Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica

Abstract A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly ( Musca domestica ) as well as other Diptera and Lepidoptera . Crystal δ-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 μg/ml, and β-exotoxin was not detected. Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination. Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cry IA(b), one cry IIA, and two cry IIB genes.     

Kinetics of membrane lysis by custom lytic peptides and peptide orientations in membrane.

To aid the development of custom peptide antibiotics, a kinetic study of membrane lysis by cecropin B (CB) and its analogs, cecropin B1 (CB1) and cecropin B3 (CB3) was carried out to determine the mechanism by which these peptides disrupt the bilayer structure of liposomes of defined composition. Disruption of the phospholipid bilayer was determined by a fluorescence assay involving the use of dithionite to quench the fluorescence of lipids labeled with N-7-nitro-2,1,3-benzoxadiazol-4-yl. Lytic peptides caused the disruption of liposomes to occur in two kinetic steps. For liposomes composed of mixtures of phosphatidylcholine and phosphatidic acid, the time constants for each kinetic step were shorter for CB and CB1 than for CB3. Oriented circular dichroism experiments showed that the peptides could exist in at least two different membrane-associated states that differed primarily in the orientation of the helical segments with respect to the bilayer surface. The results are discussed in terms of kinetic mechanisms of membrane lysis. The mode of actions of these peptides used for the interpretation of their kinetic mechanisms were supported by surface plasmon resonance experiments including or excluding the pore-forming activities.     

Quantification of tartrate resistant acid phosphatase distribution in mouse tibiae using image analysis

Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm² region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p>0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r²=0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.     

Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis

A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila. This strain of H. vermiformis, designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.     

Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.

Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.     

Structure at 2.5-A resolution of chemically synthesized human immunodeficiency virus type 1 protease complexed with a hydroxyethylene-based inhibitor

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.     

Extreme weather‐year sequences have nonadditive effects on environmental nitrogen losses

The frequency and intensity of extreme weather years, characterized by abnormal precipitation and temperature, are increasing. In isolation, these years have disproportionately large effects on environmental N losses. However, the sequence of extreme weather years (e.g., wet–dry vs. dry–wet) may affect cumulative N losses. We calibrated and validated the DAYCENT ecosystem process model with a comprehensive set of biogeophysical measurements from a corn–soybean rotation managed at three N fertilizer inputs with and without a winter cover crop in Iowa, USA. Our objectives were to determine: (i) how 2‐year sequences of extreme weather affect 2‐year cumulative N losses across the crop rotation, and (ii) if N fertilizer management and the inclusion of a winter cover crop between corn and soybean mitigate the effect of extreme weather on N losses. Using historical weather (1951–2013), we created nine 2‐year scenarios with all possible combinations of the driest (“dry”), wettest (“wet”), and average (“normal”) weather years. We analyzed the effects of these scenarios following several consecutive years of relatively normal weather. Compared with the normal–normal 2‐year weather scenario, 2‐year extreme weather scenarios affected 2‐year cumulative NO₃^? leaching (range: ?93 to +290%) more than N₂O emissions (range: ?49 to +18%). The 2‐year weather scenarios had nonadditive effects on N losses: compared with the normal–normal scenario, the dry–wet sequence decreased 2‐year cumulative N₂O emissions while the wet–dry sequence increased 2‐year cumulative N₂O emissions. Although dry weather decreased NO₃^? leaching and N₂O emissions in isolation, 2‐year cumulative N losses from the wet–dry scenario were greater than the dry–wet scenario. Cover crops reduced the effects of extreme weather on NO₃^? leaching but had a lesser effect on N₂O emissions. As the frequency of extreme weather is expected to increase, these data suggest that the sequence of interannual weather patterns can be used to develop short‐term mitigation strategies that manipulate N fertilizer and crop rotation to maximize crop N uptake while reducing environmental N losses.