Mechanism of response of potential-sensitive dyes studied by time-resolved fluorescence

The mechanism of response of two potential-sensitive dyes, diOC₂(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC₂(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC₂(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye.     

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.     

Antimicrobial activity of polyphenolic compounds from Spirulina against food-borne bacterial pathogens

Food-borne drug-resistant bacteria have adverse impacts on both food manufacturers and consumers. Disillusionment with the efficacy of current preservatives and antibiotics for controlling food-borne pathogens, especially drug-resistant bacteria, has led to a search for safer alternatives from natural sources. Spirulina have been recognized as a food supplement, natural colorant, and enriched source of bioactive secondary metabolites. The main objectives of this study were to isolate polyphenolic compounds from Spirulina and analyze their antibacterial potential against drug-resistant food-borne bacterial pathogens. We found that fraction B of methanol extract contained a high quantity of polyphenols exhibiting broad spectrum antimicrobial effects against drug-resistant food-borne bacterial pathogens. Potential secondary metabolites, such as benzophenone, dihydro-methyl-phenylacridine, carbanilic acid, dinitrobenzoate, propanediamine, isoquinoline, piperidin, oxazolidin, and pyrrolidine, were identified by gas chromatography and mass spectrophotometry (GCMS). These metabolites are active against both gram-positive and gram-negative pathogens. Our work suggests that phenolic compounds from Spirulina provide a natural and sustainable source of food preservatives for future use.     

A couple stress model of blood flow in the microcirculation

A simple mathematical model depicting blood flow in the capillary is developed with an emphasis on the permeability property of the blood vessel based on Starling's hypothesis. In this study the effect of inertia has been neglected in comparison with the viscosity on the basis of the smallness of the Reynolds number of the flow in the capillary. The capillary blood vessel is approximated by a circular cylindrical tube with a permeable wall. The blood is represented by a couple stress fluid. With such an ideal model the velocity and pressure fields are determined. It is shown that an increase in the couple stress parameter increases the resistance to the flow and thereby decreases the volume rate flow. A comparison of the results with those of the Newtonian case has also been made.     

25-hydroxycholesterol acts in the Golgi compartment to induce degradation of tyrosinase

Oxysterols play a significant role in cholesterol homeostasis. 25-Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol-binding protein and oxysterol-related proteins, the sterol regulatory element binding protein, and the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC(50) of 0.34 microM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse-chase studies of 25HC-treated cells demonstrated enhanced degradation of tyrosinase, the rate-limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis-Golgi SNARE protein complex, were also diminished in 25HC-treated melanocytes, however levels of the ER chaperone calnexin and the cis-Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4 alpha-allylcholestan-3 alpha-ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.     

Stopped-flow kinetic, 1H, and 31P NMR analysis of the intercalation of ethidium with poly d(G-C) X d(G-C)

In a low salt buffer (0.011 M Na+) stopped-flow kinetic results for the SDS driven dissociation of an ethidium-Poly d(G-C) X d(G-C) complex are 8.7, 23, and 58.5 s-1 at 20, 30, and 40 degrees C, respectively. These results predict that in NMR experiments at high field strengths, ethidium should be in slow exchange among polymer binding sites. This has been found to be the case for both 31P (109 MHz) and 1H (imino proton spectra in H2O at 270 MHz) experiments. At higher salt, and/or higher temperature, and/or lower field, the bound and free peaks are no longer resolved in the NMR spectra. Good agreement is obtained between the stopped-flow kinetic results and the coalescence temperature observed in NMR experiments. Imino protons in base pairs on both sides of the intercalated ethidium are shifted approximately one ppm upfield while only the phosphate groups at the intercalation site experience large chemical shifts.     

Anthropogenetical analysis of abnormal human alpha-globin gene cluster arrangement on chromosome 16

An earlier study of human globin gene polymorphism in two Adriatic islands of Olib and Silba showed an abnormal arrangement of alpha-globin genes in two different individuals. The next step was to determine the degree of the kinship relationship between the two probands, one with a deleted and another with triplicated alpha-globin gene on the island Silba, and to determine the stability of this disorder through generations. We reviewed the parish registers (Status Animarum) of the island of Silba, dating from the year 1527, and constructed family trees for the two probands. Restriction endonuclease mapping was performed to study the arrangement of the alpha-globin genes in the offspring of our probands. A total of 183 ancestors completed the two family trees. The kinship relationship between them was established in the 5th, 6th, and 7th generation. The analysis of alpha-globin genes in the offspring of our probands showed the triplicated alpha-globin genes in two persons. We also found alpha-globin gene triplication in other three relatives. We did not find any deleted alpha-globin genes. We determined the kinship relationship between the two probands, one with deleted and the other with triplicated alpha-globin genes. This finding enabled us to determine the stability of this gene disarrangement through generations. It also showed new possibilities in anthropogenetic research, by combining the analyses of parish registers with those of modern genetic methods, such as restriction endonuclease mapping.