Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764

We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ ¹H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 10³ M^?1 s^?1) is less than stellar.     

Inheritance of lambda-cyhalothrin resistance in the predator lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae)

Lambda-cyhalothrin is a pyrethroid insecticide widely used to control pests in various crop ecosystems, in which the lady beetle occurs naturally. Therefore, lady beetle populations are exposed to lambda-cyhalothrin sprays that may foster tolerance to this insecticide. This study was conducted to confirm the occurrence of resistance in the lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae) to lambda-cyhalothrin and to characterize the inheritance of resistance after eight progressive selection with insecticide dosages based on the LD₅₀ determined for the F1 generation. Dose–mortality curves were determined for parental populations, F1 hybrids and backcross progenies. Parameters regarding the heritability of resistance to lambda-cyhalothrin in E. connexa allow estimating 10-fold increase in the initial LD₅₀ after 54.5 generations of successive selections. The resistance of E. connexa to lambda-cyhalothrin was characterized as autosomally inherited and incompletely dominant, and influenced by a major gene with possible influence of secondary genes. Additionally, the resistance in E. connexa varies from functionally dominant to functionally recessive depending on the dose used. These findings indicate that insecticide resistance in E. connexa can be selected in the field as determined for field-collected individuals, and subsequently enhanced under laboratory conditions. Its characterization presented here is an important step toward linking biological and chemical control within pest management regarding the lady beetle and lambda-cyhalothrin targeting different pest groups.     

An electromyographic analysis of two handwriting grasp patterns

BackgroundHandwriting is a fundamental skill needed for the development of daily-life activities during lifetime and can be performed using different forms to hold the writing object. In this study, we monitored the sEMG activity of trapezius, biceps brachii, extensor carpi radialis brevis and flexor digitorum superficialis during a handwriting task with two groups of subjects using different grasp patterns.Subjects and methodsTwenty-four university students (thirteen males and eleven females; mean age of 22.04 ± 2.8 years) were included in this study. We randomly invited 12 subjects that used the Dynamic Tripod grasp and 12 subjects that used the Static Tripod grasp.ResultsThe static tripod group showed statistically significant changes in the sEMG activity of trapezium and biceps brachii muscles during handwriting when compared to dynamic tripod group’s subjects. No significant differences were found in extensor carpi radialis brevis and flexor digitorum superficialis activities among the two groups.ConclusionThe findings in this study suggest an increased activity of proximal muscles among subjects using a transitional grasp, indicating potential higher energy expenditure and muscular harm with the maintenance of this motor pattern in handwriting tasks, especially during the progression in academic life.     

Electromyographic and mechanomyographic responses across repeated maximal isometric and concentric muscle actions of the leg extensors

The purpose of the present study was to examine the patterns of responses for torque, electromyographic (EMG) amplitude, EMG mean power frequency (MPF), mechanomyographic (MMG) amplitude, and MMG MPF across 30 repeated maximal isometric (ISO) and concentric (CON) muscle actions of the leg extensors. Twelve female subjects (21.1 ± 1.4 yrs; 63.3 ± 7.4 kg) performed ISO and CON fatigue protocols with EMG and MMG signals recorded from the vastus lateralis. The relationships for torque, EMG amplitude, EMG MPF, MMG amplitude, and MMG MPF versus repetition number were examined using polynomial regression. The results indicated there were decreases (p < 0.05) across the ISO muscle actions for torque (r² = 0.95), EMG amplitude (R² = 0.44), EMG MPF (r² = 0.62), and MMG MPF (r² = 0.48), but no change in MMG amplitude (r² = 0.07). In addition, there were decreases across the CON muscle actions for torque (R² = 0.97), EMG amplitude (R² = 0.46), EMG MPF (R² = 0.86), MMG amplitude (R² = 0.44), and MMG MPF (R² = 0.80). Thus, the current findings suggested that the mechanisms of fatigue and motor control strategies used to modulate torque production were similar between maximal ISO and CON muscle actions.     

Resprouting Ability of Dry Forest Tree Species after Disturbance Does Not Relate to Propagation Possibility by Stem and Root Cuttings

Tropical dry forest tree species are recognized for their high resprouting ability after disturbance. We tested whether species that commonly produce root and stem suckers can be propagated by large stem and root cuttings, a useful method for landscape restoration programs. We performed four experiments: (1) In a greenhouse, we tested the propagation of six species using large stem cuttings collected from early successional sites. We used the following treatments: (i) dry season collection and planting; (ii) dry season collection, storage in humid soil, and wet season planting; (iii) wet season collection and planting; and (iv) wet season collection and planting after treatment with commercial NAA auxin. (2) Stem cuttings of Myracrodruon urundeuva were planted in a pasture during the rainy season after either NAA, IBA, or no auxin treatment. (3) As a control experiment, we also planted cuttings of Spondias mombin, a species known for successfully regenerating from cuttings. (4) Root cuttings of six species were collected in recently plowed pastures and planted in the greenhouse with and without treatment with NAA auxin. No root cuttings rooted. Only M. urundeuva and Astronium fraxinifolium stem cuttings rooted. Maximum success was obtained for stem cuttings collected and planted in the dry season (23%). Only 13% of M. urundeuva had sprouted by the 15th month of the field experiment. As a result, large cuttings are not recommended for propagation of the studied species. Future studies should include development of suitable methods of root harvesting and prospection of traditional knowledge for species selection.     

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.     

Cryo-electron tomography of the magnetotactic vibrio Magnetovibrio blakemorei: Insights into the biomineralization of prismatic magnetosomes

We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggest fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.