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Biomedical subjects

A Salvetti

Publications and source records attributed to A Salvetti.

At least 55 records · Page 3Linked to original sources
PubMed2000

Early impairment of coronary flow reserve and increase in minimum coronary resistance in borderline hypertensive patients.

OBJECTIVE: To evaluate relations between coronary flow velocity and myocardial oxygen demand at rest, as well as coronary vasodilator capacity and flow reserve, in asymptomatic subjects with borderline hypertension as compared to normotensive controls and patients with sustained high blood pressure (HBP) and without left ventricular hypertrophy (LVH). SUBJECTS AND METHODS: Forty-two asymptomatic males were studied: 13 healthy normotensive volunteers; 12 subjects with borderline HBP and 17 asymptomatic subjects with sustained systemic hypertension. Coronary flow velocity in left anterior descending artery and coronary flow reserve were assessed by transesophageal echo-doppler at baseline and during intravenous adenosine infusion. Left ventricular mass, peak systolic wall stress (PSWS; Pa), and midwall fractional shortening (MFS; %) were obtained from M-mode images of the left ventricle in transthoracic long-axis view and in transesophageal transgastric view. RESULTS: Coronary flow velocity at baseline was not significantly different in the three groups, despite significantly higher rate-pressure product (RPP) in the hypertensive groups as compared with controls. Only in control subjects, was resting coronary flow velocity significantly correlated with RPP (y = 4279 + 200x, r = + 0.58, P < 0.05) and PSWS (y = 17.2 + 5.1 x, r = + 0.62, P < 0.05). Coronary reserve was 3.5 +/- 0.65 in controls and significantly lower (P < 0.05) in borderline hypertensive (2.87 +/- 0.46) and in sustained hypertensive subjects (2.66 +/- 0.56). Minimum coronary resistance was significantly increased in both hypertensive groups (1.30 +/- 0.29 and 1.39 +/- 0.48 mmHg/s per cm) as compared to normotensive controls (0.93 +/- 0.20 mmHg/s per cm, P < 0.01). CONCLUSIONS: In asymptomatic subjects with borderline hypertension and without LVH, a significant reduction in coronary flow reserve is already detectable and appears almost entirely related to an impaired coronary vasodilator capacity rather than to an increased myocardial oxygen demand.

Adenosine↗
PubMed2000

Vascular effects of endothelin-1 in essential hypertension: relationship with cyclooxygenase-derived endothelium-dependent contracting factors and nitric oxide.

Endothelium plays a primary role in the local modulation of vascular function and structure by the production and release of several substances including nitric oxide and endothelins (ET). Nitric oxide is a labile substance produced from the catabolism of L-arginine and not only causes vessel relaxation, but also inhibits platelet aggregation, smooth muscle cell proliferation, monocyte adhesion, adhesion molecules expression and endothelin-1 (ET-1) production. Endothelium-derived ET-1 is a potent vasoconstrictor and has inotropic and mitogenic properties. ET-1 acts through smooth muscle ET(A) and ET(B) receptors, which mainly mediate vasoconstriction, and endothelial ET(B) receptors, which oppose ET(A)- and ET(B)-mediated vasoconstriction by stimulating nitric oxide formation. Both nitric oxide and ET-1 play a crucial role in the cardiovascular physiology and an alteration of these systems can be a promoter of or be associated with most cardiovascular diseases. Essential hypertension is a pathological condition characterized by endothelial dysfunction. In hypertensive patients nitric oxide availability is impaired because of the production of cyclooxygenase-derived vasoconstrictor substances. The latter may also mediate the vasoconstrictor response to exogenous ET-1 because in forearm circulation of essential hypertensives, but not of normotensive controls, the ET-1-induced vasoconstriction is significantly blunted by intrabrachial indomethacin. Therefore, in normotensive subjects and essential hypertensives the vasoconstrictor effect of ET-1 seems to be dependent on different mechanisms. Moreover, in the peripheral circulation of normotensive subjects, where tonic nitric oxide production is preserved, unselective ET(A/B), receptor blockade by TAK-044 causes a very modest degree of vasodilation. In contrast in essential hypertensives, where the tonic nitric oxide production is reduced, the vasodilating effect of TAK-044 is more evident, indicating that the predominant vascular effect of endogenous ET-1 is the vasoconstriction. A possible explanation for this finding, in addition to an increased production of the peptide, could be related to a reduced ET(B) receptor-mediated nitric oxide activation. These peculiar aspects of the role of ET-1 in essential hypertension could have physiopathological relevance.

Animals↗
PubMed2000

Effect of the angiotensin II type 1 receptor blocker candesartan on endothelial function in patients with essential hypertension.

Patients with essential hypertension are characterized by impaired basal and agonist-evoked nitric oxide release and increased endogenous endothelin (ET)-1-induced vasoconstriction. To assess whether candesartan, an angiotensin II type 1 receptor blocker, can improve endothelial function, we studied the changes in forearm blood flow (FBF) induced in 15 hypertensive patients and in 15 control subjects by the intrabrachial infusion of N(G)-monomethyl-L-arginine (L-NMMA), norepinephrine, the ET A/B receptor antagonist TAK 044, sodium nitroprusside, and acetylcholine. In hypertensive patients, the FBF study was repeated 2 and 12 months after the start of treatment with candesartan cilexetil (8 to 16 mg daily). Compared with controls (maximal FBF decrease, -46+/-11%), hypertensive patients showed a reduced (P<0.001) vasoconstrictor response to L-NMMA (maximal FBF decrease, -28+/-7%); the response to norepinephrine was only slightly impaired, and the response to sodium nitroprusside was similar to that of controls. Finally, TAK-044 caused greater vasodilation in hypertensive patients (maximal FBF increase, 77+/-9%) than in controls (maximal FBF increase, 17+/-10%). In hypertensive patients, candesartan cilexetil significantly enhanced vasoconstriction to L-NMMA after 2 and 12 months (maximal FBF decrease, 37+/-2% [P<0.05] and 42+/-2% [P<0.001], respectively). The responses to norepinephrine, acetylcholine, and sodium nitroprusside were not modified after 2 months. After 12 months, the responses to acetylcholine and sodium nitroprusside were significantly (P<0.05) enhanced at the highest rates. Vasodilation to TAK-044 was abolished after treatment with candesartan cilexetil; this effect is associated with a reduced plasma ET-1 concentration. This study demonstrated that the angiotensin II receptor blocker candesartan improves tonic nitric oxide release and reduces vasoconstriction to endogenous ET-1 in the forearm of hypertensive patients.

Acetylcholine↗
PubMed1999

Vasoconstriction to endogenous endothelin-1 is increased in the peripheral circulation of patients with essential hypertension.

BACKGROUND: In humans, endothelin (ET)-1 could be implicated in the pathophysiology of several cardiovascular diseases, including essential hypertension. We therefore evaluated the role of ET-1 in control of vascular tone in essential hypertension. METHODS AND RESULTS: We used strain-gauge venous plethysmography to test changes in forearm blood flow induced by intrabrachial infusion of TAK-044 (10, 30, and 100 microgram. 100 mL(-1). min(-1)), an ET(A)/ET(B) receptor antagonist, or sodium nitroprusside (1 and 2 microgram. 100 mL(-1). min(-1)), a vasodilator that acts on smooth muscle cells, in hypertensive patients and healthy controls (n=10 in each group). The NO pathway was also evaluated by infusion of N(G)-monomethyl-L-arginine, (L-NMMA; 10, 30, and 100 microgram. 100 mL(-1). min(-1)), an NO synthase inhibitor, and norepinephrine (3, 9, and 30 ng. 100 mL(-1). min(-1)) as control. Immunoreactive plasma ET-1 was measured by radioimmunoassay. In hypertensive patients, TAK-044 caused a vasodilation that was significantly (P<0.01) increased compared with normotensive subjects. Moreover, vasoconstriction to L-NMMA was significantly (P<0.01) decreased in hypertensive patients compared with controls. In contrast, the vascular responses to sodium nitroprusside and norepinephrine, as well as levels of immunoreactive plasma ET-1, were similar in hypertensive patients and controls. In the study population, vasodilation to TAK-044 and vasoconstriction to L-NMMA showed an inverse correlation (r=-0.56, P<0.05). CONCLUSIONS: These results indicate that TAK-044 caused a greater degree of vasodilation in the forearm vessels of essential hypertensive patients compared with normotensive subjects, an alteration associated with decreased tonic NO release.

Endothelin Receptor Antagonists↗
PubMed1999

Vasodilation to bradykinin is mediated by an ouabain-sensitive pathway as a compensatory mechanism for impaired nitric oxide availability in essential hypertensive patients.

BACKGROUND: In essential hypertension, endothelium-dependent vasodilation is impaired because of reduced nitric oxide (NO) availability, which is mainly caused by oxidative stress. The present study was designed to identify the mechanism(s) responsible for NO-independent vasodilation to bradykinin in patients with essential hypertension. METHODS AND RESULTS: In 16 healthy subjects (49.5+/-5.8 years; 118.6+/-3.5/78.9+/-2.9 mm Hg) and 16 patients with essential hypertension (47.9+/-4.8 years; 154.6+/-4.5/102.9+/-3.2 mm Hg), we measured modifications in forearm blood flow (strain-gauge plethysmography) during intrabrachial infusion of bradykinin (5, 15, or 50 ng/100 mL of forearm tissue per minute) in the presence of saline, N(omega)-monomethyl-L-arginine (L-NMMA; used to inhibit NO synthase; 100 microg/100 mL of forearm tissue per minute), and ouabain (to block Na(+)K(+)/ATPase and prevent hyperpolarization; 0.7 microg/100 mL of forearm tissue per minute). In healthy subjects, vasodilatation to bradykinin was significantly blunted by L-NMMA and unaffected by ouabain. In hypertensive patients, vasodilatation to bradykinin was not modified by L-NMMA, but it was significantly reduced by ouabain. In an adjunctive group of 8 hypertensive patients (49.9+/-3.8 years; 155.9+/-5.5/103.7+/-3.9 mm Hg), the response to bradykinin was repeated during the administration of intrabrachial vitamin C (a scavenger for oxygen free radicals; 8 mg/100 mL of forearm tissue per minute). In these patients, L-NMMA-induced inhibition of vasodilation to bradykinin was restored, and ouabain was no longer effective. In a final group of 6 normotensive controls (45.9+/-4.1 years; 115.1+/-2.9/79.3+/-2.1 mm Hg), vasodilation to bradykinin residual to L-NMMA blockade was further inhibited by simultaneous ouabain infusion. CONCLUSIONS: Vasodilation to bradykinin is impaired in essential hypertensive patients because of an NO-system alteration caused by oxidative stress, and it is mediated by an alternative pathway, possibly involving endothelium-dependent hyperpolarization.

Adaptation, Physiological↗
PubMed1999

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Cell Cycle↗
PubMed1999

Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

Adenoviridae↗
PubMed1999

Renin-angiotensin-aldosterone system in primary hyperparathyroidism before and after surgery.

Twenty consecutive unselected patients with proven primary hyperparathyroidism (PH), 26 essential hypertensive (EH) patients, and 13 normotensives were studied. Blood pressure (BP) and, under constant salt intake, plasma renin activity (PRA), parathyroid hormone (PTH), urinary and plasma sodium, potassium, aldosterone (ALD), creatinine, total calcium, and phosphate were measured. Patients with PH were also studied 1 and 6 months after successful surgery. In patients with PH, systolic and diastolic BP was significantly lower (P < .001) than in EH patients and higher (P < .005) than in controls. Eight patients with PH (40%) had BP levels greater than 140/90 mm Hg. PTH and plasma and urinary calcium in patients with PH were significantly (P < .01) higher than in controls, while PRA, ALD, phosphate, potassium, and sodium were superimposable in the three groups. PTH in patients with PH was weakly correlated with PRA (positively) and with plasma potassium (negatively) and was not associated with ALD, calcium, sodium, and BP levels. Surgery was followed by a significant reduction (P < .01) in PTH, calcium, and urinary phosphate and an increase (P < .02) in plasma phosphate, potassium, and sodium, whereas PRA, ALD, urinary potassium and sodium, and BP showed no change. In hypertensive patients with PH, PTH, PRA, and plasma and urinary ALD, calcium, and sodium did not differ from the values in normotensive PH patients, and variations in these humoral parameters after surgery were comparable in the two groups. In conclusion, our results show that hypertension is frequently associated with PH. However, the present data raise doubts about the assumption of a renin-mediated causal relationship between hyperparathyroidism and high BP. Indeed, as a unique finding in favor of the hypothesis of a stimulating role of PTH in renin secretion, we observed only a weak relation between PTH and PRA. Thus, unknown and/or unassessed factors related to parathyroid disease cannot be ruled out to explain the hypertension observed in some patients with PH.

Adult↗
PubMed1999

Adenosine causes the release of active renin and angiotensin II in the coronary circulation of patients with essential hypertension.

OBJECTIVES: The aim of the study was to evaluate whether adenosine infusion can induce production of active renin and angiotensin II in human coronary circulation. BACKGROUND: Adenosine can activate angiotensin production in the forearm vessels of essential hypertensive patients. METHODS: In six normotensive subjects and 12 essential hypertensive patients adenosine was infused into the left anterior descending coronary artery (1, 10, 100 and 1,000 microg/min x 5 min each) while active renin (radioimmunometric assay) and angiotensin II (radioimmunoassay after high performance liquid chromatography purification) were measured in venous (great cardiac vein) and coronary arterial blood samples. In five out of 12 hypertensive patients adenosine infusion and plasma samples were repeated during intracoronary angiotensin-converting enzyme inhibitor benazeprilat (25 microg/min) administration. Finally, in adjunctive hypertensive patients, the same procedure was applied during intracoronary sodium nitroprusside (n = 4) or acetylcholine (n = 4). RESULTS: In hypertensive patients, but not in control subjects, despite a similar increment in coronary blood flow, a significant (p < 0.05) transient increase of venous active renin (from 10.7 +/- 1.4 [95% confidence interval 9.4 to 11.8] to a maximum of 13.8 +/- 2.1 [12.2 to 15.5] with a consequent drop to 10.9 +/- 1.8 [9.7 to 12.1] pg/ml), and angiotensin II (from 14.6 +/- 2.0 [12.7 to 16.5] to a maximum of 20.4 +/- 2.7 [18.7 to 22.2] with a consequent drop to 16.3 +/- 1.8 [13.9 to 18.7] pg/ml) was observed under adenosine infusion, whereas arterial values did not change. Calculated venous-arterial active renin and angiotensin II release showed a strong correlation (r = 0.78 and r = 0.71, respectively; p < 0.001) with circulating active renin. This adenosine-induced venous angiotensin II increase was significantly blunted by benazeprilat. Finally, both sodium nitroprusside and acetylcholine did not affect arterial and venous values of active renin and angiotensin II. CONCLUSIONS: These data indicate that exogenous adenosine stimulates the release of active renin and angiotensin II in the coronary arteries of essential hypertensive patients, and suggest that this phenomenon is probably due to renin release from tissue stores of renally derived renin.

Acetylcholine↗
PubMed1999

Relationship between the circulating and vascular renin-angiotensin system and the vasodilating effect of captopril in human hypertension.

A vascular renin-angiotensin system (RAS) is present in the forearm vasculature of essential hypertensive patients and is closely related to the circulating renin profile. To test whether the haemodynamic effect of acute intrabrachial administration of captopril is related to the circulating and/or vascular RAS, 31 hypertensive patients were selected and divided into four groups according to their different circulating RAS profile (n = 7 hypertensive patients with primary aldosteronism and suppressed plasma renin activity; n = 7 low renin essential hypertensive patients; n = 8 normal renin essential hypertensive patients; n = 9 high renin renovascular hypertensive patients). The forearm net balance of active renin, plasma renin activity and angiotensin II, obtained by intrabrachial infusion of the beta-adrenergic receptor agonist isoproterenol (0.03, 0.1, 0.3 microg/100 ml/min) and calculated as the product of the venous-arterial plasma concentration gradient and forearm blood flow (FBF), was closely related to the circulating RAS. Captopril (0.25, 2.5, 25 microg/100 ml/min per 20 min each dose) unchanged basal FBF in the primary aldosteronism and low renin groups (FBF increase: from 3.9 +/- 0.4 to a maximum of 4.1 +/- 0.5 and from 3.8 +/- 0.3 to a maximum of 4.3 +/- 0.5 ml/100 ml/min, respectively), whereas it caused slight vasodilation in the normal renin group (from 3.9 +/- 0.3 to a maximum of 5.3 +/- 0.7 ml/100 ml/min), and pronounced vasodilation in the high renin group (from 4.0 +/- 0.4 to a maximum of 6.4 +/- 0.5 ml/100 ml/min). Captopril-induced vasodilation showed a significant direct correlation with the circulating and vascular RAS. The present data, while confirming the existence of a vascular RAS in the forearm of hypertensive patients indicate that the acute vasodilating effect of intrabrachial captopril is linked to a stimulated RAS, either circulating or vascular, supporting the evidence that, in acute conditions, ACE inhibitors exert their vasodilating effect through the RAS blockade.

Antihypertensive Agents↗
PubMed1999

Endogenous androgens and carotid intimal-medial thickness in women.

The influence of endogenous androgens on atherosclerotic disease in women is unknown. In this study involving 101 pre- and post-menopausal females, we evaluated the relationship between serum androgen levels and both carotid artery intimal-medial thickness (IMT) and major cardiovascular risk factors. In addition to evaluation of blood pressure, body mass index, and waist-to-hip ratio, serum dehydroepiandrosterone sulfate (DHEA-S), androstenedione (A), total testosterone (TTS), free testosterone (FTS), insulin, cholesterol (total and high density lipoproteins), triglycerides, and glucose were measured. All women underwent carotid ultrasonography. Spearman correlation coefficients showed that serum DHEA-S and A levels were negatively related (P < 0.03-0.0004) to several IMT measures. Higher tertiles of DHEA-S, A, and FTS corresponded to significantly lower measures of carotid thickness. DHEA-S, and all androgens were inversely related to age (P < 0.03 or less), showing no unfavorable association with major cardiovascular risk factors. In contrast, serum DHEA-S was negatively associated with WHR (P < 0.02), while A was negatively associated with body mass index (P < 0.02). Stepwise multiple regression analysis indicated that A and FTS showed an inverse association with IMT measures (P < 0.05-0.001). In conclusion, our data indicate that in women serum DHEA-S and androgens decline with age and that normal hormonal levels are not associated with major cardiovascular risk factors. They also show that higher DHEA-S and androgen concentrations are related to lower carotid wall thickness; for A this association is independent of cardiovascular risk factors. Our results suggest that, in the physiological range, DHEA-S and androgens in women are correlated with lower risk of carotid artery atherosclerosis.

Adult↗
PubMed1999

Renal protection and antihypertensive drugs: current status.

The renal protective effect of antihypertensive drugs is linked to 2 mechanisms. First, reduction in blood pressure (BP) is a fundamental prerequisite common to all antihypertensive drugs. The exact definition of the level to which BP should be reduced remains to be established, although there is some evidence that BP should be reduced below 130/85 mm Hg in patients with diabetic and nondiabetic nephropathies and below 125/75 mm Hg in patients with nondiabetic nephropathies and proteinuria >1 g/day. However, available data suggest that tight BP control (BP<140/80 mm Hg) can reduce the risk of cardiovascular complications in hypertensive patients with type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus; NIDDM). Secondly, intrarenal actions on mechanisms such as glomerular hypertension and hypertrophy, proteinuria, mesangial cell proliferation, mesangial matrix production and probably endothelial dysfunction, which can cause and/or worsen renal failure, are relevant for the renal protective action of some drug classes. ACE inhibitors possess such properties and also seem to lower proteinuria more than other antihypertensive drugs, despite a similar BP lowering effect. Calcium antagonists likewise exert beneficial intrarenal effects, but with some differences among subclasses. It remains to be evaluated whether angiotensin II-receptor antagonists can exert intrarenal effects and antiproteinuric actions similar to those of ACE inhibitors. While primary prevention of diabetic nephropathy is still an unsolved problem. there is convincing evidence that in patients with type 1 (insulin-dependent diabetes mellitus; IDDM) or 2 diabetes mellitus and incipient nephropathy ACE inhibitors reduce urinary albumin excretion and slow the progression to overt nephropathy. Similar effects have been reported with some long-acting dihydropyridine calcium antagonists, although less consistently than with ACE inhibitors. In patients with diabetic overt nephropathy, ACE inhibitors and nondihydropyridine calcium antagonists are particularly effective in reducing proteinuria and both drugs can slow the decline in glomerular filtration rate more successfully than other antihypertensive treatment. Available data in patients with nondiabetic nephropathies indicate that ACE inhibitors can be beneficial, principally in patients with significant proteinuria, in slowing the progression of renal failure. However, it is still unclear whether this beneficial effect of ACE inhibitors is particularly evident in patients with mild and/or more advanced renal failure and whether calcium antagonists possess a similar nephroprotective effect. Overall, data from clinical trials thus seem to indicate that ACE inhibitors and possibly calcium antagonists should be preferred in the treatment of patients with diabetic and nondiabetic nephropathies. However, further information is needed to understand renal protection.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

Expression of DjY1, a protein containing a cold shock domain and RG repeat motifs, is targeted to sites of regeneration in planarians.

Planarians are well-known for their exceptional regenerative abilities. This investigation focuses on the involvement of a Y-box protein, defined by the presence of a cold-shock domain, in regeneration-specific processes. Previous studies have shown that developmentally expressed Y-box proteins bind to mRNA molecules and regulate the timing of their translation. We have isolated and characterized a planarian Y-box gene, DjY1, which is specifically expressed at the site of regeneration, the blastema. DjY1 transcripts appear rapidly at the site of cutting and increase in number as the blastema grows. The timing and level of expression is similar irrespective of the orientation of the cut: in anterior, posterior, and lateral regenerative tissue. As regeneration nears completion, there is a general decrease in transcript level except in structures which are still differentiating, specifically in the auricles where new DjY1 transcripts are produced. A similarly modulated temporal pattern of expression throughout regeneration is seen in assaying the DjY1 protein. Within the population of blastemal cells, a subset of differentiating cells is specifically immunostained using antibodies to DjY1. The DjY1 protein contains a cold-shock domain and RG-repeat motifs, both of which are associated with RNA-binding properties: in vitro binding studies using recombinant DjY1 show that the preferred template is single-stranded RNA of heterogeneous sequence. These data provide the first direct evidence that a Y-box protein is involved in the regeneration process in planarians and implicate DjY1 in the translational regulation of differentiation-specific mRNAs.

Amino Acid Sequence↗
PubMed1998

Control of erythropoietin delivery by doxycycline in mice after intramuscular injection of adeno-associated vector.

We reported previously that controlled expression of a foreign gene in response to tetracycline derivative can be accomplished in mice by the autologous transplantation of retrovirus-modified muscle cells. Although regulated systemic delivery of therapeutic proteins from engineered tissues has potential clinical application, the transplantation of muscle cells is not currently feasible in humans. Several studies have shown that a single injection of adeno-associated virus (AAV) vectors into mouse muscle results in long-term expression of reporter genes as well as sustained delivery of proteins into the serum. Because this method is potentially applicable clinically, we constructed an AAV vector in which the expression of the mouse erythropoietin (Epo) cDNA is modulated in response to doxycycline. The vector was injected intramuscularly in normal mice. We observed that hematocrit and serum Epo concentrations could be modulated over a 29-week period in response to the presence or absence of doxycycline in the drinking water of these animals. Thus, a regulated gene expression cassette can be incorporated into a single AAV vector, such that intramuscular injection of the vector allows sustained and regulated expression of a desired gene.

Animals↗
PubMed1998

Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension.

BACKGROUND: Essential hypertension is associated with impaired endothelium-dependent vasodilation. Inactivation of endothelium-derived nitric oxide by oxygen free radicals participates in endothelial dysfunction in experimental hypertension. To test this hypothesis in humans, we evaluated the effect of antioxidant vitamin C on endothelium-dependent responses in essential hypertensive patients. METHODS AND RESULTS: In 14 healthy subjects (47.1+/-4.8 years; blood pressure, 120.6+/-4.5/80.9+/-3.5 mm Hg) and 14 essential hypertensive patients (47.3+/-5.1 years; blood pressure, 153.9+/-7.1/102.3+/-4.1 mm Hg), we studied forearm blood flow (strain-gauge plethysmography) modifications induced by intrabrachial acetylcholine (0.15, 0.45, 1.5, 4.5, and 15 microg x 100 mL(-1) x min(-1)) or sodium nitroprusside (1, 2, and 4 microg/100 mL forearm tissue per minute), an endothelium-dependent and -independent vasodilator, respectively, in basal conditions and during infusion of intrabrachial vitamin C (2.4 mg/100 mL forearm tissue per minute). In hypertensive patients but not in control subjects, vitamin C increased (P<0.01) the impaired vasodilation to acetylcholine, whereas the response to sodium nitroprusside was unaffected. Moreover, in another 14 hypertensive patients (47.1+/-5.2 years; blood pressure, 155.2+/-6.9/103.7+/-4.5 mm Hg), the facilitating effect of vitamin C on vasodilation to acetylcholine was reversed by N(G)-monomethyl-L-arginine (100 microg/100 mL forearm tissue per minute), a nitric oxide synthase inhibitor, suggesting that in essential hypertension superoxide anions impair endothelium-dependent vasodilation by nitric oxide breakdown. Finally, because in adjunctive 7 hypertensive patients (47.8+/-6.1 years; blood pressure, 155.3+/-6.8/103.5+/-4.3 mm Hg), indomethacin (50 microg/100 mL forearm tissue per minute), a cyclooxygenase inhibitor, prevented the potentiating effect of vitamin C on vasodilation to acetylcholine, it is possible that in essential hypertension a main source of superoxide anions could be the cyclooxygenase pathway. CONCLUSIONS: In essential hypertensive patients, impaired endothelial vasodilation can be improved by the antioxidant vitamin C, an effect that can be reversed by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine. These findings support the hypothesis that nitric oxide inactivation by oxygen free radicals contributes to endothelial dysfunction in essential hypertension.

Acetylcholine↗
PubMed1998

Factors influencing recombinant adeno-associated virus production.

Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.

Animals↗
PubMed1998

Effects of long-term pravastatin treatment on spermatogenesis and on adrenal and testicular steroidogenesis in male hypercholesterolemic patients.

To evaluate the influence of an hydrophilic statin, pravastatin, on adrenal and testicular steroidogenesis and spermatogenesis, eight male hypercholesterolemic patients were studied. All patients observed a hypocholesterolemic diet and received placebo for 4 weeks followed by pravastatin (20 mg/die) for 6 months. Before, during (4th-5th week) and at the end (23th-24th week) of active treatment, CRH (1 microgram i.v.), ACTH (Synacthen 250 micrograms i.v.) and human CG (HCG 3000 IU i.m.) tests were performed in addition to semen analysis. Pravastatin significantly reduced total cholesterol (20.3%), calculated LDL-cholesterol (24.6%) and apolipoprotein B (10.5%, increased apolipoprotein A1 (16.1%) and did not influence plasma HDL-cholesterol and triglycerides. Basal plasma cortisol, aldosterone, androstenedione, testosterone and oestradiol did not change under active treatment. Pravastatin administration affected neither adrenal hormone responses to CRH and ACTH or testicular response to HCG nor spermatogenesis in respect of motility, morphology and sperm count. In conclusion, long-term pravastatin treatment, at doses effective in improving lipid profile, did not influence testicular reproductive and endocrine function and did not interfere with basal and stimulated adrenal activity of male hypercholesterolemic patients.

Adrenal Glands↗