Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.     

Acute and chronic effects of insulin on lactate dehydrogenase (LDH) activity in tissues of albino rat (Wistar strain)

Male albino rats were treated with insulin for one week (acute) and four weeks (chronic). The lactate dehydrogenase (LDH) activity, lactate and pyruvate levels were estimated in the tissues of experimental and control animals. LDH activity decreased in all the tissues of acute- and chronic treated animals whereas the lactate content is elevated. Pyruvate content also showed increment except in heart and pancreas with reference to acute treatment where it is decreased. The hyperinsulinaemia effect in relation to lacticacidaemia and its influence on energy demand and ammonia secretion is discussed.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Attenuation of visible light by phytoplankton in a vertically structured ocean: solutions and applications

To find the solution to problems in applied marine optics, wedo not always need to compute the submarine irradiance at alldepths; in many cases, it is sufficient to know the averageattenuation of light for finite layers of arbitrary thickness.If multiple scattering can be neglected, the average attenuationof light in a finite layer of the water column depends onlyon the vertical distribution of the attenuating substances;in open-ocean waters, the most important of these is phytoplankton.It is shown how the average attenuation of monochromatic lightin an arbitrary layer can be determined when the vertical pigmentprofile takes one of two standardized forms: a shifted Gaussianor a triangle. The choice of efficient algorithms to computethe attenuation in a layer using these models is discussed.The extension to polychromatic light involves the selectionof a function to represent the spectral distribution of thespecific absorption coefficient for phytoplankton, as determinedby observation. Chebyshev polynomial representation is shownto be convenient for applications which require the calculationof a weighted wavelength integral. An efficient procedure forthe evaluation of these integrals, Gauss-Chebyshev quadrature,is presented and evaluated. The extension to the computationof bulk properties for discrete layers is straightforward.     

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper)

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Role of Arabidopsis BBX proteins in light signaling

Journal of Plant Biochemistry and Biotechnology - Light regulates numerous aspects of plant growth and development like seed germination, seedling de-etiolation, pigment accumulation, cotyledon...     

In-vivo dose measurements with MOSFET dosimeters during MV portal imaging

BackgroundThe purpose of this study was to investigate the feasibility of MOSFET dosimeter in measuring eye dose during 2D MV portal imaging for setup verification in radiotherapy.Materials and methodsThe in-vivo dose measurements were performed by placing the dosimeters over the eyes of 30 brain patients during the acquisition of portal images in linear accelerator by delivering 1 MU with the field sizes of 10 × 10 cm² and 15 × 15 cm².ResultsThe mean doses received by the left and right eyes of 10 out of 30 patients when both eyes were completely inside the anterior portal field were found to be 2.56 ± 0.2 cGy and 2.75 ± 0.2, respectively. Similarly, for next 10 patients out of the same 30 patients the mean doses to left and right eyes when both eyes were completely out of the anterior portal fields were found to be 0.13 ± 0.02 cGy and 0.17 ± 0.02 cGy, respectively. The mean doses to ipsilateral and contralateral eye for the last 10 patients when one eye was inside the anterior portal field were found to be 3.28 ± 0.2 cGy and 0.36 ± 0.1 cGy, respectively.ConclusionThe promising results obtained during 2D MV portal imaging using MOSFET have shown that this dosimeter is well suitable for assessing low doses during imaging thereby enabling to optimize the imaging procedure using the dosimetric data obtained. In addition, the documentation of the dose received by the patient during imaging procedure is possible with the help of an in-built software in conjunction with the MOSFET reader module.