Assessing soil microbial populations responding to crude-oil amendment at different temperatures using phylogenetic, functional gene (alkB) and physiological analyses

The effect of temperature as a determinant for selecting microbial populations associated with alkane-degradation was examined in crude oil-amended soil microcosms. After a 30-day incubation, >95% of n-alkane components in the crude-oil were depleted and approximately 40 and 60% of added [14C] hexadecane was converted to 14CO2 at 4-10 and 25 degrees C, respectively. Concomitant with crude-oil depletion, 16S rRNA gene sequence analysis revealed the emergence of a prominent Rhodococcus-like 16S rRNA sequence at all temperatures and a prominent Pseudomonas-like sequence at 4 and 10 degrees C. The diversity of alkane hydroxylase genes (alkB) associated with the amendments was examined using group-specific alkB-PCR primerstargeting phylogenetically distinct groups of alkane-degrading bacteria and subsequent cloning, denaturing gradient gel electrophoresis and sequencing analyses. Diverse Rhodococcus-alkB genes were detected at all temperatures, while a single prominent Pseudomonas-alkB genotype was detected only at lower temperatures. Two isolates obtained from the microcosms were shown to have 16S rRNA and alkB genes identical to those observed and were used to examine growth as a function of temperature. The Pseudomonas isolate exhibited a substantially higher growth rate at 4 and 10 degrees C than the Rhodococcus isolate, consistent with the inference that differences in adaptation to low temperature explain the observed shift in populations. High resolution analysis of alkB genes enabled the differentiation of distinct alkane-degrading populations responding to crude-oil amendment from other closely related, well-studied strains with different temperature adaptations.     

Use of appropriate formulas for selecting WAIS—R short forms.

Demonstrated that the simultaneous consideration of both validity and reliability in selecting the best short forms (SFs) of the WAIS—R may yield findings different from earlier studies, which were usually based on a single psychometric property. Based on the WAIS—R standardization data, results indicate that incorporating reliability as a criterion had a significant impact on the obtained best SFs. A diagram is included as a practical guide for clinicians in selecting appropriate SFs. (12 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.     

Shift of the Curie Point of Barium Titanate Ceramics with Sintering Temperature

Definite increases in the Curie point (T_C) of undoped and lanthanum- (La-) doped (<0.5 at.%) barium titanate (BaTiO₃) ceramics sintered at elevated temperatures in the range of 1300°-1450°C were observed. Both undoped and 0.3 at.% La-doped BaTiO₃ (chosen as a typical doping concentration to yield semiconducting materials) ceramics showed almost the same T_C behavior; their T_C values increased by ∼3.5°C as the sintering temperature was increased from 1300° to 1450°C. Semiconducting 0.3 at.% La-doped materials increased in room-temperature bulk resistivity and TC with increased sintering temperature. The bulk resistivity of the La-doped materials, which was obtained from complex impedance analysis, increased from ∼2 omega cm for the material sintered at 1350°C to ∼6 ω cm at 1450°C. The phenomenon of bulk resistivity increase with sintering temperature was observed in the materials with a doping concentration of ≥ 0.2 at.% La, but was not observed in those doped with <0.2 at.% La. The mechanisms of TC and the bulk resistivity increase observed in the present materials with increased sintering temperature are discussed based on various models found in the literature, particularly in terms of the defect chemistry in semiconducting BaTiO₃ ceramics and the influence of liquid phases present during sintering.     

Grain-Boundary Diffusion of Strontium in (La,Ca)CrO3 Perovskite-Type Oxide by SIMS

The grain-boundary-diffusion coefficient ( D _gb) of strontium in La_0.9Ca_0.13CrO_3-δ was determined by secondary-ion mass spectrometry (SIMS), using two different measurement modes: depth profiling from the surface and line scanning on the fracture surface. The depth profiles that were sputtered by an O₂⁺-primary-ion beam gave two slopes of strontium concentration profiles, which corresponded to grain (bulk) and grain-boundary diffusion. The depth profiles were fitted to an appropriate equation that gave the grain- (bulk-) and grain-boundary-diffusion coefficients ( D _bulk= 6.5 × 10^-20 m²/s and D _gb= 1.6 × 10^-15 m²/s in air at 1273 K, respectively). Initially, to obtain the D _gb value via the SIMS line-scanning measurement, the fracture surface of La_0.9Ca_0.13CrO_3-delta was measured by a low-energy O₂⁺-primary-ion beam. The line-scanning measurement enabled us to successfully determine the strontium concentration profiles around the grain boundary. However, the D _gb value that was obtained via the line-scanning mode was 6.0 10⁻¹³ m²/s, which was a factor of 100 greater than that which was obtained by the depth-profile mode. Comparison between the depth-profile and line-scanning modes will require additional careful examination.     

Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ Visualized by High-Speed Atomic Force Microscopy

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.     

A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe

An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.