Investigation of the ß-sheet interactions between dHP1 chromodomain and histone 3.

Methylated lysine 9 on the histone 3 (H3) tail recruits heterochromatin protein 1 from Drosophila (dHP1) via its chromodomain and results in gene silencing. The dHP1 chromodomain binds H3 K9Me3 with an aromatic cage surrounding the trimethyllysine. The sequence selectivity of binding comes from insertion of the histone tail between two ß-strands of the chromodomain to form a three-stranded ß-sheet. Herein, we investigated the sequence selectivity provided by the ß-sheet interactions and how those interactions compare to other model systems. Residue Thr6 of the histone tail forms cross-strand interactions with Ala25 and Asp62 of the chromodomain. Each of these three residues was substituted for amino acids known to have high ß-sheet propensities and/or to form favorable side chain-side chain (SC-SC) interactions in ß-sheets, including hydrophobic, H-bonding, and aromatic interactions. We found that about 50% of the chromodomain mutants resulted in equal or tighter binding to the histone tail and about 25% of the histone tail mutants provided tighter binding compared to that of the native histone tail sequence. These studies provide novel insights into the sequence selectivity of the dHP1 chromodomain for the histone tail and relates the information gleaned from model systems and statistical studies to ß-sheet-mediated protein-protein interactions. Moreover, this work suggests that the development of designer histone-chromodomain pairs for chemical biology applications is feasible.

Effect of the size of evacuated blood collection tubes on total carbon dioxide concentration in equine plasma.

OBJECTIVE: To determine whether plasma total CO(2) concentrations would vary with the size of the evacuated tube used to collect blood samples. DESIGN: Randomized crossover study. ANIMALS: Convenience sample of 20 healthy adult horses. PROCEDURES: Jugular venous blood was collected from horses in random order into 8 types of evacuated tubes: 2-mL glass, 2- or 3-mL plastic or plastic plasma separator, 4- or 6-mL plastic, and 10-mL glass or plastic. Total CO(2) concentrations in plasma were measured with a biochemistry analyzer. Data were analyzed via repeated-measures ANOVA and multivariate regression. RESULTS: The air volume-to-blood volume ratio was significantly higher and consequently, plasma total CO(2) concentration was significantly lower when blood was collected into 2-mL glass tubes and 2- or 3-mL plastic tubes than when the other 5 types of evacuated tubes were used. Concentrations in the other tube types were statistically equivalent. A linear relationship was detected between total CO(2) concentration and air volume-to-blood volume ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Blood samples should be collected into evacuated tubes with a small air volume-to-blood volume ratio whenever an accurate estimate of plasma total CO(2) concentration is required.

Effects of syringe type and storage conditions on results of equine blood gas and acid-base analysis.

OBJECTIVE: To determine effects of syringe type and storage conditions on blood gas and acid-base values for equine blood samples. SAMPLE: Blood samples obtained from 8 healthy horses. PROCEDURES: Heparinized jugular venous blood was equilibrated via a tonometer at 37°C with 12% O(2) and 5% CO(2). Aliquots (3 mL) of tonometer-equilibrated blood were collected in random order by use of a glass syringe (GS), general-purpose polypropylene syringe (GPPS), or polypropylene syringe designed for blood gas analysis (PSBGA) and stored in ice water (0°C) or at room temperature (22°C) for 0, 5, 15, 30, 60, or 120 minutes. Blood pH was measured, and blood gas analysis was performed; data were analyzed by use of multivariable regression analysis. RESULTS: Blood Po(2) remained constant for the reference method (GS stored at 0°C) but decreased linearly at a rate of 7.3 mm Hg/h when stored in a GS at 22°C. In contrast, Po(2) increased when blood was stored at 0°C in a GPPS and PSBGA or at 22°C in a GPPS; however, Po(2) did not change when blood was stored at 22°C in a PSBGA. Calculated values for plasma concentration of HCO(3) and total CO(2) concentration remained constant in the 3 syringe types when blood was stored at 22°C for 2 hours but increased when blood was stored in a GS or GPPS at 0°C. CONCLUSIONS AND CLINICAL RELEVANCE: Blood samples for blood gas and acid-base analysis should be collected into a GS and stored at 0°C or collected into a PSBGA and stored at room temperature.

Crystal structure of the HEAT domain from the Pre-mRNA processing factor Symplekin.

The majority of eukaryotic pre-mRNAs are processed by 3'-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3'-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the approximately 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 A resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3'-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.

A novel fold in the TraI relaxase-helicase c-terminal domain is essential for conjugative DNA transfer.

TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-A resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal alpha-domain connected by a proline-rich loop to a compact alpha/beta-domain. Both the globular nature of the alpha/beta-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

Expression of the serine protease inhibitor neuroserpin in cells of the human myeloid lineage.

Myeloid progenitors in the bone marrow differentiate into most of the major cell types of the immune system, including macrophages and dendritic cells. These cells play important roles in both innate and adaptive immunity. They express a number of proteases and protease inhibitors including members of the serine proteinase inhibitor or serpin superfamily. In this study we report the differential expression of neuroserpin in cells of the human myeloid lineage. Neuroserpin was highly expressed and secreted following the differentiation of monocytes to macrophages and dendritic cells. Activation of dendritic cells with lipopolysaccharide resulted in increased neuroserpin mRNA levels but no neuroserpin secretion. Confocal immunofluorescence microscopy showed neuroserpin was differentially localised in human myeloid cells. In macrophages and dendritic cells it was concentrated in vesicles located in close proximity to the plasma membrane. The majority of activated dendritic cells also exhibited an intracellular focal concentration of neuroserpin which co-localised with the lysosomal/late endosomal marker LAMP-1. As neuroserpin inhibits tissue plasminogen activator, a comparative analysis of tPA and plasminogen activator inhibitor-1 (PAI-1) expression was undertaken. This analysis revealed differential expression of PAI-1 and neuroserpin suggesting they may have different functions in human immune cells.