Non-transgenic Research Articles

Abstract P134: Identification of Tissue Inhibitor of Metalloproteinases (TIMP)-4 as a Novel PPARγ Target in Smooth Muscle Cell

Patients with PPARγ mutations develop severe early onset hypertension, type 2 diabetes and lipodystrophy. Our recent findings demonstrated that transgenic mice expressing a dominant negative (DN) PPARγ mutant in vascular smooth muscle (S-P467L) exhibited exacerbated DOCA-salt induced hypertension and vascular remodeling in conduit and resistance arteries. Here, we hypothesized that predisposition to vascular injury during hypertension in S-P467L mice is through altered expression of PPARγ target genes in smooth muscle cells (SMC). Gene expression profiling in aorta and mesenteric arteries revealed a significant loss of Tissue Inhibitor of Metalloproteinases (TIMP)-4 in S-P467L compared to non-transgenic (NT) littermates. Interference with PPARγ activity either by treatment with PPARγ inhibitor, GW9662 or expressing P467L PPARγ markedly suppressed TIMP-4 in primary SMC, suggesting that loss of TIMP-4 in S-P467L arteries is a direct result of PPARγ inhibition. Downregulation of TIMP-4 in SMC by GW9662 was correlated with a significant increase in total MMP activity (Fluorescent signal subtracted from negative control; vehicle: 1±0.5, GW9662: 2.2±0.7, p<0.05), consistent with TIMP-4 function as an endogenous inhibitor of MMPs. Overexpressing TIMP-4 in SMC significantly blunted cell migration compared to those with empty plasmid (change in open area after 8 hr; Empty: 1±0.05, TIMP-4: 0.8±0.051, p<0.05), whereas decreased TIMP-4 expression caused by mutant PPARγ resulted in increased migration (change in open area after 10 hr; GFP: 1±0.25, P467L: 1.63±0.26, p<0.05). The significance of TIMP-4 was also underscored during hypertension since the compensatory increase in TIMP-4 during DOCA-salt in NT mesenteric arteries was lost in S-P467L. We identified two highly conserved potential PPAR response elements (PPREs) close to TIMP-4 promoter region using a sequence-based model. Chromatin immunoprecipitation assay showed strong binding of PPARγ at one of the suggested PPREs in SMC (% input±sem; GFP: 37±0.6, P467L: 66±0.7, p<0.05 vs. IgG), suggesting that TIMP-4 is a direct target of PPARγ. Our findings highlight one of protective mechanisms of PPARγ during hypertension and provide a novel link between PPARγ and TIMP-4.

Characterization of transgenic Poncirus trifoliata overexpressing the ferric chelate reductase gene CjFRO2 from Citrus junos

Iron deficiency chlorosis occurs frequently in calcareous soils. The transformation of plants with ferric chelate reductase genes (FROs) provides a potential strategy to alleviate plant chlorosis under iron deficiency. A CjFRO2 gene isolated from Citrus junos Sieb. ex Tanaka was introduced into Poncirus trifoliata (L.) Raf via Agrobacterium-mediated transformation. The transgene integration and expression were confirmed by PCR, Southern blot, and real-time PCR analyses. Hydroponic- and soil-grown transgenic plants were tested for their tolerance to iron deficiency. Compared with nontransgenic (NT) P. trifoliata plants, a rhizosphere acidification capacity in the transgenic lines increased, and a ferric chelate reductase activity in roots was up to 3.39- and 2.93-fold higher in a hydroponic solution and soil, respectively. A transgenic line TO-8, which reacted similarly in hydroponics and soil, appeared tolerant to the iron deficiency. Its leaf chlorophyll and ferrous ion content was significantly higher than in NT. These results indicate that tolerance to the iron deficiency in P. trifoliata could be improved through the genetic engineering.

Abstract 376: Amino Terminal C0-C1f Region of Cardiac Myosin Binding Protein-C is Essential for Normal Cardiac Function

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N’) regions. However, it is unknown whether the first 271 residues (C0-C1f region) are necessary to regulate contractile function in vivo. Hypothesis: The N’-region of cMyBP-C is critical for proper cardiac function in vivo. Methods and Results: Transgenic mice with approximately 80% expression of mutant truncated cMyBP-C missing C0-C1f (cMyBP-C 110kDa ), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C 110kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C 110kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C 110kDa hearts. Importantly, cMyBP-C 110kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG littermates. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C 110kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C 110kDa hearts compared to NTG hearts. Conclusion: The N’-region of cMyBP-C is indispensable for maintaining normal cardiac morphology and function and loss of this region promotes contractile dysfunction both at the molecular and tissue levels.

Abstract 115: A Decrease in Mitochondrial, but Not Cytosolic, Iron Protects Against Cardiac Ischemia-reperfusion Damage Through a Reduction in Ros

Introduction: Iron is essential for the activity of several cellular proteins, but excess free iron can cause cellular damage through production of reactive oxygen species (ROS). Iron accumulation in mitochondria, the major site of cellular iron homeostasis, leads to cardiomyopathy. However, it is not known whether a reduction in baseline mitochondrial iron (as opposed to iron in other cellular compartments) can protect against ischemia-reperfusion (I/R) injury in the heart. We hypothesized that since mitochondria are the major site of iron homeostasis and that mitochondrial iron can lead to oxidative damage, a reduction in mitochondrial iron at baseline would be sufficient to protect against I/R injury. Results: Transgenic (TG) mice with cardiomyocyte-specific overexpression of the mitochondrial iron export protein ATP-binding cassette (ABC)-B8 had significantly lower mitochondrial iron in the heart than nontransgenic (NTG) littermates at baseline, but their cardiac function and the expression of key antioxidant systems were similar to NTG littermates. In response to I/R, TG mice displayed significantly less apoptosis and lipid peroxidation products and better preserved cardiac function than NTG littermates, suggesting that a reduction in mitochondrial iron protects against I/R injury. To confirm these results, we next took a pharmacological approach to assess the effects of a reduction in mitochondrial vs cytosolic iron on the response to I/R using 2,2’-bipyridyl (BPD, a mitochondria-accessible iron chelator) and deferoxamine (DFO, an iron chelator that can only reduce cytosolic iron). Mice pretreated with BPD but not DFO are protected against I/R injury. In addition, BPD but not DFO treatment in rat cardiomyoblast H9C2 cells significantly lowered chelatable mitochondrial iron and protected against H2O2 induced cell death. These results suggest that a reduction in baseline mitochondrial, but not cytosolic, iron is sufficient to protect against I/R injury. Conclusions: Our findings demonstrate that selective reduction in mitochondrial iron is protective in I/R injury. Thus, targeting mitochondrial iron with selective iron chelators may provide a novel approach for treatment of ischemic heart disease.

The barley lipid transfer protein, BLT101, enhances cold tolerance in wheat under cold stress

To understand the mechanism by which the barley plants tolerate overwintering, we analyzed the BLT101 gene expression in four different cultivars including two each of spring and winter barleys, where the BLT101 transcript level varied among the cultivars with a long-term cold acclimation. The BLT101 transcript was induced at a higher level in the crown tissues in response to low temperature treatment. The BLT101 transcript level correlated with those of the corresponding protein. BLT101-overexpressing transgenic wheat lines (BLT101ox’s) grew slowly and showed decreased expression of the genes induced by hormones (such as auxin and cytokinin), compared to non-transgenic (NT) plants. After a prolonged cold treatment, the BLT101ox leaves showed normal phenotypes, while the NT plants displayed blighted and withered leaves. A substances leakage test showed that the BLT101ox wheat retains enhanced freezing tolerance, compared to the NT plants. The transgenic lines lost less water than the NT plants, under a cold-stressed condition. Our findings suggest that tolerance to water stress and the retardation of growth in cold conditions, the essential features in overwintering barleys, involve BLT101.

Sleep-Wake Cycle Dysfunction in the TgCRND8 Mouse Model of Alzheimer's Disease: From Early to Advanced Pathological Stages.

In addition to cognitive decline, individuals affected by Alzheimer’s disease (AD) can experience important neuropsychiatric symptoms including sleep disturbances. We characterized the sleep-wake cycle in the TgCRND8 mouse model of AD, which overexpresses a mutant human form of amyloid precursor protein resulting in high levels of β-amyloid and plaque formation by 3 months of age. Polysomnographic recordings in freely-moving mice were conducted to study sleep-wake cycle architecture at 3, 7 and 11 months of age and corresponding levels of β-amyloid in brain regions regulating sleep-wake states were measured. At all ages, TgCRND8 mice showed increased wakefulness and reduced non-rapid eye movement (NREM) sleep during the resting and active phases. Increased wakefulness in TgCRND8 mice was accompanied by a shift in the waking power spectrum towards fast frequency oscillations in the beta (14-20 Hz) and low gamma range (20-50 Hz). Given the phenotype of hyperarousal observed in TgCRND8 mice, the role of noradrenergic transmission in the promotion of arousal, and previous work reporting an early disruption of the noradrenergic system in TgCRND8, we tested the effects of the alpha-1-adrenoreceptor antagonist, prazosin, on sleep-wake patterns in TgCRND8 and non-transgenic (NTg) mice. We found that a lower dose (2 mg/kg) of prazosin increased NREM sleep in NTg but not in TgCRND8 mice, whereas a higher dose (5 mg/kg) increased NREM sleep in both genotypes, suggesting altered sensitivity to noradrenergic blockade in TgCRND8 mice. Collectively our results demonstrate that amyloidosis in TgCRND8 mice is associated with sleep-wake cycle dysfunction, characterized by hyperarousal, validating this model as a tool towards understanding the relationship between β-amyloid overproduction and disrupted sleep-wake patterns in AD.

Overexpression of the genes coding ascorbate peroxidase from Brassica campestris enhances heat tolerance in transgenic Arabidopsis thaliana

Previously, the ascorbate peroxidase (APX1) activity and gene expression in Chinese cabbage (Brassica campestris, Bc) heat-tolerant cv. ASVEG2 were found to be significantly higher than in heat-sensitive cv. RN720 under a heat stress. Furthermore, BcAPX2 and BcAPX3, isoforms of BcAPX1, were cloned in this study. Our objective was to transfer BcAPX cDNA under the control of the ubiquitin promoter to Arabidopsis via Agrobacterium tumefaciens strain GV3101. We found that BcAPX genes were overexpressed in transgenic Arabidopsis, and the expression of APX, and the APX activity in transgenic lines were higher than in non-transgenic (NT) plants under high temperatures. The chlorophyll content and the germination rate were significantly higher, and the malondialdehyde content was lower in BcAPX1-3, 2-1, and 3–5 lines subjected to the heat-stress treatment than those in the NT plants. Compared to the NT plants, a lower heat-induced H2O2 accumulation was detected by diaminobenzidine staining in leaves of the transgenic lines with a high APX activity indicating that the overexpression of BcAPX in Arabidopsis could enhance heat tolerance by eliminating H2O2.

Targeted Overexpression of Astrocytic Endothelin-1 Attenuates Neuropathic Pain by Upregulating Spinal Excitatory Amino Acid Transporter-2

We previously demonstrated that endogenous endothelin-1 (ET-1) inhibits pathological pain in a transgenic mouse model with astrocyte-specific ET-1 overexpression (GET-1 mice); however, the underlying mechanism is unclear. ET-1 regulates excitatory amino acid transporter-2 (EAAT-2), a predominant subtype of glutamate transporters that plays critical role in pain modulation in spinal astrocytes. We hypothesized that astrocytic ET-1 overexpression alleviates neuropathic pain through modulating EAAT-2. GET-1 or nontransgenic (NTg) mice either received sham operation or sciatic nerve ligation (SNL) with or without ceftriaxone (CEF, an EAAT-2 inducer, for 4days before termination). In GET-1 mice, mRNA and protein expressions of EAAT-2, but not EAAT-1, were upregulated associated with reduced SNL-induced neuropathic pain. Despite that SNL induced a significant reduction of EAAT-2 mRNA expression in both genotypes of mice, post-SNL EAAT-2 mRNA expression was higher in GET-1 mice than that in NTg mice. EAAT-2 induction by CEF reduced SNL-induced neuropathic pain in both NTg and GET-1 mice. In cultured rat astrocytic cell line, overexpression of ET-1 mRNA expression also elevated EAAT-2 mRNA expression, which was reversed by ET receptor antagonists. In conclusion, overexpressed astrocytic ET-1 suppressed neuropathic pain by upregulating spinal EAAT-2 expression via ET receptors.

Food intake and appetite control in a GH-transgenic zebrafish.

The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. The regulatory network for GH is complex and includes many central and peripheral endocrine factors as well as that from the environment. It is known that GH transgenesis results in increased growth, food intake, and consequent metabolic rates in fishes. However, the manner in which GH transgenesis alters the energetic metabolism in fishes has not been well explored. In order to elucidate these consequences, we examined the effect of GH overexpression on appetite control mechanisms in a transgenic zebrafish (Danio rerio) model. To this, we analyzed feeding behavior and the expression of the main appetite-related genes in two different feeding periods (fed and fasting) in non-transgenic (NT) and transgenic (T) zebrafish as well as glycaemic parameters of them. Our initial results have shown that NT males and females present the same feeding behavior and expression of main appetite-controlling genes; therefore, the data of both sexes were properly grouped. Following grouped data analyses, we compared the same parameters in NT and T animals. Feeding behavior results have shown that T animals eat significantly more and faster than NT siblings. Gene expression results pointed out that gastrointestinal (GT) cholecystokinin has a substantial contribution to the communication between peripheral and central control of food intake. Brain genes expression analyses revealed that T animals have a down-regulation of two strong and opposite peptides related to food intake: the anorexigenic proopiomelanocortin (pomc) and the orexigenic neuropeptide Y (npy). The down-regulation of pomc in T when compared with NT is an expected result, since the decrease in an anorexigenic factor might keep the transgenic fish hungry. The down-regulation of npy seemed to be contradictory at first, but if we consider the GH's capacity to elevate blood glucose, and that NPY is able to respond to humoral factors like glucose, this down-regulation makes sense. In fact, our last experiment showed that transgenics presented elevated blood glucose levels, confirming that npy might responded to this humoral factor. In conclusion, we have shown that GT responds to feeding status without interference of transgenesis, whereas brain responds to GH transgenesis without any effect of treatment. It is clear that transgenic zebrafish eat more and faster, and it seems that it occurs due to pomc down-regulation, since npy might be under regulation of the humoral factor glucose.

Influence of feeding a high plant protein diet on growth and nutrient utilization to combined ‘all-fish’ growth-hormone transgenic diploid and triploid Atlantic salmon (Salmo salar L.)

Abstract This study assessed the capacity of non-transgenic (NTG) and growth-hormone transgenic (TG; gene construct EO-1α) Atlantic salmon (Salmo salar L.), comprised of conventional diploid (DIP) and reproductively-sterile triploid (TRIP) fish, to utilize a diet containing relatively high amounts of plant protein (PP) concomitant with lower levels of fish meal (FM) protein. Triplicate groups of full-sibling NTG/DIP, NTG/TRIP, TG/DIP and TG/TRIP salmon (initial weight, 27–35 g) were held in freshwater and fed two experimental diets until they exceeded 400% growth. Two isonitrogenous (50% crude protein), isolipidic (21% lipid) and isoenergetic (22 MJ/kg gross energy) experimental diets were tested. The control diet (FM) contained 64 and 36%, while PP diet contained 32 and 68% of total dietary protein from FM and PP, respectively. TG and NTG fish achieved the target (> 150 g) weight in 89 and 206 days, respectively. TG fish exhibited significantly higher specific growth rates (SGR) (2.48 vs 0.7%/day) and thermal growth coefficient (TGC) (3.04 vs 0.79) than NTG, regardless of ploidy or diet. TG/TRIP fish had significantly lower growth rates than DIP due to lower feed intake, while no ploidy effect was observed within the NTG group. Feed conversion ratio (FCR) was significantly better in TG/DIP and TG/TRIP fish having achieved the same target weight with 20–25% less feed due to improved protein utilization and retention efficiency compared to NTG fish. NTG fish had higher digestibility of protein (93% vs 89%), lipid (95% vs 94%) and energy (89% vs 85%) relative to their TG siblings, and was similar between DIP and TRIP fish. Nutrients' digestibility was significantly lower in TG fish fed PP diet than those fed FM diet, regardless of ploidy. At the end of the study, TG fish had significantly lower whole-body protein (56% vs 59%) and higher lipid (36% vs 34%) and energy (2746 vs 2694 kJ/100 g) content than NTG fish. However, as a result of the rapid growth rate and efficient feed utilization, nutrient gain and retention efficiencies were significantly higher in TG than NTG fish. DIP and TRIP TG Atlantic salmon have the ability to maintain accelerated growth even when fed a high PP diet (68% of dietary protein), which may have important benefits for the production of growth hormone transgenic Atlantic salmon. Statement of relevance The results from the present study are innovative and may have important benefits for the optimization and production of growth hormone transgenic Atlantic salmon using sustainable feed ingredients.

Marble-burying is enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable by handling

Translational research on behavioural and psychological symptoms of dementia (BPSD) is relevant to the study the neuropsychiatric symptoms that strongly affect the quality of life of the human Alzheimer’s disease (AD) patient and caregivers, frequently leading to early institutionalization. Among the ethological behavioural tests for rodents, marble burying is considered to model the spectrum of anxiety, psychotic and obsessive–compulsive like symptoms. The present work was aimed to study the behavioural interactions of 12 month-old male 3xTg-AD mice with small objects using the marble-burying test, as compared to the response elicited in age-matched non-transgenic (NTg) mice. The distinction of the classical ‘number of buried marbles’ but also those left ‘intact’ and those ‘changed’ of position of marbles or partially buried (the transitional level of interaction) provided new insights into the modelling of BPSD-like alterations in this AD model. The analysis revealed genotype differences in the behavioural patterns and predominant behaviors. In the NTg mice, predominance was shown in the ‘changed or partially buried’, while interactions with marble were enhanced in 3xTg-AD mice resulting in an increase of marble burying. Besides, genotype-dependent meaningful correlations were found, with the marble test pattern of 3xTg-AD mice being directly related to neophobia in the corner tests. In both genotypes, the increase of burying was reversed by chronic treatment with risperidone (1mg/kg, s.c.). In 3xTg-AD mice, the repetitive handling of animals during the treatment also exerted modulatory effects. These distinct patterns further characterize the modelling of BPSD-like symptoms in the 3xTg-AD mice, and provide another behavioural tool to assess the benefits of preventive and/or therapeutic strategies, as well as the potential action of risk factors for AD, in this animal model.

Expression of both CuZnSOD and APX in chloroplasts enhances tolerance to sulfur dioxide in transgenic sweet potato plants

We have previously reported that transgenic sweet potato (Ipomoea batatas) plants overexpressing both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of a stress-inducible SWPA2 promoter in chloroplasts (referred to as SSA plants) showed increased resistance to methyl viologen-mediated oxidative stress and chilling. To investigate whether SSA plants show enhanced tolerance to air pollutants, they were exposed to 500ppb of sulfur dioxide (SO2). SO2 caused visible damage to the leaves of sweet potato, but damage in the leaves of non-transgenic (NT) plants was more severe than in those of SSA plants. The photosynthetic activity (Fv/Fm) of the SSA plants decreased by only 7% on the 5th day after the treatment, whereas that of NT plants severely decreased by 63% after 5days of recovery. Moreover, the chlorophyll content in the oldest leaf of NT plants decreased by 69%, whereas that of SSA plants remained at a high level. APX activity in NT plants increased about three times under an SO2 stress, and in SSA plants about five times compared to the case with no stress conditions. These results suggest that the overexpression of both CuZnSOD and APX in chloroplasts reduces the oxidative stress derived from SO2.

Increased Renal Oxidative Stress in hGRK4 486V Transgenic Mice

Variants of hGRK4 are associated with human essential hypertension. hGRK4486V transgenic mice have salt-sensitive hypertension. Because renal oxidative stress is increased in some rodent models of salt-sensitive hypertension, we quantified renal expression of reactive oxygen species-related proteins of hGRK4486V transgenic and non-transgenic (NT) mice on normal (NS, 0.8%) and high (HS, 4%) NaCl diets. Systolic blood pressure was similar in hGRK4486V (89.83±2.7, mm Hg, n=9) and NT (94.7±2.5) mice on NS diet and elevated in hGRK4486V (114±6.1) but not in NT mice (94.1±2.8) on HS diet. The renal expressions of NOX1, 2, and 4, were similar in both strains on NS diet but NOX2 was decreased by HS in NT (28±7, % of NT on NS diet, n=5-6/group). On NS diet, CuZnSOD and ECSOD were similar in the two mouse strains while MnSOD (66±3%) was lower in GRK4486V than NT mice. However, on HS diet, CuZnSOD (87±7%), MnSOD (70±3%), and ECSOD (55±7%) were decreased in GRK4486V mice not in NT mice. HO-2, not HO-1, was slightly greater in GRK4486V than NT mice on NS diet (117±7%) but this difference was abolished by HS diet. Urinary 8-isoprostane was lower in GRK4486V than NT mice (57±2.5 vs 70±0.1, ng/mg of Cr) on NS diet but increased to a greater extent in GRK4486V than NT mice (197±18 vs128±6) on HS diet. Renal SOD activity and superoxide production were similar in both strains on NS diet. HS diet decreased SOD activity (81.6±2.7%) and increased superoxide (138.9±6.6%) production in GRK4486V but not in NT mice. HS diet also decreased the renal expression of NOS3, not NOS 1 and 2, in GRK4486V (49±2%) but not in NT mice. These findings suggest that the salt-sensitive hypertension of GRK4486V mice is related to renal oxidative stress due to decreased protein expression and activity of SODs and decreased NOS3 protein.

Vinexin-β exacerbates cardiac dysfunction post-myocardial infarction via mediating apoptotic and inflammatory responses.

Vinexin-β is one of the adaptor proteins that are primarily involved in signal transduction and cytoskeletal organization under various pathological conditions, including cardiac hypertrophy. However, the role of Vinexin-β in myocardial infarction (MI) remains unknown. In this study, dramatically up-regulated Vinexin-β expression was observed in both ischaemic human hearts and infarcted animal hearts. To explore the potential involvement of Vinexin-β in MI further, we induced MI injury in global Vinexin-β-knockout mice and wild-type (WT) controls as well as in mice with cardiac-specific over-expression of the human Vinexin-β gene-transgenic (TG) and -non-transgenic (NTG) littermates. Compared with that observed in WT controls, Vinexin-β deficiency significantly decreased MI-induced infarct size, concomitant with an improved cardiac function, leading to an increase in the survival rate. The myocardial apoptosis in the border zone was dramatically reduced by Vinexin-β deficiency, resulting from the altered expression of apoptotic factors. Furthermore, Vinexin-β depletion mitigated the inflammatory response, as evidenced by reduced inflammatory cell infiltration, decreased expression of cytokines and the inactivation of NF-κB (nuclear factor κB) signalling. In contrast, Vinexin-β-TG mice were much more susceptible to MI injury compared with NTG controls. Further mechanism analyses suggested that Vinexin-β exerted detrimental effects largely dependent on blocking AKT signalling. The effects and mechanisms of Vinexin-β on MI observed in vivo were further confirmed by our in vitro assays. When collected, these data demonstrate for the first time that Vinexin-β increases MI-induced mortality and worsens cardiac dysfunction through aggravation of myocardial apoptosis and inflammatory response.

Journal of Bone and Mineral Research: Volume 30, Number 3, March 2015

Abstract Cover: Transgenic mice overexpressing the mutant Ifi tm5. Skeletal preparations of mutant transgenics (Mut Tg) at E18.5 show reduced alizarin red staining and fractures which are not evident in wild type transgenic (WT Tg) or non-transgenic (NT) littermates. (see Lietman et al. beginning on page 489)

Transcription factors and anthocyanin genes related to low-temperature tolerance in rd29A:RdreB1BI transgenic strawberry

Dehydration-responsive element-binding (DREB) transcription factors play critical roles in plant stress responses and signal transduction. To further understand how DREB regulates genes expression to promote cold-hardiness, Illumina/Solexa sequencing technology was used to compare the transcriptomes of non-transgenic and rd29A:RdreB1BI transgenic strawberry plants exposed to low temperatures. Approximately 3.5 million sequence tags were obtained from non-transgenic (NT) and transgenic (T) strawberry untreated (C) or low-temperature treated (LT) leaf samples. Over 1000 genes were differentially expressed between the NT-C and T-C plants, and also the NT-C and NT-LT, as well as the T-C and T-LT plants. Analysis of the genes up-regulated following low-temperature treatment revealed that the majority are linked to metabolism, biosynthesis, transcription and signal transduction. Uniquely up-regulated transcription factors as well as anthocyanin biosynthetic pathway genes are discussed. Accumulation of anthocyanin in the stolon and the base of the petiole differed between non-treated NT and T plants, and this correlated with gene expression patterns. The differentially expressed genes that encode transcription factors and anthocyanin enzymes may contribute to the cold hardiness of RdreB1BI transgenic strawberry. The transcriptome data provide a valuable resource for further studies of strawberry growth and development and DREB-mediated gene regulation under low-temperature stress.

Transcriptome analysis reveals the role of BpGH3.5 in root elongation of Betula platyphylla × B. pendula

The GH3 family comprises a set of early auxin-responsive genes. Several previous studies have proven that GH3 family proteins play important roles in various aspects of plant growth and development. Our previous studies have found that the overexpression or suppression of BpGH3.5 in birch tree can result in shorter primary and lateral root compared to non-transgenic control, however, the molecular mechanism underlying is not clear. To further reveal the roles of BpGH3.5 in root elongation of birch (Betula platyphylla × B. pendula), we carried out transcriptome analysis on BpGH3.5-overexpressed (G1 and G5), BpGH3.5-suppressed (AG1), and non-transgenic (NT) birch. Compared with the NT line, 3,320 and 5,212 unigenes were found to be differentially expressed in the BpGH3.5 overexpressed and suppressed lines, respectively. Many differentially expressed unigenes related to cell proliferation and growth were downregulated in both the BpGH3.5-overexpressed and suppressed plants, which is consistent with the short-root phenotypes observed in these lines. We found that indole-3-acetic acid (IAA) biosynthesis and signaling pathways were affected in the BpGH3.5-overexpressed and suppressed plants. Interestingly, Free IAA levels were significantly lower in BpGH3.5-overexpressed and BpGH3.5-suppressed birch than in NT plants. This suggested that the expression levels of BpGH3.5 may play a role in maintaining the balance of free IAA in birch and the decreased IAA levels may be responsible for the short-root phenotype of the transgenic birch.

Abstract 76: Genetic Interference with PPARγ in Endothelium Accelerates Oxidative Stress, Inflammation, and Vascular Senescence With Aging

Although aging is the greatest risk factor for vascular disease and stroke, very little is known regarding mechanisms that control vascular aging. Endothelial dysfunction - a critical element of carotid artery and cerebrovascular disease - progresses with age, contributing to increased risk for ischemic stroke and cognitive impairment. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) exerts protective effects in the vasculature when pharmacologically activated. Because endothelial PPARγ may normally protect against vascular disease, we examined the hypothesis that genetic interference with PPARγ in endothelium would promote vascular aging. We studied carotid arteries from adult (11-12 mo) and old (24±1 mo) transgenic mice with endothelial specific expression of a dominant negative mutation in PPARγ driven by the vascular cadherin promoter (designated E-V290M), along with age-matched, non-transgenic (NT) littermates. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of arteries from adult NT and E-V290M mice as well as old NT mice. In contrast, responses to acetylcholine were reduced by greater than 50% in old E-V290M mice (P<0.01). Endothelial function in old E-V290M mice was not altered by an inhibitor of cyclooxygenase, but was restored to normal by a superoxide scavenger, or an inhibitor of NADPH oxidase, or inhibition of Rho kinase. Relaxation of arteries to nitroprusside, which acts directly on vascular muscle, was similar in all groups. Vascular expression of interleukin-6, a mediator of vascular disease, was increased 1.6-fold in old NT mice, but almost 9-fold in old E-V290M mice (P<0.05). Interestingly, expression of CDKN2A, a molecular marker of senescence, was increased 2.6-fold in old NT mice, but almost 6-fold in old E-V290M mice (P<0.05). These findings provide the first evidence that senescence and age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARγ through mechanisms that appear to involve oxidative stress, inflammation, and Rho kinase. This critical role for endothelial PPARγ has implications for vascular pathophysiology as well as therapeutic approaches for age-induced large and small vessel disease.

Bioactive compounds of tomato fruits from transgenic plants tolerant to drought

Advances in agricultural biotechnology bring forth the need for experimental evidence for benefits and risks of engineered crops and the quality of fruit products obtained from them. Tomato fruits from non-transgenic (NT) and BcZAT12-transformed tomato lines ZT1-ZT6 (cv. H-86, var. Kashi vishesh) tolerant to drought, were assessed for nutritional quality, changes in physico-chemical characteristics and health-related bioactive compounds. Fruits from transgenics were evaluated for size, pH, total-soluble solids, total sugars, phenolics, flavonoids, vitamin C, lycopene and β-carotene. An early ripening of tomatoes from transgenics with more red but smaller fruits, high sugar levels, elevated phenolics, flavonoids, lycopene and β-carotene with an unaltered vitamin C levels as compared to tomatoes from non-transgenics were noted. Results suggest that although differences between tomatoes from transgenics and non-transgenics do exist, yet tomato fruits from transgenic plants have relatively improved antioxidant capacity than those from non-transgenics and therefore may be products safe for consumption.

Cardiac Myosin Essential Light Chain N-Terminus Regulates Motor Step-Size

Slow ventricular cardiac myosin (βmys) has three distinct unitary step-sizes, nominally 3, 5, and 8 nm, that move actin with varying step-frequencies. Ventricular cardiac myosin essential light chain (vELC) has a 43 residue N-terminal extension that binds actin. The basis for the three unitary steps in cardiac myosin was proposed to involve actin binding of the vELC extension wherein the predominant 5 nm step does not engage the actin/vELC linkage, the slightly less likely 8 nm step engages actin/vELC linkage facilitating an extra ∼19 degrees of lever-arm rotation, and the minor 3 nm step is the unlikely conversion of the 5 nm step to the full actin/vELC bound 8 nm step. The hypothesis was tested using several cardiac myosin constructs including a 17 residue N-terminal truncated vELC in porcine mys (Δ17) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43). Mouse myosin heavy chain is the fast isoform (αmys). Cardiac myosin step-size and relative step-frequency were measured using a high throughput Qdot super-resolution in vitro motility assay. Δ17 showed a significant increase to the 5 nm step-frequency and coincident reduction to the 3 and 8 nm step-frequencies implying a reduction in probability for forming vELC/actin linkage. Nontransgenic (NTg) and human vELC expressed αmys in transgenic mouse heart had three unitary step-sizes like those in βmys although step-frequencies in NTg were redistributed to favor the 8 nm step. Δ43 showed a significant increase to the 5 nm step-frequency and a significant reduction to the combined 3 and 8 nm step-frequency again indicating lower overall probability for forming vELC/actin linkage. Data strongly supports the hypothesis that the vELC extension is the basis for three step-sizes in cardiac myosin. Supported by NIH grants R01AR049277, R01HL095572, and R01HL108343.