Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa.

AIMS: The aim of this study was to determine if endophytes from wild and ancient Zea plants (corn family) have anti-fungal activities, specifically against the most important fungal pathogen (Sclerotinia homoeocarpa) of creeping bentgrass, a relative of Zea, used here as a model grass. METHODS AND RESULTS: A library of 190 bacterial endophytes from wild, ancient and modern Zea plants were tested for their ability to suppress S. homoeocarpa in vitro, followed by in planta testing of candidates using greenhouse trials. Three endophytes could suppress S. homoeocarpa, originating from wild maize and an ancient Mexican landrace, consistent with our hypothesis. 16S phylogenetic analysis and BOX-PCR DNA fingerprinting suggest that the anti-fungal endophytes are distinct strains of Burkholderia gladioli. One strain (3A12) was confirmed to colonize creeping bentgrass using green fluorescent protein (GFP) tagging. Evans blue vitality staining demonstrated that the bacterial endophytes exhibited fungicidal activities against the pathogen. The endophytes inhibited a wide spectrum of plant-associated fungi including diverse crop pathogens. CONCLUSIONS: The results support the hypothesis that wild and ancient Zea genotypes host bacterial endophytes that can control fungal pathogen(s). SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that wild and ancient crops may be an unexplored reservoir of anti-fungal bacterial endophytes.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. EXPERIMENTAL APPROACH: A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. KEY RESULTS: Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

C1 catecholamine neurons form local circuit synaptic connections within the rostroventrolateral medulla of rat.

C1 catecholamine neurons reside within the rostroventrolateral medulla (RVLM), an area that plays an integral role in blood pressure regulation through reticulospinal projections to sympathetic preganglionic neurons in the thoracic spinal cord. In a previous investigation we mapped the efferent projections of C1 neurons, documenting supraspinal projections to cell groups in the preautonomic network that contribute to the control of cardiovascular function. Light microscopic study also revealed putative local circuit connections within RVLM. In this investigation we tested the hypothesis that RVLM C1 neurons elaborate a local circuit synaptic network that permits communication between C1 neurons giving rise to supraspinal and reticulospinal projections. A replication defective lentivirus vector that expresses enhanced green fluorescent protein (EGFP) under the control of a synthetic dopamine beta hydroxylase (DßH) promoter was used to label C1 neurons and their processes. Confocal fluorescence microscopy demonstrated thin varicose axons immunopositive for EGFP and tyrosine hydroxylase that formed close appositions to C1 somata and dendrites throughout the rostrocaudal extent of the C1 area. Dual-labeled electron microscopic analysis revealed axosomatic, axodendritic and axospinous synaptic contacts with C1 and non-C1 neurons with a distribution recapitulating that observed in the light microscopic analysis. Labeled boutons were large, contained light axoplasm, lucent spherical vesicles, and formed asymmetric synaptic contacts. Collectively these data demonstrate that C1 neurons form a synaptic network within the C1 area that may function to coordinate activity among projection-specific subpopulations of neurons. The data also suggest that the boundaries of RVLM should be defined on the basis of function criteria rather than the C1 phenotype of neurons.

Desflurane and sevoflurane attenuate oxygen and glucose deprivation-induced neuronal cell death.

Neuronal cell death may occur via two pathways: those causing necrosis or those causing apoptosis. Apoptosis can be activated during periods of stress such as oxygen and glucose deprivation. Anesthetic agents such as desflurane or sevoflurane can attenuate early neuronal necrotic death, but their effect on oxygen and glucose deprivation-induced apoptosis has not been investigated. Neuronal cell cultures were prepared from neonatal rat cortex and were used between 10 and 14 days in vitro. The neuronal cell cultures were pretreated 30 minutes prior to oxygen and glucose deprivation with either desflurane or sevoflurane (N = 18). Three concentrations of each anesthetic were evaluated. The cultures were then deprived of oxygen and glucose for 30, 60, or 90 minutes. Treatment with desflurane or sevoflurane was continued during the period of oxygen and glucose deprivation. Forty-eight hours after exposure, the cells were examined for apoptosis using TUNEL and DNA gel electrophoresis. Comparisons were made to neuronal cortical cell cultures exposed to oxygen and glucose deprivation alone (N = 9). This in vitro model of oxygen and glucose deprivation was successful in producing neuronal cell death during the exposure times examined. During 30-, 60-, and 90-minute periods of oxygen and glucose deprivation, both desflurane and sevoflurane significantly ( approximately 98%) attenuated neuronal cell death regardless of concentration.

Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells.

There is increasing evidence that angiotensin II influences thrombogenesis by regulating the expression of plasminogen activator inhibitor-1 (PAI-1). In this study, the effects of angiotensin II and its receptors on the expression and release of PAI-1 and tissue-type plasminogen activator (t-PA) were examined in human coronary artery endothelial cells (HCAECs). As control, cells were treated with angiotensin IV. HCAECs incubated with angiotensin II increased the expression of PAI-1 mRNA in a concentration (10-9-10-5 M)- and time (6-24 h)-dependent manner. PAI-1 protein release was also increased in the culture medium of HCAECs treated with angiotensin II. The effects of angiotensin II (10-6 M) were blocked completely by the AT1 receptor blocker losartan (10-6 M) but not by the AT2 receptor blocker PD123319 (10-6 M). Angiotensin II pretreatment also slightly, but significantly, increased t-PA mRNA expression. This effect of angiotensin II on t-PA mRNA was blocked by losartan but not by PD123319. HCAECS treated with angiotensin II revealed large amounts of the lipid peroxidation product, malonaldehyde (MDA). The effects of angiotensin II on PAI-1 expression and MDA release were blocked by pretreatment of cells with alpha-tocopherol (10-5 M). In control experiments, treatment of HCAECs with angiotensin IV markedly increased PAI-1 mRNA expression and protein release. This effect of angiotensin IV was blocked by the AT4 receptor blocker divalinal (10-6 M). These observations indicate that AT1 receptor activation plays an important role in the stimulation of PAI-1 expression and release in response to angiotensin II. Upregulation of t-PA gene may reflect autoregulation in response to PAI-1 release. Angiotensin II-mediated activation of oxidation pathways may relate to uupregulation of PAI-1. This study also confirms that angiotensin IV upregulates PAI-1 expression in HCAECs.

Oxygen and glucose deprivation-induced neuronal apoptosis is attenuated by halothane and isoflurane.

UNLABELLED: Both in vitro and in vivo evidence supports the reduction of early ischemic, both global and focal, brain injury by volatile anesthetics. However, the protection afforded by volatile anesthetics in later neuronal death, i.e., apoptosis, caused by global ischemia has not been investigated. We induced oxygen and glucose deprivation in neuronal cortical cell cultures prepared from newborn rats on in vitro Days 10-14. This hypoxic (PO2 <50 mm Hg) condition was maintained continuously (30, 60, and 90 min). In a separate experiment, the neuronal cell cultures were exposed to isoflurane (1.13%, 2.3%, or 3.3%) or halothane (1.7%, 3.4%, or 5.1%) before oxygen and glucose deprivation, with continued exposure to isoflurane or halothane during oxygen and glucose deprivation. After 48 h, neuronal apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling and DNA gel electrophoresis. Oxygen and glucose deprivation (30, 60, and 90 min) caused significant apoptosis of cerebral cortical cultured neurons. However, pretreatment and continued treatment during the period of oxygen and glucose deprivation with halothane or isoflurane resulted in a concentration-dependent attenuation of oxygen and glucose deprivation-induced neuronal apoptosis. IMPLICATIONS: This is the first investigation to evaluate the effect of volatile anesthetics on oxygen and glucose deprivation-induced neuronal apoptosis. Oxygen and glucose deprivation-induced neuronal apoptosis can be decreased by prior and continued administration of halothane or isoflurane.

Novel role of macrophage migration inhibitory factor in angiotensin II regulation of neuromodulation in rat brain.

Previously we determined that angiotensin II (Ang II) activates neuronal AT(1) receptors, located in the hypothalamus and the brainstem, to stimulate noradrenergic pathways. To link Ang II to the regulation of norepinephrine metabolism in neurons cultured from newborn rat hypothalamus and brainstem we have used cDNA arrays for high throughput gene expression profiling. Of several genes that were regulated, we focused on macrophage migration inhibitory factor (MIF), which has been associated with the modulation of norepinephrine metabolism. In the presence of the selective AT(2) receptor antagonist PD123,319 (10 microM), incubation of cultures with Ang II (100 nM; 1-24 h) elicited an increase in MIF gene expression. Western immunoblots further revealed that Ang II (100 nM; 1-24 h) increased neuronal MIF protein expression. This effect was inhibited by the AT(1) receptor antagonist losartan (10 microM), the PLC inhibitor U-73122 (10 or 25 microM), the PKC inhibitor chelerythrine (10 microM), and the Ca(2+) chelator 1,2-bis-[2-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (10 microM). Taken together with our observation that MIF is expressed in the terminal fields of noradrenergic neurons (hypothalamus) and that Ang II increases the expression of MIF in this region in vivo, our data may suggest a novel role of Ang II in norepinephrine metabolism.

Gene therapy attenuates the elevated blood pressure and glucose intolerance in an insulin-resistant model of hypertension.

OBJECTIVE: Fructose feeding in male Sprague-Dawley (SD) rats results in a mild hypertension and glucose intolerance. Although the mechanism of this glucose intolerance and hypertension is not completely understood, a role for the renin-angiotensin system (RAS) has been proposed. In the current study our aim was to test the hypothesis that intervention of the RAS with a gene therapy approach would be effective in preventing the development of hypertension and glucose intolerance in this animal model. DESIGN AND METHODS: Five-day-old SD rats were administered either an empty retroviral vector (LNSV) or retroviral vector containing AT1 receptor antisense DNA (AT1R-AS). The virus (25 microl, 8 x 10(9) CFU/ml) was injected into the heart and the animals were returned to their mothers. After weaning, half the animals from each group were placed on breeder's chow or a 60% fructose diet. Indirect blood pressures (BP) were determined and an oral glucose tolerance test (OGTT) was performed when the animals had been on the respective diets for 2 months. RESULTS: Fructose-fed animals developed mild hypertension (145 +/- 3 versus 132 +/- 4 mmHg) by 6 weeks of dietary intervention. This increase in BP was prevented by AT1R-AS treatment (125 +/- 3 mmHg). At 2 months of age, fasting blood glucose was comparable among the four groups; however, the glucose excursion during the OGTT was significantly greater and more prolonged in the LNSV-treated, fructose-fed group than the other three groups. AT1R-AS treatment significantly prevented glucose intolerance in the fructose rat to levels observed in the controls. CONCLUSIONS: Early fructose dietary treatment results in moderate hypertension and glucose intolerance, which is prevented by a single neonatal treatment with AT1R-AS. These results suggest that the RAS is involved in the glucose intolerance associated with fructose feeding and that genetic intervention is effective in this rat model.

Somatic and germinal mobility of the RescueMu transposon in transgenic maize.

RescueMu, a Mu1 element containing a bacterial plasmid, is mobilized by MuDR in transgenic maize. Somatic excision from a cell-autonomous marker gene yields >90% single cell sectors; empty donor sites often have deletions and insertions, including up to 210 bp of RescueMu/Mu1 terminal DNA. Late somatic insertions are contemporaneous with excisions, suggesting that "cut-and-paste" transposition occurs in the soma. During reproduction, RescueMu transposes infrequently from the initial transgene array, but once transposed, RescueMu is suitable for high throughput gene mutation and cloning. As with MuDR/Mu elements, heritable RescueMu insertions are not associated with excisions. Both somatic and germinal RescueMu insertions occur preferentially into genes and gene-like sequences, but they exhibit weak target site preferences. New insights into Mu behaviors are discussed with reference to two models proposed to explain the alternative outcomes of somatic and germinal events: a switch from somatic cut-and-paste to germinal replicative transposition or to host-mediated gap repair from sister chromatids.

Characterization of signal transduction pathway in neurotropic action of angiotensin II in brain neurons.

Interaction of angiotensin II with the neuronal angiotensin type 1 receptor stimulates the PI3K signaling pathway. Our objective in this study was to investigate the hypothesis that the PI3K cascade regulates the neurotropic actions of angiotensin II in rat brain neurons. We followed growth associated protein-43 expression and neurite extension as markers of neurotropic activity. Angiotensin II, through its interaction with the angiotensin type 1 receptor, increased growth associated protein-43 expression and neurite extension. These effects were abolished by pretreatment of neurons with wortmannin and rapamycin, but not by PD 98059. Antisense oligonucleotides specific for p70(S6) kinase also inhibited angiotensin II-stimulated neurotropic activity. These data confirm the involvement of PI3K and p70(S6) kinase in angiotensin II-mediated neurotropic action. Further support for this was provided by the observation that angiotensin II caused a time-dependent stimulation of p70(S6) kinase by an angiotensin type 1 receptor-mediated process. We also found that the neurotropic actions of angiotensin II are mediated by plasminogen activator inhibitor-1. Evidence for this includes 1) angiotensin II-stimulated neuronal plasminogen activator inhibitor-1 gene expression, 2) potent neurotropic action of exogenous plasminogen activator inhibitor-1, and 3) inhibitory neurotropic effect of angiotensin II by antisense oligonucleotide-mediated depletion of plasminogen activator inhibitor-1. Finally, we found that the neurotropic action of plasminogen activator inhibitor-1 is not blocked by either angiotensin type 1 receptor antagonist or inhibitors of PI3K or p70(S6) kinase, indicating that the plasminogen activator inhibitor-1 step is downstream from the p70(S6) kinase. These observations demonstrate that angiotensin II is a neurotropic hormone that engages a distinct PI3K-p70(S6) kinase-plasminogen activator inhibitor-1 signaling pathway for this action.

Gene therapy to control hypertension: current studies and future perspectives.

Hypertension is a complex pathophysiological state that leads to serious complications, including heart failure, coronary artery disease, and abnormal renal function. While traditional therapies can be effective in controlling the effects of hypertension, they offer no long-term cure and often lead to patient noncompliance, thereby diminishing their effectiveness. These reasons, coupled with the recent developments in gene transfer and somatic cell gene delivery, led researchers to explore alternative options that can produce long-term control of hypertension. Gene therapy offers the potential to yield lasting antihypertensive effects by influencing the genes associated with hypertension. In this review, we will discuss the merits of sense versus antisense strategies in controlling hypertension. We also discuss the advantages and disadvantages of both viral and nonviral vector types for the systemic delivery of genes for hypertension research. Results of our research group on the retrovirus-mediated delivery of the angiotensin type I receptor-antisense on the prevention of hypertension and related cardiovascular pathophysiology will be summarized. Finally, we discuss the future of this gene therapy approach in the reversal and long-term control of hypertension.

ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-alpha.

It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from newborn rat hypothalamus and brain stem in a protein kinase C (PKC)- and Ca2+-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of "physiological" activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl-sn-glycerol (1 micromol/l, n = 7) and 1-oleoyl-2-acetyl-sn-glycerol (1 micromol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 micromol/l, n = 5) or by chelation of internal Ca2+ (n = 8). PKC antisense (AS) oligodeoxynucleotides (2 micromol/l) against Ca2+-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-alpha-AS (n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl-sn-glycerol on Kv current, whereas PKC-beta-AS (n = 4) and PKC-gamma-AS (n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-alpha.

A maize MuDR transposon promoter shows limited autoregulation.

Transgenic maize expressing luciferase under the control of the mudrB terminal inverted repeat promoter (TIRB) of the MuDR transposon was assayed for transgene expression in active and inactive Mutator lines. We find that active MuDR elements increase TIRB-luciferase expression by 2- to 10-fold, relative to nonMuDR or silenced MuDR lines, in embryonic leaves in 75% of plants tested. However, this increase does not persist in juvenile and adult leaves. In pollen, TIRB-luciferase expression is up to 100-fold higher than in leaves but is unaffected by the presence or absence of active MuDR. Because the MuRA transposase binds to a motif within TIRB, we hypothesize that MURA may act as a weak transcriptional activator of TIRB or may partly inhibit host-induced silencing of TIRB in active Mutator lines during the early stages of somatic growth. Our results contrast with those for the maize transposon Spm, in which the TNPA transposase acts as a repressor of the Spm promoter in active Spm lines.

Angiotensin II increases vesicular trafficking in brain neurons.

Our previous studies have established that angiotensin (Ang) II stimulates the release, uptake, and synthesis of norepinephrine (NE) in brain neurons involving distinct signal transduction pathways. However, little is known if this NE neuromodulatory effect is a result of Ang II activation of vesicular trafficking in the catecholaminergic neurons. Thus, the aim of this study was to determine if Ang II influences movement of vesicles in live neurons. Dopamine-beta-hydroxylase (DbetaH) fused to green fluorescence protein (GFP) has been used to trace vesicular movement in live neurons by confocal microscopy. Transfection by a plasmid containing GFP-DbetaH resulted in the expression of green fluorescence, representing approximately 100 kDa GFP-DbetaH fusion protein. The fluorescence was predominantly localized in the perinuclear region of the neuronal soma, with a few neurites also depicting the fluorescence. Ang II caused a dramatic redistribution of fluorescence. The fluorescence was translocated to the neurites in a time-dependent manner. As a result, the number of neurites depicting fluorescence was significantly increased. The translocation was blocked by losartan, an Ang II type 1 receptor subtype-specific antagonist and not by PD123319, an Ang II type 2 receptor subtype antagonist. High-magnification confocal microscopic examination revealed that Ang II treatment resulted in a distal movement of certain fluorescent clusters in the neurites at an average rate of 0.84+/-0.2 micrometer/s. These observations suggest increased vesicular trafficking is a key signaling event in Ang II stimulation of NE neuromodulation.

The future of hypertension therapy: sense, antisense, or nonsense?

Hypertension is a debilitating disease with significant socioeconomic and emotional impact. Despite recent success in the development of traditional pharmacotherapy for the management of hypertension, the incidence of this disease is on the rise and has reached epidemic proportions by all estimates. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau, and novel approaches for the treatment and control of hypertension must be explored. We have begun to investigate the possibility of treating and/or curing hypertension by using genetic means. In this review, we will provide evidence in favor of targeting of the renin-angiotensin system by antisense gene therapy as an effective strategy for the lifelong prevention of hypertension in the spontaneously hypertensive rat model. In addition, we will discuss the properties of an ideal vector for the systemic delivery of genes and the potential experimental hurdles that must be overcome to take this innovative approach to the next level of evaluation.

The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs.

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

Gene expression profiling of rat brain neurons reveals angiotensin II-induced regulation of calmodulin and synapsin I: possible role in neuromodulation.

Angiotensin (Ang II) activates neuronal AT(1) receptors located in the hypothalamus and the brainstem and stimulates noradrenergic neurons that are involved in the control of blood pressure and fluid intake. In this study we used complementary DNA microarrays for high throughput gene expression profiling to reveal unique genes that are linked to the neuromodulatory actions of Ang II in neuronal cultures from newborn rat hypothalamus and brainstem. Of several genes that were regulated, we focused on calmodulin and synapsin I. Ang II (100 nM; 1-24 h) elicited respective increases and decreases in the levels of calmodulin and synapsin I messenger RNAs, effects mediated by AT(1) receptors. This was associated with similar changes in calmodulin and synapsin protein expression. The actions of Ang II on calmodulin expression involve an intracellular pathway that includes activation of phospholipase C, increased intracellular calcium, and stimulation of protein kinase C. Taken together with studies that link calmodulin and synapsin I to axonal transport and exocytotic processes, the data suggest that Ang II regulates these two proteins via a Ca(2+)-dependent pathway, and that this may contribute to longer term or slower neuromodulatory actions of this peptide.

Systemic Delivery of aTransgene in Intact Animals by a Retroviral Vector.

Essential hypertension is a chronic cardiovascular disease that effects over 50 million people in the United States. It is a complex pathophysiological state that is primarily characterized by a sustained elevation in blood pressure (BP). If untreated, this chronically elevated BP can affect major target organs of the body including the heart, kidney, brain, and vascular system. As a consequence of the sustained high BP, there is an increased risk of mortality and morbidity that is characterized by myocardial infarction, congestive heart failure, stroke, end-stage renal failure, and peripheral vascular disease (1-3). Because hypertension is basically an asymptomatic disease, it confounds effective treatment and makes compliance a major issue in the treatment of this disease. Pharmacological agents currently utilized have to be administered daily in an attempt to control BP, and there are no agents available to cure essential hypertension. Thus, more effective therapeutic intervention is required in order to have a significant impact in alleviating this chronic disease and its lethal cardiovascular sequel.