Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Protein kinase Cepsilon contributes to regulation of the sarcolemmal Na(+)-K(+) pump

A reduction in angiotensin II (ANG II) in vivo by treatment of rabbits with the angiotensin-converting enzyme inhibitor, captopril, increases Na(+)-K(+) pump current (I(p)) of cardiac myocytes. This increase is abolished by exposure of myocytes to ANG II in vitro. Because ANG II induces translocation of the epsilon-isoform of protein kinase C (PKCepsilon), we examined whether this isozyme regulates the pump. We treated rabbits with captopril, isolated myocytes, and measured I(p) of myocytes voltage clamped with wide-tipped patch pipettes. I(p) of myocytes from captopril-treated rabbits was larger than I(p) of myocytes from controls. ANG II superfusion of myocytes from captopril-treated rabbits decreased I(p) to levels similar to controls. Inclusion of PKCepsilon-specific blocking peptide in pipette solutions used to perfuse the intracellular compartment abolished the effect of ANG II. Inclusion of psiepsilonRACK, a PKCepsilon-specific activating peptide, in pipette solutions had an effect on I(p) that was similar to that of ANG II. There was no additive effect of ANG II and psiepsilonRACK. We conclude that PKCepsilon regulates the sarcolemmal Na(+)-K(+) pump.     

Chemical Composition and in Vitro Cytotoxic Screening of Sixteen Commercial Essential Oils on Five Cancer Cell Lines

The in vitro cytotoxic activity on human cancer cell lines of sixteen commercial EOs such as Aloysia citriodora, Boswellia sacra, Boswellia serrata, Cinnamomum zeylanicum, Cistus ladanifer, Citrus × aurantium, Citrus limon, Citrus sinensis, Cymbopogon citratus, Foeniculum vulgare, Illicium verum, Litsea cubeba, Satureja montana, Syzygium aromaticum, Thymus capitatus and Thymus vulgaris was performed using the MTT reduction assay. The screening was carried out on human cancer cells of breast adenocarcinoma (MCF7, T47D and MDA‐MB‐231), chronic myelogenous erythroleukemia (K562) and neuroblastoma cell lines (SH‐SY5Y). C. zeylanicum and L. cubeba EOs were the most active on almost all the cell lines studied and thus could be promising as an anticancer agent. These two species showed a difference in their composition even though they belong to the Lauraceae family. Almost 57 % of the true cinnamon composition was made of (E)‐cinnamaldehyde, while L. cubeba showed citral as the major compound (68.9 %). The K562 cells were the most sensitive to these oils with an IC₅₀ ranging from 5.2 parts‐per million (ppm) (C. zeylanicum) to 11.1 ppm (L. cubeba). The latter oil also showed an important cytotoxicity on MDA‐MB‐231 (13.4 ppm).     

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa⁺-K⁺pump for Na⁺. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa⁺. When pipette solutions wereK⁺ free, pump current(I_p) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in I_pmeasured with pipettes containingK⁺. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in I_p wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofI_p. We concludethat ANG II regulates the pump's selectivity for intracellularNa⁺ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade.

     

Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺/K⁺ pump to intracellular Na⁺, whereas a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺/K⁺ current (I_p) when pipette solutions were K⁺ free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p, whereas a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in K_K. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na⁺/K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the -isoform of protein kinase C (PKC) mediates effects of angiotensin II on the pump, we included specific PKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition. partial reactions; protein kinase C; angiotensin converting enzyme inhibitors; arteriosclerosis; insulin resistance     

Huntington's chorea (HD) in Malta: Epidemiology and origins

Since 1994 a Genetic Counselling Center devoted to assisting to subjects at risk from HD has been operative in Malta. Data evaluated in 1994 by this Center, represents, on the whole, 23 family nuclei. Forty living subjects with HD were found (26 males and 14 females) as well as 185 subjects at risk from HD. It appears that Malta is an HD “focus”, with a disease incidence of 11.8 out of 100.000 inhabitants. This value appears to be high with respect to the world average incidence of 5.0 (Tibben, 1993) and to other values found in the Mediterranean area. The origin of the disease on the island would appear to be “local”. The founder(s) can be traced far back in time, and probably brought this disease to the island through the maritime traffic, even if the concomitance of local mutations is not excluded.     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmalNa⁺ transport, we measuredouabain-sensitive electrogenicNa⁺-K⁺pump current(I_p) involtage-clamped ventricular myocytes and intracellularNa⁺ activity(aⁱ_Na) in right ventricularpapillary muscles. Aldosterone (10 nM) induced an increase in bothI_p and the rateof rise of aⁱ_Na duringNa⁺-K⁺pump blockade with the fast-acting cardiac steroid dihydroouabain. Thealdosterone-induced increase inI_p and rate ofrise of aⁱ_Na was eliminated bybumetanide, suggesting that aldosterone activates Na⁺ influx through theNa⁺-K⁺-2Clcotransporter. To obtain independent support for this, theNa⁺,K⁺, andCl concentrations in thesuperfusate and solution of pipettes used to voltage clamp myocyteswere set at levels designed to abolish the inward electrochemicaldriving force for theNa⁺-K⁺-2Clcotransporter. This eliminated the aldosterone-induced increase inI_p. We concludethat in vitro exposure of cardiac myocytes to aldosterone activates theNa⁺-K⁺-2Clcotransporter to enhance Na⁺influx and stimulate theNa⁺-K⁺pump.

     

Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump

A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa⁺-K⁺ pump current (I_p)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured I_p of myocytes voltageclamped with wide-tipped patch pipettes. I_p ofmyocytes from captopril-treated rabbits was larger thanI_p of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedI_p to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on I_p that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa⁺-K⁺ pump.