Contrasting growth responses in lamina and petiole during neighbor detection depend on differential auxin responsiveness rather than different auxin levels.

Foliar shade triggers rapid growth of specific structures that facilitate access of the plant to direct sunlight. In leaves of many plant species, this growth response is complex because, although shade triggers the elongation of petioles, it reduces the growth of the lamina. How the same external cue leads to these contrasting growth responses in different parts of the leaf is not understood. Using mutant analysis, pharmacological treatment and gene expression analyses, we investigated the role of PHYTOCHROME INTERACTING FACTOR7 (PIF7) and the growth-promoting hormone auxin in these contrasting leaf growth responses. Both petiole elongation and lamina growth reduction are dependent on PIF7. The induction of auxin production is both necessary and sufficient to induce opposite growth responses in petioles vs lamina. However, these contrasting growth responses are not caused by different auxin concentrations in the two leaf parts. Our work suggests that a transient increase in auxin levels triggers tissue-specific growth responses in different leaf parts. We provide evidence suggesting that this may be caused by the different sensitivity to auxin in the petiole vs the blade and by tissue-specific gene expression.

Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation.

To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots.

Ecosystem architecture design: endogenous and exogenous structural properties

The aim of this dissertation is to bridge and synthesize the different streams of literature addressing ecosystem architecture through a multiple‐lens perspective. In addition, the structural properties of and processes to design and manage the architecture will be examined. With this approach, the oft‐neglected actor‐structure duality is addressed and both the position and structure, and action and process are under scrutiny. Further, the developed framework and empirical evidence offer valuable insights on how firms collectively create value and individually appropriate value. The dissertation is divided into two parts. The first part comprises a literature review, as well as the conclusions of the whole study, and the second part includes six research publications. The dissertation is based on three different reasoning logics: abduction, induction and deduction; related qualitative and quantitative methodologies are utilized in the empirical examination of the phenomenon in the information and communication technology industry. The results suggest firstly that there are endogenous and exogenous structural properties of the ecosystem architecture. Out of these, the former ones can be more easily influenced by a particular actor whereas the latter ones are taken more or less for granted. Secondly, the exogenous ecosystem design properties influence the value creation potential of the ecosystem whereas the endogenous ecosystem design properties influence the value appropriation potential of a particular actor in the ecosystem. Thirdly, the study suggests that there is a relationship between endogenous and exogenous structural properties in that the endogenous properties can be leveraged to create and reconfigure the exogenous properties whereas the exogenous properties prose opportunities and restrictions on the use of endogenous properties. In addition, the study suggests that there are different emergent and engineered processes to design and manage ecosystem architecture and to influence both the endogenous and exogenous structural properties of ecosystem architecture. This study makes three main contributions. First, on the conceptual level, it brings coherence and direction to the fast growing body of literature on novel inter‐organizational arrangements, such as ecosystems. It does this by bridging and synthetizing three different streams of literature, namely the boundary, design and orchestration conception. Secondly, it sets out a framework that enhances our understanding of the structural properties of ecosystem architecture; of the processes to design and manage ecosystem architecture; and of their influence on the value creation potential of the ecosystem and the value capture potential of a particular firm. Thirdly, it offers empirical evidence of the structural properties and processes.

Protective effect of policosanol on atherosclerotic lesions in rabbits with exogenous hypercholesterolemia

Policosanol is a mixture of higher aliphatic alcohols purified from sugar cane wax, with cholesterol-lowering effects demonstrable in experimental models and in patients with type II hypercholesterolemia. The protective effects of policosanol on atherosclerotic lesions experimentally induced by lipofundin in rabbits and rats and spontaneously developed in stumptail monkeys have been described. The present study was conducted to determine whether policosanol administered orally to rabbits with exogenous hypercholesterolemia also protects against the development of atherosclerotic lesions. Male New Zealand rabbits weighing 1.5 to 2 kg were randomly divided into three experimental groups which received 25 or 200 mg/kg policosanol (N = 7) orally for 60 days with acacia gum as vehicle or acacia gum alone (control group, N = 9). All animals received a cholesterol-rich diet (0.5%) during the entire period. Control animals developed marked hypercholesterolemia, macroscopic lesions and arterial intimal thickening. Intima thickness was significantly less (32.5 ± 7 and 25.4 ± 4 µm) in hypercholesterolemic rabbits treated with policosanol than in controls (57.6 ± 9 µm). In most policosanol-treated animals, atherosclerotic lesions were not present, and in others, thickness of fatty streaks had less foam cell layers than in controls. We conclude that policosanol has a protective effect on the atherosclerotic lesions occurring in this experimental model.

Cyclic AMP increases the survival of ganglion cells in mixed retinal cell cultures in the absence of exogenous neurotrophic molecules, an effect that involves cholinergic activity

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 µM forskolin induced a two-fold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 µM H89 (an inhibitor of protein kinase A), 1.25 µM chelerythrine chloride (an inhibitor of protein kinase C), 50 µM PD 98059 (an inhibitor of MEK), 25 µM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 µM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 µM atropine or 1 µM telenzepine also blocked the effect of forskolin. When we used 25 µM BAPTA, an intracellular calcium chelator, as well as 20 µM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic amacrine cells and glial cells in the increase of RGC survival elicited by forskolin treatment.

Exogenous normal lymph alleviates microcirculation disturbances and abnormal hemorheological properties in rats with disseminated intravascular coagulation

Disturbances of the microcirculation and abnormal hemorheological properties are important factors that play an important role in disseminated intravascular coagulation (DIC) and result in organ dysfunction or failure. In the present study, we established an animal model of DIC using intravenous Dextran 500 in rats, and used exogenous normal lymph corresponding to 1/15 of whole blood volume for injection through the left jugular vein. We found that normal lymph could improve the blood pressure and survival time of rats with DIC. The results regarding the mesenteric microcirculation showed that the abnormality of the diameter of mesenteric microvessels and micro-blood flow speed in the DIC+lymph group was significantly less than in the DIC+saline group. Whole blood viscosity, relative viscosity, plasma viscosity, hematocrit (Hct), erythrocyte sedimentation rate (ESR), and electrophoresis time of erythrocytes were significantly increased in the DIC+saline group compared to the control group. The electrophoretic length and migration of erythrocytes from the DIC+saline and DIC+lymph groups were significantly slower than the control group. Blood relative viscosity, Hct, ESR, and electrophoretic time of erythrocytes were significantly increased in the DIC+lymph group compared to the control group. Whole blood viscosity, relative viscosity and reduced viscosity were significantly lower in the DIC+lymph group than in the DIC+saline group, and erythrocyte deformability index was also significantly higher than in the DIC+saline and control groups. These results suggest that exogenous normal lymph could markedly improve the acute microcirculation disturbance and the abnormal hemorheological properties in rats with DIC induced by Dextran 500.

Exogenous normal lymph reduces liver injury induced by lipopolysaccharides in rats

The liver is one of the target organs damaged by septic shock, wherein the spread of endotoxins begins. This study aimed to investigate the effects of exogenous normal lymph (ENL) on lipopolysaccharide (LPS)-induced liver injury in rats. Male Wistar rats were randomly divided into sham, LPS, and LPS+ENL groups. LPS (15 mg/kg) was administered intravenously via the left jugular vein to the LPS and LPS+ENL groups. At 15 min after the LPS injection, saline or ENL without cell components (5 mL/kg) was administered to the LPS and LPS+ENL groups, respectively, at a rate of 0.5 mL/min. Hepatocellular injury indices and hepatic histomorphology, as well as levels of P-selectin, intercellular adhesion molecule 1 (ICAM-1), myeloperoxidase (MPO), and Na+-K+-ATPase, were assessed in hepatic tissues. Liver tissue damage occurred after LPS injection. All levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma as well as the wet/dry weight ratio of hepatic tissue in plasma increased. Similarly, P-selectin, ICAM-1, and MPO levels in hepatic tissues were elevated, whereas Na+-K+-ATPase activity in hepatocytes decreased. ENL treatment lessened hepatic tissue damage and decreased levels of AST, ALT, ICAM-1, and MPO. Meanwhile, the treatment increased the activity of Na+-K+-ATPase. These results indicated that ENL could alleviate LPS-induced liver injury, thereby suggesting an alternative therapeutic strategy for the treatment of liver injury accompanied by severe infection or sepsis.

Behavioral effects of endogenous or exogenous estradiol and progesterone on cocaine sensitization in female rats

Cocaine sensitization is a marker for some facets of addiction, is greater in female rats, and may be influenced by their sex hormones. We compared the modulatory effects of endogenous or exogenous estradiol and progesterone on cocaine-induced behavioral sensitization in 106 female rats. Ovariectomized female rats received progesterone (0.5 mg/mL), estradiol (0.05 mg/mL), progesterone plus estradiol, or the oil vehicle. Sham-operated control females received oil. Control and acute subgroups received injections of saline, while the repeated group received cocaine (15 mg/kg, ip) for 8 days. After 10 days, the acute and repeated groups received a challenge dose of cocaine, after which locomotion and stereotypy were monitored. The estrous cycle phase was evaluated and blood was collected to verify hormone levels. Repeated cocaine treatment induced overall behavioral sensitization in female rats, with increased locomotion and stereotypies. In detailed analysis, ovariectomized rats showed no locomotor sensitization; however, the sensitization of stereotypies was maintained. Only females with endogenous estradiol and progesterone demonstrated increased locomotor activity after cocaine challenge. Estradiol replacement enhanced stereotyped behaviors after repeated cocaine administration. Cocaine sensitization of stereotyped behaviors in female rats was reduced after progesterone replacement, either alone or concomitant with estradiol. The behavioral responses (locomotion and stereotypy) to cocaine were affected differently, depending on whether the female hormones were of an endogenous or exogenous origin. Therefore, hormonal cycling appears to be an important factor in the sensitization of females. Although estradiol increases the risk of cocaine sensitization, progesterone warrants further study as a pharmacological treatment in the prevention of psychostimulant abuse.

Exogenous abscisic acid inhibits the water-loss of postharvest romaine lettuce during storage by inducing stomatal closure

Abstract Postharvest lettuce often lose water, thus affecting both its market value and consumer acceptance. However, the mechanism of the water-loss is still waiting well exploration. The aim of the present study was to investigate the effect of a foliar application of ABA on the fresh weight-loss and the chlorophyll content of postharvest lettuce as well as its association with the regulation of stomata. The present data demonstrated that exogenously application of ABA, in a concentration range of 0 to 100 µM, significantly lowered the fresh weight-loss of postharvest lettuce. ABA also delayed chlorophyll reduction during ambient storage, but this protective effect was ABA concentration-dependent. Among the tested ABA concentrations, 50 µM or lower ABA produced an inhibition effect on chlorophyll degradation in postharvest lettuce leaves. The results demonstrated that the exogenous ABA treatment can obviously reduce the transpiration rate of lettuce leaves by promoting the stomatal closure of postharvest lettuce, therefore eventually delay fresh weight-loss. The present study primarily showed that the application of exogenous ABA, which originated from a naturally-produced phytohormone, has a great potential in retaining the freshness of postharvest lettuce that is stored in an ambient condition, although possible practical application still need to be further evaluated.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the advent of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at hight titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.

Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems

A series of in vitro experiments was carried out to examine the impact of enzyme application rate and incubation medium pH on the rate and extent of fermentation of alfalfa stems. In Experiment 1, a commercial enzyme product (Liquicell 2500, Specialty Enzyme and Biochemicals, Fresno, CA, USA) was added to alfalfa stems at six levels: 0, 0.51, 1.02, 2.55, 5.1, and 25.5 mu l/g (control and L1-L5, respectively) to forage DM in a completely randomized design, with a factorial arrangement of treatments. Rate and extent of fermentation and apparent organic matter degradation (OMD) were determined in vitro, using a gas production technique. Addition of enzyme linearly increased (P < 0.01) gas production for up to 12 h (68.9, 70.9, 67.6, 67.9, 71.9, and 74.9 ml/g OM for control, L1-L5, respectively) and OMD for up to 19 h incubation (0.425, 0.444, 0.433, 0.446, 0.443, and 0.451 for control, L1-L5, respectively), but no increases (P > 0.05) were detected thereafter. In Experiment 2, the effect of the same enzyme as used previously (added at 0.51 mu l/g forage DM, directly into the incubation medium), and buffer pH were examined using the ANKOM system, in a completely randomized design. Incubation medium pH was altered using 1 M citric acid, in order to obtain target initial pH values of 6.8 (control, no citric acid added), 6.2, 5.8, and 5.4. Actual initial pH values achieved were 6.72, 6.50, 6.20, and 5.72. Lowering the pH decreased (P < 0.01) dry matter disappearance (DMD) at 18 h incubation (0.339, 0.341, 0.314, and 0.291 for 6.72, 6.50, 6.20, and 5.72, respectively), whereas enzyme addition increased (P < 0.05) DMD at 24 h (0.363 versus 0.387 for control and enzyme-treated, respectively). Addition of enzyme increased (P < 0.05) neutral detergent fibre (NDF), acid detergent fibre (ADF), and hemicellulose (HC) degradation at pH 6.50 (0.077 versus 0.117; 0.020 versus 0.051; 0.217 versus 0.270 for control and enzyme-treated NDF, ADF and hemicellulose degradation, respectively) and 6.72 (0.091 versus 0.134; 0.041 versus 0.079; 0.205 versus 0.261 for control and enzyme-treated NDF, ADF and HC degradation, respectively). It is concluded that the positive effects of this enzyme product were independent of the pre-treatment period, but pH influenced the responses to enzyme supplementation. Under the conditions of this experiment, exogenous fibrolytic enzymes seemed to work better at close to neutrality ruminal pH conditions. (C) 2006 Elsevier B.V. All rights reserved.

The regulation of sweet cherry fruit abscission by polar auxin transport

Inconsistency of cropping is an important problem for UK sweet cherry production. Premature fruit abscission in Prunus can reduce yields severely, however, the environmental cues and hormonal signals that trigger abscission have not been identified. Auxin (IAA) is known to delay abscission by reducing the sensitivity of cells in the abscission zone to ethylene, a promoter of abscission. Therefore, the capacity for polar auxin transport (PAT) through sweet cherry pedicels was examined in relation to fruit abscission. Cherry ‘spurs’ (short shoots) with similar leaf areas and different fruit numbers were phloem-girdled to restrict assimilate movement. Abscission from spurs with many fruit (eight or more) occurred within 14 days of girdling, whereas abscission from spurs with few (two) fruit was minimal. The pedicels’ capacity for PAT in spurs with different fruit numbers was determined 1, 3 and 9 days after girdling (DAG). Fruit were analysed for endogenous IAA concentration 3, 5, 7 and 9 DAG. PAT inhibitors 2,3,5-triiodobenzoic acid or 1-N-naphthylphtalamic acid were applied to pedicels of fruit not expected to abscise, i.e. on spurs with few fruit. The effect of these inhibitors on fruit abscission was determined 14 DAG. The proportion of the transported [3H]-IAA was lower from the outset in pedicels from spurs with many fruit. By 9 DAG, symptoms of fruit abscission were apparent and 40% less [3H] -IAA was transported through pedicels on spurs with many fruit. Fruit endogenous IAA concentrations were similar in the two groups of spurs. Application of PAT inhibitors shortly after girdling increased fruit abscission by 30%. The results suggest that although a decline in PAT is not the only cause of fruit abscission, the maintenance of PAT contributes to fruit retention.