Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Correlation between the parameters of contingent negative variation and characteristics of variational pulsometry in Parkinsonian patients

In patients suffering from Parkinson's disease (PD), we analyzed correlations between the parameters of contingent negative variation (CNV) and data of variational pulsometry (according to the measurements of R-R ECG intervals). Studies were carried out on 35 patients (group PD), 49 to 74 years old, with the stage of disease of 1.5 to 3.0 according to the Hoehn-Yahr international classification. In the course of CNV recording (i.e., in the state of a certain functional loading), we observed significant negative correlations between the integral magnitude (area) of this potential and indices of variational pulsometry (RMSSD, SDNN, C. var, and HF) that characterize the intensity of parasympathetic (respiratory) influences on the cardiovascular system. In the control group, such correlations were absent. We found significant correlations between the autonomic balance, CNV magnitude, and stage of PD reflecting the level of generalization of the pathological process. In the subgroup of patients with the PD stage 1.5 to 2.0, significant changes in the mean values of indices of parasympathetic influences during recording of the CNV were not observed, while in another subgroup (the PD stage 2.5 to 3.0), these values increased significantly (P < 0.05 and P < 0.01). If the estimates of the PD stage were low, the CNV area demonstrated greater values (P < 0.01). The disturbance of coordination of muscle-to-muscle interactions in the PD group is, probably, an important factor responsible for parasympathetic dysregulation and suppression of the CNV generation. We found positive correlation between the intensity of parasympathetic influences in the course of CNV recording and the level of postural disorders (r = 0.37, P < 0.05). On the contrary, the CNV magnitude demonstrated a negative correlation with the intensity of these disorders (r = −0.36, P < 0.05), as well as with the level of postural instability (r = −0.55, P < 0.001). We hypothesize that alterations of the autonomic balance and the activity of those cerebral structures, which are responsible for the motor readiness, result, to a significant extent, from weakening of the activity of the noradrenergic system due to degenerative processes developing in cells of the locus coeruleus. The impairment of the latter structure, together with degeneration of neurons of the substantia nigra and a decrease in the level of nigro-striatal dopamine, underlies the pathomorphological pattern of PD. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 242–253, May–June, 2008.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

Senescence in cut carnation flowers: Temporal and physiological relationships among water status, ethylene, abscisic acid and membrane permeability

Changes in water status, membrane permeability, ethylene production and levels of abscisic acid (ABA) were measured during senescence of cut carnation flowers ( Dianthus caryophyllus L. cv. White Sim) in order to clarify the temporal sequence of physiological events during this post-harvest period. Ethylene production and ABA content of the petal tissue rose essentially in parallel during natural senescence and after treatment of young flowers with exogenous ethylene, indicating that their syntheses are not widely separated in time. However, solute leakage, reflecting membrane deterioration, was apparent well before the natural rise in ethylene and ABA had begun. In addition, there were marked changes in water status of the tissue, including losses in water potential (ψ_w), and turgor (ψ_p), that preceded the rise in ABA and ethylene. As senescence progressed, ψ_w continued to decline, but ψ_p returned to normal levels. These temporal relationships were less well resolved when senescence of young flowers was induced by treatment with ethylene, presumably because the time-scale had been shortened. Thus changes in membrane permeability and an associated water stress in petal tissue appear to be earlier symptoms of flower senescence than the rises in ABA or ethylene. These observations support the contention that the climacteric-like rise in ethylene production is not the initial or primary event of senescence and that the rise in ABA titre may simply be a response to changes in water status.     

Virtual brain endocast of Antifer (Mammalia: Cervidae), an extinct large cervid from South America

A diverse fossil record of Cervidae (Mammalia) has been documented in the South American Pleistocene, when these animals arrived during the Great American Biotic Interchange. Using computed tomography-scanning techniques, it is possible to access the endocranial morphology of extinct species. Here, we studied the brain endocast of the extinct late Pleistocene cervid Antifer ensenadensis from southern Brazil, one of the largest forms that lived on this continent, using comparative morphology, geometric morphometrics, and encephalization quotients. The analyzed endocasts demonstrate that A. ensenadensis had a gyrencephalic brain, showing a prominent longitudinal sinus (=sagittal superior sinus), which is also observed in the large South American cervid Blastocerus dichotomus. The encephalization quotient is within the variation of extant cervids, suggesting maintenance of the pattern of encephalization from at least the late Pleistocene. Geometric morphometric analysis suggested a clear and linear allometric trend between brain endocast size and shape, and highlights A. ensenadensis as an extreme form within the analyzed cervids regarding brain morphology.     

The incidence, distribution and expression of Australian grapevine yellows, restricted growth and late season leaf curl diseases in selected Australian vineyards

Surveys were conducted in four Chardonnay vineyards for 3 to 6 years and one Shiraz vineyard for 3 years to determine the yearly percentage of grapevines affected by Australian grapevine yellows disease (AGYd), restricted growth disease (RGd) and late season leaf curl disease (LSLCd). In each of the Chardonnay vineyards in each year, all three diseases were characterised by remission of disease in some grapevines, recurrence of disease in other grapevines and new observations of disease in previously unaffected grapevines. The pattern of temporal incidence of each disease was different between vineyards for the survey period. Although Koch's postulates have not been fulfilled, phytoplasmas are considered to be the most likely cause of AGYd. While some grapevines exhibited a combination of AGYd and RGd or AGYd and LSLCd, both RGd and LSLCd can occur independently of AGYd. Statistical analyses using log-linear models also indicated that RGd and LSLCd were not always associated with AGYd. Thus, it is possible that phytoplasmas are not the cause of RGd or LSLCd and their association is coincidental. Expression of AGYd in Shiraz grapevines occurred later in the season compared to Chardonnay. Very little recurrence of AGYd was observed in the Shiraz grapevines indicating that the variety Shiraz responds differently to phytoplasma infections, assuming that AGYd in Shiraz is a phytoplasma caused disease. RGd and LSLCd were not observed on any grapevines in the Shiraz vineyard.     

Nitrogen mineralization rates in salinevs. salt-amended soils

Studies were conducted to compare N mineralization rates in salt-amended nonsaline soils to naturally-occurring saline soils. NaCl, CaCl₂, and Na₂SO₄ were added to nonsaline soils at rates that produced electrical conductivities of the saturation extracts (EC_e) of 5, 10, 15, and 20 dS m⁻¹. Saline soils with similar properties were leached to the same EC_c levels. N mineralization in the Chino soil was inhibited by salt addition, particularly with sodium and calcium chlorides. In the Domino soil there was some inhibition of N mineralization with the chloride salts, but enhancement with Na₂SO₄ was observed. Nitrification in both soils was more sensitive to salt addition than ammonification. N mineralization occurred more slowly in both leached saline soils compared to the salt-amended soils. Leached saline soils often accumulated greater amounts of inorganic N compared to their native saline counterparts, particularly with the 5 dS m⁻¹ Chino soil (native, 44 dS m⁻¹) and with the 5, 10, 15 and 20 dS m⁻¹ Domino soils (native, 32 dS m⁻¹). Kinetic parameters were estimated by the linear least squares (LLS) and the nonlinear least squares (NLLS) methods. Generally, the LLS transformation estimated greater values of potentially mineralizable N (N_o) and lower rate constants (k). With the NLLS equation, N_o values for the leached saline soils were usually lower, and k values usually higher than in the salt-amended soils. The nonsaline controls generally had the highest N_o and lowest k estimates. Average LLS rate constants for the salt-amended and leached saline soils were 0.055 and 0.083 for the Chino, and 0.104 and 0.137 week⁻¹, respectively, for the Domino soils. With the NLLS equation, average k values for the salt-amended and leached saline soils were 0.087 and 0.089 for the Chino, and 0.181 and 0.387 week⁻¹, respectively, for the Domino soils. These results suggest that N mineralization rates obtained in salt-amended nonsaline soils may not be representative of those in naturally-occurring saline soils.     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.