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.     

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%.     

Marked cell-type-specific differences in glycosylation of human interleukin-6

Human interleukin-6 (IL-6) secreted by cytokine- or endotoxin-induced fibroblasts, monocytes, keratinocytes, endometrial stromal cells, and endothelial cells, when analyzed under denaturing and reducing conditions, consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kD (a set of at least three O-glycosylated 23- to 25-kD species and a set of at least three N- and O-glycosylated 28- to 30-kD species). The 23- to 25-kD and 28- to 30-kD fibroblast-derived IL-6 species have been separately purified to homogeneity with the use of a combination of lectin and immunoaffinity chromatography. Glycosidase digestion experiments on such purified preparations confirmed that almost all human fibroblast-derived IL-6 species were O-glycosylated; additionally, the 28- to 30-kD species were N-glycosylated. Amino acid sequencing revealed that the major amino terminus in the fibroblast-derived 23- to 25-kD O-glycosylated IL-6 was at Ala28 whereas the major amino terminus in the 28- to 30-kD N- and O-glycosylated IL-6 was at Val30, suggesting that targeting of newly synthesized IL-6 polypeptides into the two different processing pathways in fibroblasts may be keyed to differences in the signal peptide cleavage site. Unexpectedly, IL-6 "constitutively" secreted by the Epstein-Barr virus (EBV)-infected human and primate (tamarin) B-cell lines designated sfBJAB and sfBT, respectively, consisted of a major apparently unglycosylated 21-kD species and a minor 25-kD N-glycosylated species.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.