After AbMole beta-Eudesmol self-crossing, a portion of the seeds, specifically, 25% of the seeds, will lose the transgene. Several self-crosses and generations are needed to obtain transgene-homozygous plants and seeds. This can be time and labor consuming. Also, the amount of seeds developed from a plant is often limited. A large number of plants need to be grown to obtain sufficient seeds for research, especially commercial uses. This needs large spaces and land, long periods of time and extensive labor input. In addition, seed development in some plant species is naturally impaired due to various reasons and thus transgenes may not be passed to the next generation and transgenic materials can be lost. Moreover, perennial plants and woody plants need a much longer time to produce seeds. As such, the breeding process to pass transgenes to the next generation in these types of plants can be very slow. Plants have unique characteristics that allow various cells, after certain induction, to reprogram and develop into somatic embryos. Somatic embryos have the same morphology and structure as zygotic embryos and can germinate and develop into full and fertile plants. Somatic embryogenesis has been developed in a large number of plant species and the system has been used widely for producing transgenic plants for molecular biology and functional genomics research and in biotechnology for plant trait improvement. Somatic embryos, after certain treatments such as abscisic acid, sucrose and heat shock, can acquire tolerance to water loss. They can be dried to contain less than 15% water, similar to the water content in true seeds, and still remain viable under ambient environment. After rehydration, the somatic embryos can germinate and develop into full plants. Dried somatic embryos can be intact as they are produced or encapsulated and they are collectively called artificial seeds or synthetic seed. Artificial seeds can be stored for long periods of time and still possess propagation ability. These embryos can be handled or shipped as true seeds. Artificial seeds indeed are a true analog of conventional seeds and can be used for germplasm and genetic material preservation. Artificial seed technology and artificial seedrelated technology have been reported in various plant species. Induction of somatic embryos from transgenic plants and the use of artificial seeds may provide a new system for transgene preservation. Here, we report stable transgene preservation and faithful expression of a transgene in plants developed from dried somatic embryos in alfalfa. The new system can be used to preserve transgenic materials for research use and preserve transgenic germplasm for applications in different plant species. Fluorometric analysis was conducted to measure GUS enzyme activity in leaf tissues as described by Jefferson et al.. Protein content in the extract was determined spectrophotometrically according to Bradford using a commercially available Bradford Reagent dye. Measurements of the enzyme activity were repeated 2�C4 times after incubation lasting from 15 min to 24 hours depending on the levels of sample fluorescence. GUS activity was expressed as pM 4-MU per mg protein per minute. Somatic embryos were induced from different and independent transgenic plants using petioles as explants. The procedure was the same as the transformation method but without Agrobacterium infection. Cotyledonary-staged somatic embryos of different transgenic alfalfa lines were randomly divided into two groups. One group was used for desiccation treatment and the other was used as controls. Transgenic alfalfa plants were obtained via Agrobacteriummediated transformation using the method well established in our laboratory. Plant transformation was confirmed by PCR using uid gene primers, histochemical analysis and Southern blot analysis.

Even when viable cells are present, the newly synthesized matrix may not be sufficiently cross-linked to the native tissue. This study aims to overcome all of these factors by supplying viable cells to the interface via engineered neocartilage to mitigate the issues of cell death and lack of cell migration at the wound edge by exogenously inducing cross-links. One way to deliver cells at an interface may be via the use of constructs engineered using the self-assembling process, which is an established method for generating tissue with abundant cells at the construct edge. This method has also generated neocartilage with properties approaching those of native tissue. Maintenance of cartilage with normal functional properties requires sustaining cell density; large areas of cell death would undoubtedly result in biomechanically inferior matrix or none at all. Thus, this study seeks to use tissue engineered constructs created via chondrocyte self-assembly to deliver a higher cell density to the wound edge to enhance integration. Another suggested mechanism for the enhancement of integration is collagen pyridinoline cross-links. PYR crosslinks have been shown to be a major factor in determining the stiffness of connective tissues. PYR naturally forms within cartilage and other musculoskeletal tissues during development and aging via the enzyme lysyl oxidase, a metalloenzyme that converts amine side-chains of lysine and hydroxylysine into aldehydes. In vivo, LOX is most active at sites of growing collagen fibrils. A potential method for inducing collagen cross-linking across cartilage interfaces is thus the exogenous application of this enzyme. Since LOX is a small-sized molecule, at roughly,50 kDa, and since cross-link formation occurs over several weeks, exogenous LOX can be applied to in vitro cultures on a continuous basis to ensure full penetration via diffusion and to allow sufficient time for cross-link formation. By employing LOX, one would expect the formation of “anchoring” sites, composed of PYR cross-links in the collagen network of the engineered tissue as well as of the native tissue, to bridge the two tissues together. Thus, LOX application combined with the delivery of high cell numbers to the wound edge are expected to promote tissue integration. Using the self-assembling process, the objective of this study was to determine whether LOX can alter the integration of native-toconstruct and native-to-native tissue systems through two experiments. It was hypothesized that application of LOX would enhance integration, as evidenced through tensile measurements. The first experiment sought to examine whether LOX would promote integration between native cartilage and neocartilage and to determine time and duration of application. The second experiment sought to determine whether the results from the first experiment can be replicated in a native-to-native cartilage micrornas influence processes negative regulation binding targets system. Motivated by the as-of-yet unsolved issue of cartilage integration, the objective of this study was to examine the hypothesis that LOX would induce cartilage integration. This enzyme naturally occurs in cartilage and promotes PYR cross-links in collagen, thereby holding potential for strengthening cartilage-to-cartilage interfaces. The hypothesis was proven to be correct as evidenced by the biomechanical and histological data. At the dosage applied, this naturally occurring enzyme did not alter cellular response with respect to collagen and GAG production. Engineered tissues, formed using a self-assembling process, were integrated to native tissue explants by applying LOX to a ring-and-implant assembly. Additionally, LOX was applied to native-to-native cartilage interfaces to examine whether this novel integration method can also be applicable to cases where there is not an abundance of cells at the wound edge.

Because KORs are expressed in moderately high levels in the hippocampus, here we studied a potential role for hippocampal KORs in the renewal of extinguished fear. We found a dissociation in the contribution of hippocampal KORs, such that KORs in the VH, but not the DH, mediate the renewal of fear. The results from Experiment 1 showed that antagonizing KORs in the VH impaired the renewal of fear. Although recent research has clearly demonstrated a role for the VH in the return of fear following extinction, the DH has also been shown to mediate renewal. Furthermore, the DH in the rat projects to the dorsal region of the mPFC, and contains KORs. These experiments investigated the role of hippocampal KORs in the renewal of extinguished fear. We demonstrated that: 1) intra-VH microinfusions of the KOR antagonist norBNI significantly attenuated renewal using a within-subjects design, 2) both a 5 mg and 10 mg dose were equally effective at reducing CSfreezing in the training context, and 3) intra-DH microinfusions of norBNI had no effect on the expression of renewal. Together these experiments reveal a dissociation in hippocampal contributions to renewal, where KORs in the VH, but not the DH, contribute to the renewal of extinguished fear. These findings are consistent with previous studies demonstrating involvement of the hippocampus in fear renewal. However unlike previous studies, here we show a clear dissociation in the contribution of distinct hippocampal regions. Prior functional studies investigating the neural circuitry underlying renewal have used lesions or temporary inactivation methods to target either the DH or VH. Such studies found that inactivating either hippocampal region stabilizing reproductive division labor maintaining link physiological state foraging behavior prevented renewal. Clearly both the DH and VH are essential components of the circuitry mediating the return of fear following extinction, yet here we demonstrate that the contributions of these regions are distinct. Specifically, we demonstrated that only the VH involvement in renewal relies on activation of KORs, at least in part. Recently, Orsini end colleagues demonstrated that the VH mediates renewal via direct projections to the mPFC and BA. This raises the question of how KORs within the VH are acting on this circuit to mediate renewal. One possibility is through the extensive projections from the VH to the BA. Renewal increases Fos expression in BA-projecting neurons in the VH, and results in increased firing in a population of BA neurons receiving input from the VH. This suggests that recruitment of this VH-BA pathway is activated during renewal. Although activation of KORs in the hippocampus has been shown to inhibit excitatory transmission, in regions of the caudal hippocampus KORs are also located on GABAergic interneurons. As such, the attenuation of renewal seen here in Experiment 1 is potentially due to norBNI acting on these GABAergic interneurons to prevent the disinhibition of pyramidal neurons, reducing activation of the VHBA pathway and thus diminishing the response of fear neurons in the BA. Recently however, it was demonstrated that individual VH neurons send convergent projections to both the BA and mPFC, including the prelimbic cortex. This is of note considering the role of the PL in renewal and the expression of conditioned fear. For example, the PL shows significant neuronal activation during renewal, and CS-evoked firing which correlates with learned freezing behavior. Such findings raise the possibility that the attenuation of renewal by infusions of norBNI into the VH was due to simultaneous reduction in activity in both PL and BA. Of course it is important to note that KORs are widely distributed in the hippocampus, including on granule cell mossy fibres and perforant path terminals, and hence could exert numerous effects on hippocampal neurons.

As a result of limited therapeutic accumulation within mitochondria, targeting the mitochondria with antioxidants or therapeutics has been a major interest especially for cardiovascular disease and cancer. Small molecules can permeate through the mitochondrial outer membrane but fail to cross the inner membrane. Taking advantage of the high inner membrane potential gradient, lipophilic cations can easily accumulate within the mitochondria as well as permeate the phospholipid bilayers. Vitamin E conjugated to TPP + can accumulate into the mitochondria, where it decreases ROS more effectively than vitamin E alone, and is able to ameliorate oxidative stress-mediated disease. While conjugating vitamin E to TPP + has been previously described, our goal was to conjugate the vitamin E metabolite, a-CEHC, to TPP+ and to design a fast and efficient synthetic method using a lysine linker and solid phase synthesis. This method does not require isolation of synthetic intermediates, while reagents and by-products are washed away after each step. In addition, similar to trolox, a-CEHC contains the a-tocopherol ring structure but have a truncated side chain with one carbon longer than trolox. The chroman ring of vitamin E becomes redox active at the mitochondria, where it forms semiquinone after detoxifying a free radical via hydrogen donation. The semiquinone is further reduced by intramitochondrial ascorbic acid or by electron donation. The chroman ring is still intact in aCEHC when conjugated to TPP +. A lysine linker with two protecting groups was used, which enabled the conjugation of TPP + and a-CEHC. The masked lysine was coupled onto the Rink Amide MBHA resin. HBTU and HOBt were used to enhance the coupling rate. The Fmoc was then deprotected to allow for TPP + conjugation through its carboxylic acid group forming an amide bond. The Mtt protecting group was then removed. The removal of the protecting group enabled the carboxylic acid on a-CEHC side chain to form an amide bond with the lysine linker. The final product, TPP + -Lysine-a-CEHC, was then released from the resin via treatment with 95% TFA. The final product was characterized by MALDI-TOF mass spectrometry. The molecular weight peak was at 736.39, which corresponds to the expected peak for the MitoCEHC generated by ChemDraw software. The mass spectrometry data also shows virtually no trace of by-products, reagents or synthetic intermediates. The ability of final product to diminish oxidative stress was examined in vitro. Oxidative stress is defined as the overproduction of oxidizing chemical species and the failure to eradicate their excess by enzymatic or non-enzymatic antioxidants. Elevation in ROS production is a factor in the etiology of cardiovascular disease by modifying lipids, proteins, and nucleic acids. To further explore the antioxidant activity of the conjugated MitoCEHC, the oxidation of CM-H2DCFDA was measured. The H2DCFDA derivative with a thiol-reactive chloromethyl group was used due to its better retention in live cells than H2DCFDA. This derivative is retained better in cells because of its ability to bind covalently to intracellular components. BAEC were incubated with low and high glucose concentrations. The cells incubated under hyperglycemic conditions showed an increase in ROS production, which is mainly in the mitochondria. Flow cytometry data also showed decrease in ROS production in the hyperglycemic cells treated with MitoCEHC.

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Therefore, we speculated that it may be resulted from the increased activity of cholinergic receptors, the promoted release of Ach and the decreased expression of Ach. The underlying mechanism for the DCP improvement may be due to increases of cAMP in bladder, which could modulate the signaling pathways of neurotransmitter and receptors and increases of CGRP expression in bladder wall and DRG, leading to the enhancement of the contractility of the detrusor and sense of bladder filling. We have established a Rel report transgenic mouse model which can be used to monitor the endogenous Rel expression. Using in vivo bioluminescence imaging detection system, we demonstrated that luciferase activity in B6-Tg8Mlit mice was dramatically induced after i.p injection of LPS. The result of ex vivo experiment showed that the luciferase expression was induced in the heart, liver, spleen, lung, kidney, intestine, stomach, thymus and macrophages after the treatment with LPS, especially in the heart, liver, spleen, intestine and stomach. The data were in consistent with the change of endogenous murine Rel mRNA expression induced by LPS treatment. Meanwhile, the patterns of luciferase expression in LPS-treated B6-Tg8Mlit mice were also comparable to the results of Rel expression reported previously. The fold change did not match exactly between the luciferase activity and endogenous Rel mRNA expression. However, this is understandable that the protein level is often not in linear correlation with the endogenous mRNA expression in cells. Dexamethasone and aspirin, two well-known antiinflammatory drugs, suppressed the induction of luciferase expression and endogenous Rel expression in LPS treated B6-Tg8Mlit mice. These data demonstrate that the transgenic mice are faithful for monitoring Rel expression in vivo in c-Rel involved physiological or pathological processes and for evaluating the effects of anti-inflammatory drugs. LPS could induce Rel expression in monocytes and macrophages. We also collected macrophages from the abdomen fluid of the transgenic mouse. These specific cells manifested a good response of luciferase expression to LPS stimulus in vitro and could be used for high-throughput screening and studying of anti-inflammatory drugs at cellular level. Zymosan, a cell wall particle derived from Saccharomyces cerevisiae, is also widely used to induce inflammation in various relative experiments. However, the receptors for zymosan are distinct from those for LPS. LPS, the part of the outer cell wall of Gram negative bacteria, is detected by TLR4, while zymosan is recognized by TLR2 and TLR6. Although LPS and zymosan activates different triggers of target cells, the following inflammatory cascades seem to be similar. Our data also support the conclusion, for both LPS and zymosan could induce similar luciferase expression profiles in the B6-Tg8Mlit mice. The mouse EAE model is routinely used to study molecular mechanisms and signaling pathways of inflammatory regulation in Multiple sclerosis. It was reported that c-Rel-deficient mouse was resistant to the development of EAE due to its defective in the IL-12 and IFN-c induction and in the Th1 responses. It suggested that Rel expression was involved in the disease process of EAE. In our experiments, the luciferase expression in EAE group increased significantly at the eighth day after MOG injection before the loss of body weight and clinical symptoms occurred.

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