Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.     

RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), a selective alpha 1A-adrenoceptor antagonist, displays low affinity for functional alpha 1-adrenoceptors in human prostate: implications for adrenoceptor classification

Hereditary developmental abnormalities of the upper or lower limbs in humans are easily recognizable phenotypes that can be used in the mapping and cloning of genes involved in normal human development. We studied a large Indian pedigree (UR002) with an autosomal dominant triphalangeal thumb (TPT) and polysyndactyly (PSD). The abnormalities were present only in the upper limbs, and the phenotype was fully penetrant. The expression of the phenotype was variable and ranged from unilateral TPT to bilateral TPT, preaxial du-, tri-, or quadruplication of the thumb, or syndactyly of multiple thumbs. There were 112 affected individuals in the pedigree. Previous linkage analyses on apparently similar phenotypes have identified a locus at 7q36 [Heutink et al., 1994, Nature Genet 6:287-291; Tsukurov et al., 1994]. To map the gene responsible for the TPT-PSD in family UR002, we performed linkage analysis in DNA from 47 affected and 7 normal individuals. Marker D7S550, located at 7q36, yielded a maximum LOD score of 11.31 at theta = 0.00. Additional markers in the region also showed no recombination. These data indicate that the gene responsible for the hand abnormality in pedigree UR002 maps to the same region as that in previous pedigrees with similar phenotype. Further analyses of recombinants among all the linked families by using new polymorphic markers will narrow the critical genomic region and facilitate positional cloning of the elusive gene.     

Changes in energy expenditure and work during response acquisition in rats.

The principle of least effort predicts that behavior will tend to maximum efficiency. To test this prediction, changes in the energy expended (VO?) and work performed per reinforcement were monitored continuously as rats learned to press a beam with a criterion force for liquid food rewards. All 12 Ss exhibited significant decreases in energy expended per reinforcement over the 16 days of observation. Of these, 10 Ss also decreased the work performed per reinforcement. Analyses of motor performance were undertaken to determine how motor programs for changing efficiency were generated. The 10 Ss showing decreased work reinforcement also exhibited significant decreases in the variability of temporal and kinetic response features and in mean response magnitude as a function of practice. Adjustments in work output were primarily accomplished by modifying temporal response features. The kinetic features remained relatively constant for these animals. The remaining 2 Ss differed in that response recruitment increased after Day 9. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dose-rate effects in silicon-implanted gallium arsenide from low to high doses

For implantation of silicon dopant into gallium arsenide, sheet resistance and damage increase as the ion dose rate increases in the high-dose regime (>5.0 × 10¹³ cm⁻²). But, in the low-dose regime (<5.0 × 10¹² cm⁻²), although damage still increases with dose rate, the sheet resistance decreases. This qualitative difference implies that there must be a crossover point between the low- and high-dose regimes in the effect of damage and defect formation on dopant activation. This paper describes experiments in which damage and silicon dose were independently varied through the crossover point. Thermal wave, ion channeling, Hall effect measurements, and transmission electron microscopy were used to characterize structural and electrical changes that occur near the crossover. In GaAs implanted with silicon (²⁹Si⁺) at doses between 3 × 10¹² and 6 × 10¹³cm⁻², it is shown that electrical activation for low dose rates first begins to exceed that for high dose rates at a dose of 2 × 10¹³ cm⁻². Rapid growth of Type I dislocations also begins near this same dose, suggesting that there may be a link between defect formation and the crossover to negative dose-rate effects in the high-dose regime.     

Ligand efficacy and potency at recombinant alpha2 adrenergic receptors: agonist-mediated [35S]GTPgammaS binding

Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) > guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation.     

Flexural strength of lapped and oxidized siliconized silicon carbide

A siliconized silicon carbide composite has been microstructurally characterized, oxidized in air at 1350 °C for times up to 1079 h, air-cooled, and tested in four-point bending in the lapped condition at various temperatures up to 1425 °C, and in the pre-oxidized condition at room temperature and at 1300 °C. The strength of the lapped specimens increased by 25% at temperatures up to 1350 °C. Oxidation always decreased the strength of the material. After 315 h oxidation, the strength at room temperature and 1300°C was reduced by 50% and 40%, respectively. Preferential oxidation of the inter-grain regions formed pits up to 50 m deep. Hot salt corrosion increased the amount of oxidation by nearly 800%, and formed pits about 100 m deep. Microstructural details of the oxidation and fracture processes are presented, and the possible mechanisms of failure discussed.     

Cephalopod‐Inspired High Dynamic Range Mechano‐Imaging in Polymeric Materials

Cephalopods, such as squid, cuttlefish, and octopuses, use an array of responsive absorptive and photonic dermal structures to achieve rapid and reversible color changes for spectacular camouflage and signaling displays. Challenges remain in designing synthetic soft materials with similar multiple and dynamic responsivity for the development of optical sensors for the sensitive detection of mechanical stresses and strains. Here, a high dynamic range mechano‐imaging (HDR‐MI) polymeric material integrating physical and chemical mechanochromism is designed providing a continuous optical read‐out of strain upon mechanical deformation. By combining a colloidal photonic array with a mechanically responsive dye, the material architecture significantly improves the mechanochromic sensitivity, which is moreover readily tuned, and expands the range of detectable strains and stresses at both microscopic and nanoscopic length scales. This multi‐functional material is highlighted by creating detailed HDR mechanographs of membrane deformation and around defects using a low‐cost hyperspectral camera, which is found to be in excellent agreement with the results of finite element simulations. This multi‐scale approach to mechano‐sensing and ‐imaging provides a platform to develop mechanochromic composites with high sensitivity and high dynamic mechanical range.     

Limitations in predicting dyebath exhaustion using optical spectroscopy

There are practical limitations in predicting dyebath concentrations and dyebath exhaustion using absorbance measurements from optical spectroscopy. The purpose of this paper is to examine the common assumptions of prediction models that cause prediction errors, and especially when multiple dye combinations are used. If a linear model is used to map absorbance to concentration, then five assumptions must hold: measurement repeatability, linear scaling, spectral additivity, linear independence of the constituent spectra and the absence of spectral morphing. Violation of one or more of these assumptions will lead to errors in predicting the concentrations of dyes in a dyebath and subsequent exhaustion calculations.