A prospective study of concomitant GLP-1 analogue and insulin use in type 2 diabetes in clinical practice.

BACKGROUND: A small number of studies have shown a significant reduction in HbA1c, weight and total daily insulin dose when a glucagon-like-peptide-1 (GLP-1) analogue was added in type 2 diabetes patients already on insulin treatment. Therefore, in a clinical setting, we investigated the effect of adding GLP-1 analogues in patients with type 2 diabetes already using insulin with respect to glycaemic control, body weight and insulin dose. METHODS: In this prospective hospital-based study, we included 125 patients suffering from type 2 diabetes, treated with insulin and with a body mass index ≥ 35 kg/m2, who had started on GLP-1 analogues (liraglutide/exenatide). HbA1c, body weight, daily insulin dose, and side effects were registered at baseline, and after three, six and 12 months. RESULTS: HbA1c and weight decreased significantly at all the timepoints (p ≤ 0.001 compared with baseline; HbA1c: -5.5 mmol/mol (-0.5%) and weight: -14.3 kg after 12 months), with the largest decrease in the first three months. No significant correlation was found between weight loss and HbA1c reduction, and between duration of diabetes and both weight loss and HbA1c reduction. After six and 12 months, the total daily insulin dose decreased significantly (p < 0.001, -75.4 IU after 12 months). Moreover, 34% of the patients were able to stop using insulin therapy after 12 months. CONCLUSION: By adding a GLP-1 analogue in obese patients with type 2 diabetes already on insulin therapy, a significant reduction of HbA1c levels and body weight, and a significant reduction in insulin dose or complete discontinuation of insulin can be achieved.

Seasonal quality profile and production of foliage from trees grown on degraded cropland in arid Uzbekistan, Central Asia.

Feed shortages hamper livestock rearing and thus impede the development of rural livelihoods in Central Asia. The production and in vitro quality of foliage from Ulmus pumila, Elaeagnus angustifolia and Populus euphratica on degraded cropland were examined to determine the potential of these species to supplement diary cattle diets. Leaf dry matter (DM) production of the species, respectively, averaged 6, 8 and 17 t DM/ha, 4 years after planting. Over seasons and years, crude protein concentrations (g/kg DM) ranged within 151-257 for E. angustifolia, 70-241 for U. pumila and 92-187 for P. euphratica. The metabolizable energy concentrations (MJ/kg DM) were the highest in U. pumila and ranged within 9-10, followed by 7-10 of E. angustifolia and 7-9 of P. euphratica. The organic matter digestibility (%) ranged within 58-70, 54-66, and 51-66, respectively, for these species. These indicators combined denoted a medium-to-good feed quality of E. angustifolia and U. pumila leaves as a cheap protein supplement to roughages. The foliage of P. euphratica was the least suitable. The seasonal profile of in vitro indicators revealed the highest feed quality in spring but early fall seems most appropriate for forage collection given the peak leaf production and an adequate quality.

Identification by a differential proteomic approach of the induced stress and redox proteins by resveratrol in the normal and diabetic rat heart.

A recent study showed cardioprotective effects of resveratrol on the diabetic heart. The present study sought to compare the protein profiles of the normal versus diabetic hearts after resveratrol treatment using differential proteomic analysis. Rats were randomly divided into two groups: control and diabetic. Both groups of rats were fed resveratrol (2.5 mg/kg/day) for 7 days, and then the rats were sacrificed, hearts were isolated and cytoplasmic fraction from left ventricular tissue was collected to carry out proteomic profiling as well as immunoblotting. Compared to normal hearts, diabetic hearts show increased myocardial infarct size and cardiomy-ocyte apoptosis upon ex vivo global ischaemia of 30 min. followed by 2 hrs of reperfusion. Resveratrol reduced infarct size and apop-totic cell death for both the groups, but the extent of infarct size and apoptosis remained higher for the diabetic group compared to the normal group. The left ventricular cytoplasmic proteins were analysed by 2D-DIGE and differentially displayed bands were further analysed by nano Liquid Chromatography-Mass Spectroscopy (LC-MS/MS). The results showed differential regulation of normal versus diabetic hearts treated with resveratrol of many proteins related to energy metabolism of which several were identified as mitochondrial proteins. Of particular interest is the increased expression of several chaperone proteins and oxidative stress and redox proteins in the diabetic group including Hsc70, HSPp6, GRP75, peroxiredoxin (Prdx)-1 and Prdx-3 whose expression was reversed by resveratrol. Western blot analysis was performed to validate the up- or down-regulation of these stress proteins. The results indicate the differential regulation by resveratrol of stress proteins in diabetic versus normal hearts, which may explain in part the beneficial effects of resveratrol in diabetic induced cardiovascular complications.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

Nucleic acid-based modulation of cardiac gene expression for the treatment of cardiac diseases. Approaches and perspectives.

During the past few years major conceptual and technical advances have been made towards the therapeutic modulation of cardiac gene expression for the treatment of cardiac diseases. Among these are 1) the identification of new molecular therapy targets in cardiac disorders, often derived from genetic animal models. 2) A better understanding of the molecular and cellular determinants of cardiac gene transfer in vivo, in animal models and in first clinical trials. 3) The development of novel regulatable and long-term stable vector systems. This review is focused on nucleic acid-based modulation of cardiac calcium homeostasis as a paradigm for the new gene therapeutic approaches, since recent landmark papers have suggested this to be a molecular target of key importance in heart failure. In particular, the development of severe heart failure in the genetic MLP(-/-) animal model could be completely abolished by the targeted ablation of phospholamban (PL), a key regulator of cardiac calcium homeostasis. This impressive effect of permanent germline PL ablation provides-in conjunction with former important work on disturbed calcium handling in the failing human heart-a rationale for the gene therapeutic approach of ad hoc suppression of PL by antisense strategies (antisense RNAs, ribozymes, RNA interference) or PL variants. Based on the broad spectrum of methods employed to characterize this general strategy, PL-targeted approaches may be considered as a paradigm of future genetic treatments of cardiac disorders, although the differences between animal models and humans must be kept in mind. High safety of any such therapy will be a prerequisite for any possible clinical application and therefore novel methods to improve control are being devised: 1) The regulation of gene therapy vectors by biochemical abnormalities associated with the target disease itself (" Induction-by-Disease" gene therapy). 2) External control of vector activity by the employment of drug-sensitive promotors. In addition, the important goal of cardiac long-term stability of the therapeutic vectors has recently been achieved in animal models using vectors derived from adeno-associated viruses (AAVs).

Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs.

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05

A comparative study of the effects of carbamazepine and the NMDA receptor antagonist remacemide on road tracking and car-following performance in actual traffic.

RATIONALE: Antiepileptic drugs are known to produce side effects which may impair driving performance. Performance effects, however, may differ substantially between individual antiepileptic drugs. OBJECTIVE: To compare the effects of carbamazepine, remacemide, and placebo on actual driving performance during a 12-day incremental dosing regimen. METHODS: Twenty-two healthy volunteers participated in a three-way, double-blind, cross-over driving study. Treatment effects were assessed in two actual driving tests carried out on days 8, 10, and 12 of each treatment period. The Road Tracking Test involved driving an instrumented vehicle at a constant speed and steady lateral position between the delineated lane boundaries. Standard deviation of lateral position (SDLP) was measured to indicate precision of road tracking control. The Car-Following Test involved driving the same vehicle behind a leading car and maintaining that distance while the latter executed a series of deceleration/acceleration maneuvers. Time to speed adaptation (TSA) and brake reaction time were the primary measures. RESULTS: Remacemide did not affect the subjects' driving performance. Carbamazepine increased SDLP throughout treatment and lengthened TSA on day 8. Changes in SDLP relative to placebo were comparable to those previously seen in drivers conducting the same test with blood alcohol concentrations of 0.05 g/dl. CONCLUSION: Remacemide, at the given dose regimen, does not affect driving performance. Carbamazepine, at the given dose regimen, can produce mild but sufficient impairment to put epileptic patients at risk when driving, at least during initiation therapy.

The Eriksen flanker effect revisited.

Four studies are reported on the potential role of perceptual interference in a standard Eriksen flanker task. In the first study, incongruent flanker letters showed the usual effect on choice reaction time (CRT) to the target letter but had no effect on the visual fixation time (VFT) needed to distinguish target and flankers. In the second experiment, the effect of incongruent flankers was studied in the context of a same-different response in regard to the target letter and a subsequently presented single letter. The effect of incongruent flankers vanished at an interstimulus interval of 200 ms. In Experiment 3, the same-different task was used in the paradigm of the functional visual field with a target-flankers combination as stimulus on the left (SL) and a single letter as stimulus on the right side (SR) of the visual field. Flankers did neither affect VFT nor the same-different CRT suggesting that target selection may proceed during the saccade from SL to SR. In Experiment 4 effects were studied of flanker-to-target and target-to-single-letter similarity. Flanker-to-target similarity did neither affect VFT nor same-different CRT but target-to-single-letter similarity prolonged same-different CRT. Together, the results suggest parallel perceptual processing of target and flankers, followed by competition of responses to the target and to the incongruent flankers. In line with earlier research, processes of response selection and response competition appear not to be tied to VFT but to proceed in parallel with the saccade from SL to SR.

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes.

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. CONCLUSIONS: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Oxygen wastage of stunned myocardium in vivo is due to an increased oxygen cost of contractility and a decreased myofibrillar efficiency.

OBJECTIVE: We investigated whether an increased oxygen cost of contractility and/or a decreased myofibrillar efficiency contribute to oxygen wastage of stunned myocardium. Because Ca(2+)-sensitizers may increase myofibrillar Ca(2+)-sensitivity without increasing cross-bridge cycling, we also investigated whether EMD 60263 restores myofibrillar efficiency and/or the oxygen cost of contractility. METHODS: Regional fiber stress and strain were calculated from mesomyocardially implanted ultrasound crystals and left ventricular pressure in anesthetized pigs (n=18). Regional myocardial oxygen consumption (MVO(2)) was measured before contractility (end-systolic elastance, E(es)) and total myofibrillar work (stress-strain area, SSA) were determined from stress-strain relationships. Atrial pacing at three heart rates and two doses of dobutamine were used to vary SSA and E(es), respectively. After stunning (two times 10-min ischemia followed by 30-min reperfusion), measurements were repeated following infusion of saline (n=8) or EMD 60263 (1.5 mg.kg(-1) i.v., n=10). Linear regression was performed using: MVO(2)=alpha.SSA+beta.E(es)+gamma.HR(-1) (alpha(-1), myofibrillar efficiency; beta, oxygen cost of contractility; and gamma, basal metabolism/min). RESULTS: Stunning decreased SSA by 57% and E(es) by 64%, without affecting MVO(2), while increasing alpha by 71% and beta by 134%, without affecting gamma. From the wasted oxygen, 72% was used for myofibrillar work and 18% for excitation-contraction coupling. EMD 60263 restored both alpha and beta. CONCLUSIONS: Oxygen wastage in stunning is predominantly caused by a decreased myofibrillar efficiency and to a lesser extent by an increased oxygen cost of contractility. Considering that EMD 60263 reversed both causes of oxygen wastage, it is most likely that this drug increases myofibrillar Ca(2+)-sensitivity without increasing myofibrillar cross-bridge cycling.

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors.

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin.

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium.

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.

Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter.

BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun.

The transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75-1.50x10(5)cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- beta-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10(-8)M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.

Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping.

ABSTRACT A new method for the control of soilborne plant pathogens was tested for its efficacy in two field experiments during two years. Plots were amended with fresh broccoli or grass (3.4 to 4.0 kg fresh weight m(-2)) or left nonamended, and covered with an airtight plastic cover (0.135 mm thick) or left noncovered. In plots amended with broccoli or grass and covered with plastic sheeting, anaerobic and strongly reducing soil conditions developed quickly, as indicated by rapid depletion of oxygen and a decrease in redox potential values to as low as -200 mV. After 15 weeks, survival of Fusarium oxysporum f. sp. asparagi, Rhizoctonia solani, and Verticillium dahliae in inoculum samples buried 15 cm deep was strongly reduced in amended, covered plots in both experiments. The pathogens were not or hardly inactivated in amended, noncovered soil or nonamended, covered soil. The latter indicates that thermal inactivation due to increased soil temperatures under the plastic cover was not involved in pathogen inactivation. The results show the potential for this approach to control various soilborne pathogens and that it may serve as an alternative to chemical soil disinfestation for high-value crops under conditions where other alternatives, such as solarization or soil flooding, are not effective or not feasible.