By the combined application of these filter criteria to the test set, it was possible to increase the percentage of predictions with a global RMSD < 1 A to over 50% among the first five ranks, with average global RMSD values for the first rank candidate that are between 1.3 and 2.2 A for different loop lengths. Compared to other examples described in the literature, our large numbers of test cases are not self-predictions, where loops are placed in a protein after a peptide loop has been cut out, but are attempts to predict structural changes that occur in evolution when a protein is affected by insertions and deletions.     

The Helmholtz Network for Bioinformatics: an integrative web portal for bioinformatics resources

SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de     

Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenulatus

The His-tagged lipase BTL2 from Bacillus thermocatenulatus was expressed in Escherichia coli and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography. The success of protein separation and purification was pH-dependent and increased with decreasing pH. The purified BTL2 lipase showed a strong tendency to aggregate upon concentration, which prevented a reproducible crystallization. Aggregation studies using dynamic light-scattering (DLS) analysis were performed to improve the purification and concentration of BTL2 lipase. Different chemical classes of additives were tested to manipulate the aggregation behaviour of BTL2 lipase with the aim of obtaining a monodisperse sample to use for crystallization. For the process of concentration of BTL2 lipase in monomeric form, the alcohol 2-propanol and the ionic detergent dodecyl dimethylamine-N-oxide (LDAO) were found to be necessary. For the concentrated lipase, the availability of 5% 2-propanol was sufficient to hold the lipase in monomeric form and no additional detergent was needed.