A self-assembling ß-peptide hydrogel for neural tissue engineering.

We report a new class of ß-peptide based hydrogel for neural tissue engineering. Our ß-peptide forms a network of nanofibres in aqueous solution, resulting in a stable hydrogel at physiological conditions. The hydrogel shows excellent compatibility with neural cells and provides a suitable environment for cells to adhere and proliferate.

Hormonal therapy with estradiol and drospirenone improves endothelium-dependent vasodilation in the coronary bed of ovariectomized spontaneously hypertensive rats.

Drospirenone (DRSP) is a progestin with anti-aldosterone properties and it reduces blood pressure in hypertensive women. However, the effects of DRSP on endothelium-dependent coronary vasodilation have not been evaluated. This study investigated the effects of combined therapy with estrogen (E2) and DRSP on endothelium-dependent vasodilation of the coronary bed of ovariectomized (OVX) spontaneously hypertensive rats. Female spontaneously hypertensive rats (n=87) at 12 weeks of age were randomly divided into sham operated (Sham), OVX, OVX treated with E2 (E2), and OVX treated with E2 and DRSP (E2+DRSP) groups. Hemodynamic parameters were directly evaluated by catheter insertion into the femoral artery. Endothelium-dependent vasodilation in response to bradykinin in the coronary arterial bed was assessed using isolated hearts according to a modified Langendorff method. Coronary protein expression of endothelial nitric oxide synthase and estrogen receptor alpha (ER-α) was assessed by Western blotting. Histological slices of coronary arteries were stained with hematoxylin and eosin, and morphometric parameters were analyzed. Oxidative stress was assessed in situ by dihydroethidium fluorescence. Ovariectomy increased systolic blood pressure, which was only prevented by E2+DRSP treatment. Estrogen deficiency caused endothelial dysfunction, which was prevented by both treatments. However, the vasodilator response in the E2+DRSP group was significantly higher at the three highest concentrations compared with the OVX group. Reduced ER-α expression in OVX rats was restored by both treatments. Morphometric parameters and oxidative stress were augmented by OVX and reduced by E2 and E2+DRSP treatments. Hormonal therapy with E2 and DRSP may be an important therapeutic option in the prevention of coronary heart disease in hypertensive post-menopausal women.

Hormonal therapy with estradiol and drospirenone improves endothelium-dependent vasodilation in the coronary bed of ovariectomized spontaneously hypertensive rats

Drospirenone (DRSP) is a progestin with anti-aldosterone properties and it reduces blood pressure in hypertensive women. However, the effects of DRSP on endothelium-dependent coronary vasodilation have not been evaluated. This study investigated the effects of combined therapy with estrogen (E2) and DRSP on endothelium-dependent vasodilation of the coronary bed of ovariectomized (OVX) spontaneously hypertensive rats. Female spontaneously hypertensive rats (n=87) at 12 weeks of age were randomly divided into sham operated (Sham), OVX, OVX treated with E2 (E2), and OVX treated with E2 and DRSP (E2+DRSP) groups. Hemodynamic parameters were directly evaluated by catheter insertion into the femoral artery. Endothelium-dependent vasodilation in response to bradykinin in the coronary arterial bed was assessed using isolated hearts according to a modified Langendorff method. Coronary protein expression of endothelial nitric oxide synthase and estrogen receptor alpha (ER-α) was assessed by Western blotting. Histological slices of coronary arteries were stained with hematoxylin and eosin, and morphometric parameters were analyzed. Oxidative stress was assessed in situ by dihydroethidium fluorescence. Ovariectomy increased systolic blood pressure, which was only prevented by E2+DRSP treatment. Estrogen deficiency caused endothelial dysfunction, which was prevented by both treatments. However, the vasodilator response in the E2+DRSP group was significantly higher at the three highest concentrations compared with the OVX group. Reduced ER-α expression in OVX rats was restored by both treatments. Morphometric parameters and oxidative stress were augmented by OVX and reduced by E2 and E2+DRSP treatments. Hormonal therapy with E2 and DRSP may be an important therapeutic option in the prevention of coronary heart disease in hypertensive post-menopausal women.

Hormone therapy with tamoxifen reduces plasma levels of NT-B-type natriuretic peptide but does not change ventricular ejection fraction after chemotherapy in women with breast cancer

The objective of this study was to evaluate the effect of tamoxifen on the plasma concentration of NT-pro-B-type natriuretic peptide (NT-proBNP) in women undergoing chemotherapy for breast cancer and to correlate changes in NT-proBNP with the left ventricular ejection fraction (LVEF). Over a period of 12 months, we followed 60 women with a diagnosis of breast cancer. The patients were separated into a group that received only chemotherapy (n=23), a group that received chemotherapy + tamoxifen (n=21), and a group that received only tamoxifen (n=16). Plasma levels of NT-proBNP were assessed at 0 (T0), 6 (T6), and 12 (T12) months of treatment, and echocardiography data were assessed at T0 and T12. Plasma NT-proBNP levels were increased in the chemotherapy-only group at T6 and T12, whereas elevated NT-proBNP levels were only found at T6 in the chemotherapy + tamoxifen group. At T12, the chemotherapy + tamoxifen group exhibited a significant reduction in the peptide to levels similar to the group that received tamoxifen alone. The chemotherapy-only group exhibited a significant decrease in LVEF at T12, whereas the chemotherapy + tamoxifen and tamoxifen-only groups maintained levels similar to those at the beginning of treatment. Treatment with tamoxifen for 6 months after chemotherapy significantly reduced the plasma levels of NT-proBNP and did not change LVEF in women with breast cancer.

Hormone therapy with tamoxifen reduces plasma levels of NT-B-type natriuretic peptide but does not change ventricular ejection fraction after chemotherapy in women with breast cancer.

The objective of this study was to evaluate the effect of tamoxifen on the plasma concentration of NT-pro-B-type natriuretic peptide (NT-proBNP) in women undergoing chemotherapy for breast cancer and to correlate changes in NT-proBNP with the left ventricular ejection fraction (LVEF). Over a period of 12 months, we followed 60 women with a diagnosis of breast cancer. The patients were separated into a group that received only chemotherapy (n=23), a group that received chemotherapy + tamoxifen (n=21), and a group that received only tamoxifen (n=16). Plasma levels of NT-proBNP were assessed at 0 (T0), 6 (T6), and 12 (T12) months of treatment, and echocardiography data were assessed at T0 and T12. Plasma NT-proBNP levels were increased in the chemotherapy-only group at T6 and T12, whereas elevated NT-proBNP levels were only found at T6 in the chemotherapy + tamoxifen group. At T12, the chemotherapy + tamoxifen group exhibited a significant reduction in the peptide to levels similar to the group that received tamoxifen alone. The chemotherapy-only group exhibited a significant decrease in LVEF at T12, whereas the chemotherapy + tamoxifen and tamoxifen-only groups maintained levels similar to those at the beginning of treatment. Treatment with tamoxifen for 6 months after chemotherapy significantly reduced the plasma levels of NT-proBNP and did not change LVEF in women with breast cancer.

Effect of tamoxifen on the coronary vascular reactivity of spontaneously hypertensive female rats.

Tamoxifen has been associated with a reduction in the incidence of myocardial infarction. However, the effects of tamoxifen on coronary reactivity have not been fully elucidated. The objective of this study was to determine the effects of chronic treatment with tamoxifen on coronary vascular reactivity in spontaneously hypertensive rats (SHR). Female SHR were divided into four groups (N = 7 each): sham-operated (SHAM), sham-operated and treated with tamoxifen (10 mg/kg) by gavage for 90 days (TAMOX), ovariectomized (OVX), and ovariectomized and treated with tamoxifen (OVX+TAMOX). Mean arterial pressure (MAP), heart rate (HR), coronary perfusion pressure (CPP), and coronary vascular reactivity were measured. MAP and HR were reduced (9.42 and 11.67%, respectively) in the OVX+TAMOX group compared to the OVX group (P < 0.01). The coronary vascular reactivity of the OVX+TAMOX group presented smaller vasoconstrictor responses to acetylcholine (2-64 µg) when compared to the OVX group (P < 0.01) and this response was similar to that of the SHAM group. The adenosine-induced vasodilator response was greater in the TAMOX group compared to the SHAM and OVX groups (P < 0.05). Baseline CPP was higher in OVX+TAMOX and TAMOX groups (136 ± 3.6 and 130 ± 1.5 mmHg) than in OVX and SHAM groups (96 ± 2 and 119 ± 2.3 mmHg; P < 0.01). Tamoxifen, when combined with OVX, attenuated the vasoconstriction induced by acetylcholine and increased the adenosine-induced vasodilatory response, as well as reducing the MAP, suggesting beneficial effects of tamoxifen therapy on coronary vascular reactivity after menopause.

Effect of tamoxifen on the coronary vascular reactivity of spontaneously hypertensive female rats

Tamoxifen has been associated with a reduction in the incidence of myocardial infarction. However, the effects of tamoxifen on coronary reactivity have not been fully elucidated. The objective of this study was to determine the effects of chronic treatment with tamoxifen on coronary vascular reactivity in spontaneously hypertensive rats (SHR). Female SHR were divided into four groups (N = 7 each): sham-operated (SHAM), sham-operated and treated with tamoxifen (10 mg/kg) by gavage for 90 days (TAMOX), ovariectomized (OVX), and ovariectomized and treated with tamoxifen (OVX+TAMOX). Mean arterial pressure (MAP), heart rate (HR), coronary perfusion pressure (CPP), and coronary vascular reactivity were measured. MAP and HR were reduced (9.42 and 11.67 percent, respectively) in the OVX+TAMOX group compared to the OVX group (P < 0.01). The coronary vascular reactivity of the OVX+TAMOX group presented smaller vasoconstrictor responses to acetylcholine (2-64 µg) when compared to the OVX group (P < 0.01) and this response was similar to that of the SHAM group. The adenosine-induced vasodilator response was greater in the TAMOX group compared to the SHAM and OVX groups (P < 0.05). Baseline CPP was higher in OVX+TAMOX and TAMOX groups (136 ± 3.6 and 130 ± 1.5 mmHg) than in OVX and SHAM groups (96 ± 2 and 119 ± 2.3 mmHg; P < 0.01). Tamoxifen, when combined with OVX, attenuated the vasoconstriction induced by acetylcholine and increased the adenosine-induced vasodilatory response, as well as reducing the MAP, suggesting beneficial effects of tamoxifen therapy on coronary vascular reactivity after menopause.

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray.

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

Ochratoxin a: comparison of extraction methods from grapes and quantitative determination by different competitive enzyme-linked immunosorbent assay kits.

The European Community has recently established a maximum limit for ochratoxin A (OTA) concentration in grapevine products, but many practical difficulties remain concerning the establishment of optimum cost-effective methods of quantification. The performance of four extraction procedures and three commercial competitive enzyme-linked immunosorbent assays (cELISAs) for grapes were compared. Results differed for the extractions and the cELISA kits. The advantage of using immunoaffinity columns (IACs) in the extraction was the excellent detection limit, which was between 0.06 and 0.0075 ng ml(-1) depending on the cELISA kit used. Despite lower sensitivity (between 1.2 and 0.15 ng ml(-1) depending on the cELISA kit), an extraction method in liquid phase, which was simple and inexpensive, was confirmed as suitable for quantifying OTA at levels estimated to be dangerous for human health. Two of the three cELISA kits produced satisfactory results. When these two cELISAs were coupled with IAC extraction, the lower quantification limits were 0.010 and 0.0075 ng ml(-1), respectively, and the dynamic ranges were 50 and 27, respectively. The most reliable procedures were then compared with the reference method, high-performance liquid chromatography plus fluorescent detection coupled with an IAC. The results were very similar, although the cELISAs generally provided slightly higher values than did the chromatography method. The IAC method coupled with the cELISA was four times more sensitive than was the IAC method coupled with the chromatography method. The cELISA detection techniques were excellent alternatives to the already established chromatographic protocols, especially for mass screening and for determining concentrations of OTA as low as 0.010 ng ml(-1).

Structural and electrophysiological effects of local anesthetics and of low temperature on myelinated nerves: implication of the lipid chains in nerve excitability.

X-ray scattering and electrophysiological experiments performed on toad sciatic nerves as a function of the exposure to either low temperature or tetracaine yielded the following results: (i) the main structural effect is to thicken the individual membranes, thus to stiffen the acyl chains and increase the repeat distance of the one-dimensional lattice, phenomena that are typical of lipid-containing systems with disordered chains; (ii) the electrophysiological effect is to decrease the amplitude and velocity of the compound action potential; (iii) the structural and physiological effects of the two agents are practically identical. Since the structural and the electrophysiological parameters have different origins in the nerves (the structure regards the myelin sheath, the electrical signals originate at the nodes of Ranvier) it is inferred that tetracaine and low temperature exert similar effects on the membranes of both the myelin sheath and the nodes of Ranvier. Also, since local anesthetics act by inhibiting the Na+ channels, these observations suggest that the acyl chain conformation modulates the channel function and thus the generation of action potential.

The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve.

X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic. Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.

Order-disorder phenomena in myelinated nerve sheaths. VI. The effects of quaking, jimpy and shiverer mutations: an X-ray scattering study of mouse sciatic and optic nerves.

We report the X-ray scattering study of sciatic and optic nerve myelin from shiverer, jimpy and quaking mice mutants and from the corresponding controls. These three mutations are known to affect dramatically central nervous system (CNS) myelin and to induce comparatively minor alterations in peripheral nervous system (PNS) myelin. Scattering experiments and data reduction were carried out using the techniques and algorithms developed in our laboratory and previously applied to several problems involving the structure of myelin. In sciatic nerve the fraction of myelin elementary pairs of membranes (total myelin) decreases in shiverer and quaking nerves (by approximately 30%) but not in jimpy nerves; in all three mutants the fraction of myelin membrane pairs that are not regularly stacked in the sheaths (loose myelin), the average number of membranes per sheath and the packing disorder are the same as in the control nerves; the repeat distance D and the membrane distance Dcyt across the cytoplasmic space increase in shiverer and decrease in jimpy; in quaking, D also decreases and the decrease is smaller than in jimpy and is not specific for Dcyt; small changes are also observed in the electron density profiles. As for the optic nerve the myelin content decreases dramatically in the three mutants; the very weak signal attests to a tiny amount of pairs of membranes structurally similar to normal CNS myelin. It is surprising that the structure of CNS myelin should be almost normal in the absence of the major structural components, namely myelin basic protein (MBP) for shiverer of proteolipid protein (PLP) for jimpy. The question arises whether the composition of the residual pairs of membranes, operationally identified as myelin in the X-ray scattering experiments, mirrors the composition determined by chemical means on the fraction of nerve tissue histologically identified as myelin, or whether in all circumstances it remains approximately the same.

Order-disorder phenomena in myelinated nerve sheaths. IV. The disordering effects of high levels of local anaesthetics on rat sciatic and optic nerves.

Sequences of 15 minute X-ray scattering spectra were recorded with rat sciatic and optic nerves, superfused with tetracaine-containing Ringer solutions. The spectra were analysed using the algorithm advocated in this series of papers. The main results, as a function of the time of exposure to tetracaine, were: the mean value of the repeat distance increases; its variance decreases; the average number of membrane pairs per coherent domain decreases; the fraction of isolated membrane pairs increases. Eventually, the spectra were observed to give way to the continuous intensity curve of a single, isolated membrane pair. At all stages of the experiment the continuous intensity curves were found to differ from one type of nerve to the other, and to be invariant, for each type of nerve, with respect to the tetracaine treatment. The X-ray scattering study clearly identified the nature of the structural differences between the two types of myelin sheaths: in that of native sciatic nerves, packing disorder preferentially affects the cytoplasmic space of the membrane pair, and tetracaine disrupts the packing in that space; in the myelin of optic nerves it is the external space that is preferentially affected by packing disorder and disrupted by tetracaine. The time-course of the structure parameters showed that, at any stage of the experiment, tetracaine acts preferentially on the more highly disordered regions of the structure and totally disrupts them. These results corroborate earlier conclusions reported in the previous papers of this series. An electron microscope study was also performed on tetracaine-treated nerves: the results, in close agreement with those of the X-ray scattering study, neatly confirm the conclusions given above. In a more general way, the remarkable agreement between the results of the analysis of the X-ray scattering spectra and the electron microscope observations strongly supports the validity of the physical model used in this series of papers and the correctness of the mathematical treatment that we advocate. Finally, the relations between this work and the work of others are discussed. It must be stressed that the present work bears on the toxic rather than on the anaesthetic effects of tetracaine.

Order-disorder phenomena in myelinated nerve sheaths. III. The structure of myelin in rat optic nerves over the course of myelinogenesis.

An X-ray scattering study was performed on optic nerves dissected from rats aged from ten days to one year. The spectra were analysed using the procedure described in the previous papers of this series. Each experiment yields the values of a variety of parameters: the average D and the variance sigma D of the repeat distance, the average number mean value of N of motifs per crystallite, the fraction alpha loose of myelin that does not belong to the compact sheaths, the sets [idiff(h/D)] and [imotif(k/2D)] that suffice to define, respectively, the spurious scattering and the continuous intensity curve of the elementary membrane pair. A surprising result is that, in the native optic, as previously found in the swollen sciatic nerves, the stacking disorder affects the external space, whereas in native sciatic nerves the disorder affects the cytoplasmic space. An analysis of the evolution of the structure parameters as a function of the age of the animal and a comparison with the results previously obtained with rat sciatic nerves led to the following conclusions: the structure of the elementary membrane pair is constant throughout myelinogenesis; mean value of N is much smaller in optic than in sciatic nerves; mean value of N and the degree of myelination increase with age in the two types of nerve; D is smaller in optic than in sciatic nerves; in optic nerves, D decreases slightly with age, but in sciatic nerves it increases; sigma D is strongly age-dependent in optic nerves, but almost age-independent in sciatic nerves. In contrast to sciatic, the structure of optic nerve myelin was found to be almost insensitive to hypertonic solutions. Finally, a pair of electron density profiles was selected, quite similar to those selected previously in sciatic nerves, one corresponding to Caspar & Kirschner's the other to Worthington & McIntosh's proposals, neither of which can be ruled out according to the criteria used in this work.

Order-disorder phenomena in myelinated nerve sheaths. II. The structure of myelin in native and swollen rat sciatic nerves and in the course of myelinogenesis.

The algorithm described in the accompanying paper was applied to X-ray scattering experiments performed with rat sciatic nerves, either as a function of the age of the animal (4 to 30 days), or with adult nerves swollen in non-isotonic media. The results were all consistent with the model of disorder used in the theoretical treatment. The algorithm leads, in one step, from the data to the numerical values of the parameters, avoiding all intermediate manipulation. For each experiment a variety of parameters was determined: the average D and the variance sigma 2D of the repeat distance, the average number [N] of motifs per crystallite, the set [idiff(h/D)], which defines the diffuse scattering, the fraction alphaloose of myelin that does not belong to the compact sheaths, and the set [imotif (k/2D)], which suffices to define the continuous intensity curve of the motif imotif(s). Note the remarkable wealth of information, especially by contrast with conventional analyses which, as a rule, only yield the values of D and of the set [imotif(h/D)] (insufficient to determine the function imotif(s]. The function imotif(s) and the parameters D and sigma D (and thus the local structure of the myelin sheaths) were shown to be almost invariant in the course of myelinogenesis; what varies is mainly the total amount of myelin in the nerve and the number of membranes per sheath. Swelling agents have a dramatic influence on the X-ray scattering spectra, but in spite of the conspicuous variation of D, sigma D and [N] the structure of the motif is invariant. The structure of the motif was shown to be quite different in the native and in the swollen samples; the stacking disorder appears to involve mainly the cytoplasmic space in native myelin, the external space in swollen nerves. The very notion of electron density profile, when disorder is present, is discussed. Two criteria were proposed to select the "best" signs of the reflections: two sets came out at almost the same rank, one corresponding to Caspar & Kirschner's the other to Worthington & McIntosh's proposals, neither of which can be ruled out according to the criteria used in this work.