Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds

Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase -subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium. Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V. To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106. Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography–mass spectroscopy. Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G). The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound. Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring. Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed. Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions. Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.     

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803

Proteome analysis by gel-free "shotgun" proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse-phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre-digestion protein separation was performed by strip-based isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre-RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight-tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non-redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF-IEF protein and peptide fractionation prior to RP gave the best overall performance.     

Thematic review series: the immune system and atherogenesis. The unusual suspects:an overview of the minor leukocyte populations in atherosclerosis

Atherosclerosis is a complex inflammatory disease process involving an array of cell types and interactions. Although macrophage foam cells and vascular smooth muscle cells constitute the bulk of the atherosclerotic lesion, other cell types have been implicated in this disease process as well. These cellular components of both innate and adaptive immunity are involved in modulating the response of macrophage foam cells and vascular smooth muscle cells to the retained and modified lipids in the vessel wall as well as in driving the chronic vascular inflammation that characterizes this disease. In this review, the involvement of a number of less prominent leukocyte populations in the pathogenesis of atherosclerosis is discussed. More specifically, the roles of natural killer cells, mast cells, neutrophils, dendritic cells, gammadelta T-cells, natural killer T-cells, regulatory T-cells, and B-cells are addressed.     

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Ectoparasite and hemoparasite infection in a diverse temperate lizard assemblage at Macraes Flat, South Island, New Zealand

A lizard assemblage at Macraes Flat, New Zealand, comprising the common skinks Oligosoma maccanni and Oligosoma nigriplantare polychroma, the endangered species Oligosoma grande and Oligosoma otagense, and the common gecko Hoplodactylus maculatus, was studied to determine the prevalence of ectoparasites and hemoparasites. The mites Odontacarus lygosomae and Ophionyssus scincorum infected all Oligosoma spp. only, and the mite Neotrombicula naultini infected only H. maculatus. The hemoparasite Hepatozoon lygosomarum infected all Oligosoma skinks, except O. n. polychroma. Oligosoma otagense had the highest infection levels of all parasites by several orders of magnitude. For all lizard species, there was no difference in mite prevalence between adult males and adult females, but juveniles were less often infected. For all skink species, there was a significant relationship between presence of the hemoparasite He. lygosomarum and infection intensity of the supposed vector, O. scincorum. It is unclear if patterns of parasite infection reflect species-specific susceptibility, host-parasite species-specific spatial ecology, or environmentally induced host physiological impairment. Considering the threatened nature of O. otagense, evidence of high parasitemia should stimulate further investigation.     

花曲柳窄吉丁的空间分布

2004年4月~9月,在天津市大港区官港森林公园对花曲柳窄吉丁Agrilus planipennis的空间分布和垂直分布进行了调查研究,应用扩散系数(C)等6种指数法分析测定,确定了该虫在林地中的空间分布呈聚集分布,分布的基本成分是个体群。扩散蔓延规律为聚集型扩散。花曲柳窄吉丁幼虫在树干的垂直分布主要区间在50~150 cm之间。对幼虫垂直分布与树干高度的关系进行分析,其符合立方曲线,曲线方程为:Y=-5.142 9+17.943 7X+21.662 3X2-14.545X3;相关系数R2=0.915,P=0.013。     

Effects of long-term storage on potency of TM Biocontrol-1, the registered viral insecticide of Orgyia pseudotsugata (Lepidoptera: Lymantriidae)

The article presents the results of bioassaying 39 samples of TM Biocontrol-1, a viral insecticide, from 10 different lots and various sizes of vacuum-sealed packages that were stored at -10 degrees C for 5-15 yr. This is the first study to present potency data for a registered virus product stored for this length of time. Laboratory bioassays in insects from the same colony from which the TM Biocontrol-1 was produced showed that the stored viral insecticide is still effective, although it lost approximately 30% of its effectiveness during storage. A direct correlation of this loss with the length of time in storage is not apparent. Bioassays also showed that there are significant differences in the susceptibility of Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough), larvae from different geographic regions to OpMNPV (family Baculoviridae, genus Nucleopolyhedrovirus) infection. Package size did not affect the potency of stored TM Biocontrol-1. There were no clear, significant differences in the effectiveness among lots of TM Biocontrol-1 processed by different organizations.     

Postexercise hypotension causes a prolonged perturbation in esophageal and active muscle temperature recovery

We examined the effect of two levels of exercise-induced hypotension on esophageal (Tes) and active and nonactive muscle temperatures during and following exercise. Seven males performed an incremental isotonic test on a Kin-Com isokinetic apparatus to determine their peak oxygen consumption during bilateral knee extensions (VO2sp). This was followed on separate days by 15-min of isolated bilateral knee extensions at moderate (60% VO2sp) (MEI) and high (80% VO2sp) (HEI) exercise intensities, followed by 90 min of recovery. Muscle temperature was measured with an intramuscular probe inserted in the left vastus medialis (Tvm) and triceps brachii (Ttb) muscles under ultrasound guidance. The deepest sensor (tip) was located approximately 10 mm from the femur and deep femoral artery and from the superior ulnar collateral artery and humerus for the Tvm and Ttb, respectively. Additional sensors were located 15 and 30 mm from the tip with an additional sensor located at 45 mm for the Tvm measurements only. Following exercise, mean arterial pressure (MAP) remained significantly below preexercise rest for the initial 60 min of recovery after MEI and for the duration of the postexercise recovery period after HEI (P< or =0.05). After HEI, significantly greater elevations from preexercise rest were recorded for Tes and all muscle temperatures paralleled a greater decrease in MAP compared with MEI (P< or =0.05). By the end of 90-min postexercise recovery, MAP, Tes, and all muscle temperatures remained significantly greater after HEI than MEI. Furthermore, a significantly shallower muscle temperature profile across Tvm, relative to preexercise rest, was observed at the end of exercise for both HEI and MEI (P< or=0.05), and for 30 min of recovery for MEI and throughout 90 min of recovery for HEI. No significant differences in muscle temperature profile were observed for Ttb. Thus we conclude that the increase in the postexercise hypotensive response, induced by exercise of increasing intensity, was paralleled by an increase in the magnitude and recovery time of the postexercise esophageal and active muscle temperatures.     

Development of a Fiber Optic Enzymatic Biosensor for 1,2-dichloroethane

There is a significant need for devices capable of measuring water contaminant concentrations in situ––continuously, rapidly, and without reagents, extraction, or other pretreatment. Toward this goal, we constructed and tested fiber optic biosensors for measurement of 1,2-dichloroethane (DCA) in aqueous solutions. The biocomponent was the haloalkane dehalogenase, DhlA, in whole cells of Xanthobacter autotrophicus GJ10. These cells were immobilized in calcium alginate on the tip of a fiber optic fluoresceinamine-based pH optode. The resulting biosensor could quantify DCA at 11 mg/l and had a linear response up to at least 65 mg/l. Total signal change was reached in 8–10 min, and measurements were reproducible (SE <9%). The sensor’s small size, potential for remote operation, and low cost make it of interest for further development.