Cry2 Is Critical for Circadian Regulation of Myogenic Differentiation by Bclaf1-Mediated mRNA Stabilization of Cyclin D1 and Tmem176b

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Rapid,efficient production of homozygous transgenic tobacco plants withagrobacterium tumefaciens: A seed-to-seed protocol

An optimized complete protocol was developed forAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T₁ flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T₂ seedlings in 6 to 7 months from the time of cocultivation withAgrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified “whole-leaf” transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T₁ and T₂ seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3∶1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.     

Mechanosensitivity below Ground: Touch-Sensitive Smell-Producing Roots in the Shy Plant Mimosa pudica

The roots of the shy plant Mimosa pudica emit a cocktail of small organic and inorganic sulfur compounds and reactive intermediates into the environment, including SO₂, methanesulfinic acid, pyruvic acid, lactic acid, ethanesulfinic acid, propanesulfenic acid, 2-aminothiophenol, S-propyl propane 1-thiosulfinate, phenothiazine, and thioformaldehyde, an elusive and highly unstable compound that, to our knowledge, has never before been reported to be emitted by a plant. When soil around the roots is dislodged or when seedling roots are touched, an odor is detected. The perceived odor corresponds to the emission of higher amounts of propanesulfenic acid, 2-aminothiophenol, S-propyl propane 1-thiosulfinate, and phenothiazine. The mechanosensitivity response is selective. Whereas touching the roots with soil or human skin resulted in odor detection, agitating the roots with other materials such as glass did not induce a similar response. Light and electron microscopy studies of the roots revealed the presence of microscopic sac-like root protuberances. Elemental analysis of these projections by energy-dispersive x-ray spectroscopy revealed them to contain higher levels of K⁺ and Cl⁻ compared with the surrounding tissue. Exposing the protuberances to stimuli that caused odor emission resulted in reductions in the levels of K⁺ and Cl⁻ in the touched area. The mechanistic implications of the variety of sulfur compounds observed vis-à-vis the pathways for their formation are discussed.Plant roots are known to exude a diversity of both small and macromolecular chemicals that mediate antimicrobial, antiquorum sensing, allelopathic, and other effects (De-la-Peña et al., 2012). However, the machinery associated with the synthesis and extrusion of these compounds is not well understood. One of the most intriguing but least studied of these is the emission of volatile and reactive organosulfur compounds such as the foul and toxic gas carbonyl sulfide (COS) and volatile carbon disulfide (CS₂). Both are reportedly released by numerous plants and are proposed to make a significant contribution to the environmental sulfur burden (Haines et al., 1989). As a case in point, the Central American rainforest plant Stryphnodendron exelsum (Mimosaceae) is a sufficiently strong sulfur emitter that its location in the forest can be determined by odor (Haines et al., 1989). Furthermore, 40 taxa from nine genera within the subfamily Mimosoideae revealed that 29 taxa from six genera produced CS₂ and 19 of the 40 taxa produced COS (Piluk et al., 2001). It has been proposed that the COS and CS₂ are derived from a putative Cys lyase-mediated cleavage of djenkolic acid, an amino acid previously isolated from the plant (Piluk et al., 1998), but this has not been confirmed.We used Mimosa pudica (Leguminosae), a perennial shrub endemic to Brazil but now pantropical in its distribution (Howard, 1988), as a model to begin investigations of how this and related plants emit these highly reactive and corrosive compounds without themselves incurring tissue damage. Its various colloquial names, such as sensitive plant, touch-me-not, shy plant, and humble plant, among many others (Holm, 1977), derive from its seismonastic movements: in response to touch, water, shaking, wind, or warming, its leaves quickly close, slowly opening after an average of about 10 min (Song et al., 2014). It also displays nyctinasty, with its leaves closing or “sleeping” with the onset of darkness. These curious characteristics coupled with its small size have made the plant a convenient and popular attraction in schools, greenhouses, and other learning environments, where it is used to illustrate seismonasty.Our studies show that by using direct analysis in real time high-resolution mass spectrometry (DART-HRMS; Cody et al., 2005), it is possible to detect the compounds emitted by plant roots in situ. Using this method, it was revealed that both M. pudica plants germinated aseptically on agar and those germinated in soil emitted a variety of small molecules into the atmosphere at levels that were not detectable by human subjects. However, an odor detectable by humans could be sensed when the plant root was disturbed, with odor emission being dependent on the nature of the stimulus. Analysis of the chemical contributors to the odor revealed that, although the array of compounds observed to be produced by the roots was the same both before and after stimulation, emission of a subset of organosulfur compounds was increased when the roots were stimulated. Light and scanning electron microscope imaging studies revealed the presence of sac-like protuberances dotted along M. pudica seedling root shafts that collapsed when the roots were exposed to stimuli that elicited odor emission. The detection by energy-dispersive x-ray spectroscopy (EDS) of relatively high levels of K⁺ and Cl⁻ prior to root stimulation, on the one hand, and reductions in the levels of these species, on the other hand, implicate the involvement of these ions in the observed mechanostimulatory behavior.     

Perimenopausal risk of falling and incidence of distal forearm fracture.

A postal survey of 2000 women and 2000 men sampled from the electoral roll in Oxford was undertaken to ascertain whether changes with age in the risk of falling might explain the stepwise increases in age specific incidence rates of distal forearm fracture which occur in women at around the age of 50. Corrected response rates were 83% for women and 72% for men. In women, but not in men, there was a rise in the risk of falling from 45 years, peaking in the 55-59 year age group, and sinking to a nadir at ages 70-74. In both sexes rates rose in extreme old age. These variations were not attributable to preferential response from people who had suffered a fracture. It is concluded that changes in the risk of falling interact with osteoporosis to produce a perimenopausal rise in the incidence of forearm fractures and contribute to the fluctuations in incidence of these fractures in old age.     

The effect of temperature,irradiance and animal size on incorporation rates of Simocephalus vetulus

Carbon incorporation rates of Simocephalus vetulus were measured to study the effects of the physical state of the animals, size of the animal, varying temperature and light conditions. Physical state of the animal showed little effect on incorporation rates after the first hour. Incorporation rates increased in proportion to the third power of animal size. Experimental animals collected at temperatures of 12, 20 or 25°C fed maximally at 10, 15 and 25°C respectively, when subjected to a feeding temperature range of 5 to 30°C. We have interpreted this as an indication that S. vetulus is able to acclimate and incorporate maximally at various temperatures after prolonged exposure to that temperature. When fed over an irradiation range of 0 to 4.68 × 10^–3 cal cm^–2 s^–1 incorporation rates were indirectly proportional to irradiance. This suggests a response to decreased irradiance in the weedy, littoral habitat of these animals.     

Abscisic acid and mannitol promote early development, maturation and storage protein accumulation in somatic embryos of interior spruce

Low levels of mannitol (2–6%) promoted the formation of globular embryos in embryogenic cultures of interior spruce ( Picea glauca engelmanni complex). However, these concentrations of mannitol were inhibitory to the formation of cotyledonary embryos. A short (1 week) pulse of mannitol in combination with abscisic acid doubled the production of late cotyledonary somatic embryos compared with the standard abscisic acid treatment. Higher levels of mannitol (13 and 20%) were required to inhibit precocious germination of spruce somatic embryos. These concentrations of mannitol promoted the accumulation of storage proteins during cotyledon maturation, but were not as effective as abscisic acid. Furthermore, 13 and 20% mannitol treatments did not substitute for abscisic acid in promoting the formation of cotyledonary embryos. Pre-treatment of late cotyledonary embryos with mannitol (13–25%) did not increase the frequency of germination compared with germination in non-treated embryos (approximately 10% germinated) although dehydration with high relative humidity treatment increased germination to 83%.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

The Effects of Inhibitors of Polyamine and Ethylene Biosynthesis on Senescence, Ethylene Production and Polyamine Levels in Cut Carnation Flowers

The role of polyamine metabolism in the regulation of senescenceand ethylene evolution was examined in cut carnations. Endogenousconcentrations of spermine and spermidine did not change asflowers aged, but putrescine, their immediate biosynthetic precursor,increased dramatically and paralleled a sharp rise in ethylene.When D-arginine, difluoromethylarginine and methylglyoxal bis(guanylhydrazone)were used to inhibit specific steps in polyamine synthesis,ethylene production and the onset of senescence were promoted.In contrast, inhibition of ethylene by aminooxyacetic acid increasedthe level of spermine, presumably by increasing the availabilityof aminopropyl groups derived from S-adenosylmethionine (SAM)and required for the synthesis of polyamines from putrescine.These results suggest that the ethylene and polyamine biosyntheticpathways compete for SAM during senescence of carnation flowers. (Received September 1, 1983; Accepted January 7, 1984)     

Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million‐fold improvements in binding affinity by alternating rounds of diversification and flow cytometry‐based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads—allowing isolation of extremely weak binders from billions of non‐binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000‐fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin‐biotin that do not cross‐react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009