Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation.

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analogue of histidine with a significantly reduced pK(a) ( approximately 1), into PA and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged.

Gene duplication and environmental adaptation within yeast populations.

Population-level differences in the number of copies of genes resulting from gene duplication and loss have recently been recognized as an important source of variation in eukaryotes. However, except for a small number of cases, the phenotypic effects of this variation are unknown. Data from the Saccharomyces Genome Resequencing Project permit the study of duplication in genome sequences from a set of individuals within the same population.

Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3).

Structural, NMR spectroscopic, and computational investigation of hemin loading in the hemophore HasAp from Pseudomonas aeruginosa.

When challenged by low-iron conditions several Gram-negative pathogens secrete a hemophore (HasA) to scavenge hemin from its host and deliver it to a receptor (HasR) on their outer membrane for internalization. Here we report results from studies aimed at probing the structural and dynamic processes at play in the loading of the apo-hemophore secreted by P. aeruginosa (apo-HasAp) with hemin.

An integrated view of molecular coevolution in protein-protein interactions.

Protein-protein interactions effectively mediate molecular function. They are the result of specific interactions between protein interfaces and are maintained by the action of evolutionary pressure on the regions of the interacting proteins that contribute to binding. For the most part, selection restricts amino acid replacements, accounting for the conservation of binding interfaces.

A dose-escalation phase I trial of nimotuzumab, an antibody against the epidermal growth factor receptor, in patients with advanced solid malignancies.

Purpose: Nimotuzumab is a humanized monoclonal antibody which inhibits the ligand-dependent activation of epidermal growth factor receptor (EGFR). We conducted a phase I trial to assess the pharmacodynamic (PD) effects of escalating doses of nimotuzumab administered alone in patients with advanced solid cancers patients.

Experimental Design: Patients were treated with escalating doses of weekly intravenous nimotuzumab at doses ranging between 100 and 800 mg.

Interactions between Kar2p and its nucleotide exchange factors Sil1p and Lhs1p are mechanistically distinct.

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p.

Structural studies of bacterioferritin B from Pseudomonas aeruginosa suggest a gating mechanism for iron uptake via the ferroxidase center .

The structure of recombinant Pseudomonas aeruginosa bacterioferritin B (Pa BfrB) has been determined from crystals grown from protein devoid of core mineral iron (as-isolated) and from protein mineralized with approximately 600 iron atoms (mineralized). Structures were also obtained from crystals grown from mineralized BfrB after they had been soaked in an FeSO(4) solution (Fe soak) and in separate experiments after they had been soaked in an FeSO(4) solution followed by a soak in a crystallization solution (double soak). Although the structures consist of a typical bacterioferritin fold comprised of a nearly spherical 24-mer assembly that binds 12 heme molecules, comparison of microenvironments observed in the distinct structures provided interesting insights.

Correlation of microsynteny conservation and disease gene distribution in mammalian genomes.

Background: With the completion of the whole genome sequence for many organisms, investigations into genomic structure have revealed that gene distribution is variable, and that genes with similar function or expression are located within clusters. This clustering suggests that there are evolutionary constraints that determine genome architecture. However, as most of the evidence for constraints on genome evolution comes from studies on yeast, it is unclear how much of this prior work can be extrapolated to mammalian genomes.

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH.

Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 A crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein.

Analysis of synthetic lethality reveals genetic interactions between the GTPase Snu114p and snRNAs in the catalytic core of the Saccharomyces cerevisiae spliceosome.

Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are regulated by eight ATPases and one GTPase Snu114p. The Snu114p guanine state regulates U4/U6 unwinding during spliceosome activation and U2/U6 unwinding during spliceosome disassembly through the ATPase Brr2p. We investigated 618 genetic interactions to identify an extensive genetic interaction network between SNU114 and snRNAs.

Evolution in protein interaction networks: co-evolution, rewiring and the role of duplication.

Molecular function is the result of proteins working together, mediated by highly specific interactions. Maintenance and change of protein interactions can thus be considered one of the main links between molecular function and mutation. As a consequence, protein interaction datasets can be used to study functional evolution directly.

Binding of Pseudomonas aeruginosa apobacterioferritin-associated ferredoxin to bacterioferritin B promotes heme mediation of electron delivery and mobilization of core mineral iron.

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues.

Quantitative analysis and prediction of curvature in leucine-rich repeat proteins.

Leucine-rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein-protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature.

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer.

Background: With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript.

Results: In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer.

Artefacts and biases affecting the evaluation of scoring functions on decoy sets for protein structure prediction.

Motivation: Decoy datasets, consisting of a solved protein structure and numerous alternative native-like structures, are in common use for the evaluation of scoring functions in protein structure prediction. Several pitfalls with the use of these datasets have been identified in the literature, as well as useful guidelines for generating more effective decoy datasets. We contribute to this ongoing discussion an empirical assessment of several decoy datasets commonly used in experimental studies.

The effect of sequence evolution on protein structural divergence.

The complex constraints imposed by protein structure and function result in varied rates of sequence and structural divergence in proteins. Analysis of sequence differences between homologous proteins can advance our understanding of structural divergence and some of the constraints that govern the evolution of these molecules. Here, we assess the relationship between amino acid sequence and structural divergence.

Comparing the full-threshold and Swedish interactive thresholding algorithms for short-wavelength automated perimetry.

Purpose: To compare the Swedish interactive thresholding algorithm (SITA) with the full-threshold (FT) strategy for short-wavelength automated perimetry (SWAP).

Methods: One eye of 286 patients with glaucomatous optic neuropathy (GON) and 289 age-matched participants without GON from the Diagnostic Innovations in Glaucoma Study (DIGS) and the African Descent and Glaucoma Evaluation Study (ADAGES) were classified with optic disc stereophotographs taken within 6 months of visual field testing, conducted within a 3-month period. Six parameters were derived per test, including pattern standard deviation (PSD) and the number of pattern deviation plot (PDP) points triggered at <1%.

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS).

The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments.

Tiny stowaways: analyzing the economic benefits of a U.S. Environmental Protection Agency permit regulating ballast water discharges.

The U.S. Environmental Protection Agency has proposed permitting ballast water discharges--a benefit of which would be to reduce the economic damages associated with the introduction and spread of aquatic invasive species.

Phosphate coordination and movement of DNA in the Tn5 synaptic complex: role of the (R)YREK motif.

Bacterial DNA transposition is an important model system for studying DNA recombination events such as HIV-1 DNA integration and RAG-1-mediated V(D)J recombination. This communication focuses on the role of protein-phosphate contacts in manipulating DNA structure as a requirement for transposition catalysis. In particular, the participation of the nontransferred strand (NTS) 5' phosphate in Tn5 transposition strand transfer is analyzed.

What keeps some program directors in their, positions so long? An examination of job longevity among anesthesia program directors.

The turnover rate of nurse anesthesia faculty has been monitored by the Council on Accreditation of Nurse Anesthesia Educational Programs for a number of years. In 2005, the turnover rate remained high, at 22%, indicating a problem with faculty retention. This article reports the results of a study in which 25 long-time nurse anesthesia program directors were interviewed to gain insights into faculty retention.

Services for seniors in small-town Canada: the paradox of community.

There is a prevailing argument that what small towns lack in formal services they make up for in close ties among rural people and a shared understanding of the notion of community. Drawing on research undertaken in 9 small towns across Canada, the authors examine how the concept of community operates with respect to the provision ofin-home and community care for seniors. The analysis is based on interviews with 55 key informants from local governments, health and social care agencies, voluntary sector organizations, and community groups.