In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

Summary We investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10^–4 M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophage's tumoricidal properties generated by liposome-encapsulated FMLP.     

Purification and characterization of 3 alpha-hydroxysteroid dehydrogenase from chicken hepatic cytosol

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 3 alpha-hydroxysteroid dehydrogenase was purified to an apparently homogeneous state by differential precipitation with ammonium sulfate, followed by column chromatographies with DE 51, DEAE-Toyopearl, and Sephadex G-100. Finally the dehydrogenase was purified 103-fold on the basis of the cytosol fraction. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS) revealed that molecular weight of the purified enzyme was 66 kDa, while that of the native dehydrogenase in the absence of SDS was estimated as 660 kDa or more from the peak of the enzyme in elution profile from Sephacryl S-200 column chromatography. The dehydrogenase required NADPH specifically for reduction of 3-oxo group of 5 beta-androstanedione (Km = 1.6 microM). Optimal temperature for 3-oxo reduction was 50 C in incubation for 10 min.     

Nicotiana suaveolens accessions with different ploidy levels exhibit different reproductive isolation mechanisms in interspecific crosses with Nicotiana tabacum

Journal of Plant Research - Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit...     

High-performance liquid chromatography with ultraviolet detection for real-time therapeutic drug monitoring of meropenem in plasma

A simple, rapid, and precise high-performance liquid chromatography (HPLC) method using ultrafiltration to remove plasma protein was developed to determine meropenem concentrations in human plasma in a clinical setting. Plasma was separated by centrifugation at 4 degrees C from blood collected in heparinized vacuum tubes, and meropenem was stabilized by immediately mixing the plasma with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Ultrafiltration was used for plasma deproteinization. Meropenem was detected by ultraviolet absorbance at 300 nm with no interfering plasma peak. The calibration curve of meropenem in human plasma was linear from 0.05 to 100 microg/mL. Intraday and interday precision was less than 7.17% (CV), and accuracy was between 97.7% and 106.3% over 0.05 microg/mL. The limit of detection was 0.01 microg/mL. The assay has been clinically applied to a real-time therapeutic drug monitoring in pediatric patients and pharmacokinetic studies.     

Visualization of microtubule-mediated transport of influenza viral progeny ribonucleoprotein

We developed a unique monoclonal antibody, mAb61A5, using the nucleoprotein (NP) of influenza virus A/Puerto Rico/8/34 (PR8) strain. Truncation and alanine substitution experiments showed that mAb61A5 recognized the NP fragment with residues 17 to 123 in which a conformational epitope formed by the beta1 sheet and the linker region between the alpha1 and alpha2 helices. Variations in the epitope or nearby can partly account for the poor mAb61A5 reactivity with the NP of A/Aichi/2/68 or A/duck/Pennsylvania/10128/84 strains. Interestingly, immunoprecipitation analysis revealed that mAb61A5 preferentially interacted with viral ribonucleoprotein complexes, composed of RNA polymerase, negative/positive sense RNA and NP, rather than exogenously added NP. Immunofluorescence microscopy using mAb61A5 showed a punctate staining in the cytoplasm during the late phase of infection. The punctate NPs accumulated at the microtubule organizing center and co-localized with microtubules. The treatment with leptomycin B to block a CRM1-dependent nuclear export failed to produce the punctate NP. The treatment with nocodazole, a microtubule-depolymerizing agent, showed random distribution of the punctate NP in the cytoplasm. These results suggest that microtubule networks, although were not required for the formation of punctate structures, were responsible for the polarized distribution of the punctate NP antigens, most likely viral progeny ribonucleoprotein complexes.     

Structure-function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf‐type (Nii1 and Nii3), and root‐type (Nii2 and Nii4). The high‐resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3‐M175G, Nii3‐M175E, and Nii3‐M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3‐Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild‐type (Nii3‐WT) and the Met175 variants showed that the hydrophobic side‐chain of Met175 facilitated enzyme efficiency (k_cat/K_m). The Nii4‐WT has Lys449 at the equivalent position of Gln448 in Nii3‐WT. The enzyme activity assay revealed that the turnover number (k_cat) and Michaelis constant (K_m) of Nii4‐WT were lower than those of Nii3‐WT. However, the k_cat/K_m of Nii4‐WT was about 1.4 times higher than that of Nii3‐WT. A comparison of the kinetics of the Nii3‐Q448K and Nii4‐K449Q variants revealed that the change in k_cat/K_m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low‐affinity and Nii4 is the high‐affinity aNiR.     

Structure-specific DNA nucleases: structural basis for 3D-scissors

Structure-specific DNA nucleases play important roles in various DNA transactions such as DNA replication, repair and recombination. These enzymes recognize loops and branched DNA structures. Recent structural studies have provided detailed insights into the functions of these enzymes. Structures of Holliday junction resolvase revealed that nucleases are broadly diverged in the way in which they fold, however, are required to form homodimers with large basic patches of protein surfaces, which are complementary to DNA tertiary structures. Many nucleases maintain structure-specific recognition modes, which involve particular domain arrangements through conformal changes of flexible loops or have a separate DNA binding domain. Nucleases, such as FEN-1 and archaeal XPF, are bound to proliferating cell nuclear antigen through a common motif, and thereby actualize their inherent activities.