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.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

20-Hydroxyecdysone increases levels of transient gene expression in transfected Drosophila cells.

Methods for increasing the transient level of expression of transfected DNA in cultured Drosophila cells have been examined. Here we show that cells exposed to the steroid hormone 20-hydroxyecdysone for 48h prior to transfection show an 4 to 5 fold increase in the levels of expression of transfected DNA. By analysis, in situ, this increase appears to be due to an increase in the number of cells expressing the transfected DNA. The stimulation in levels of expression is not correlated to any specific DNA sequence, nor does it occur if cells are exposed to hormone post-transfection.     

Cerebral glycine content and phosphoserine phosphatase activity in hyperaminoacidemias

Chronic hyperphenylalaninemia maintained with the aid of a suppressor of phenylalamine hydroxylase, -methylphenylalanine, increases the glycine concentration and the phosphoserine phosphatase activity of the developing rat brain but not that of liver or kidney. Similar increases occur after daily injections with large doses of phenylalanine alone, while tyrosine, isoleucine, alanine, proline, and threonine, were without effect. Treatment with methionine, which increases the phosphoserine phosphatase activity of the brain and lowered that of liver and kidney, left the cerebral glycine level unchanged. When varying the degrees of gestational or early postnatal hyperphenylalaninemia, a significant linear correlation was found between the developing brains' phosphoserine phosphatase and glycine concentration. Observations on the uptake of injected glycine and its decline further indicate that coordinated rises in the brain's phosphoserine phosphatase and glycine content associated with experimental hyperphenylalaninemia denote a direct impact of phenylalanine on the intracellular pathway of glycine synthesis in immature animals.     

Ultrastructure of carotenoid deprivation in photoreceptors of Manduca sexta: myeloid bodies and intracellular microvilli

Summary The ultrastructural effects of carotenoid (vitamin A) deprivation were studied in the adult photoreceptors of the tobacco hornworm moth Manduca sexta. Moths were reared on a deprived diet, which lacked the carotenoid sources of the photopigment chromophore, 3-hydroxy retinal, or on a control, fortified diet, containing ample carotenoid. The latter supported normal levels of visual function, whereas visual pigment and sensitivity were reduced by at least 3 log units in moths reared on the deprived diet. Myeloid bodies, massed cisternae of hypertrophied smooth endomembrane, filled the cytoplasm in the receptors of deprived animals. The myeloid bodies assumed various configurations that included lamellate stacks of parallel cisternae, and tubular networks in a paracrystalline form. Freeze-fracture preparations of myeloid membranes revealed a high density of P-face particles. Vacuoles containing microvilli similar to those of the rhabdomere were also present in deprived photoreceptors. We suggest that the elaboration of smooth endoplasmic reticulum as myeloid bodies in chromophore-deprived photoreceptors may stem from the hypertrophy of a biochemical system for processing the chromophore or the interruption of the intracellular pathway that normally carries visual pigment to the rhabdomere.     

Carbon-13 NMR of glycogen: hydration response studied by using solids methods

The carbon-13 NMR spectra of glycogen are reported by using the methods of magic-angle sample spinning and high-power proton decoupling to provide a dynamic report on the glucose monomer behavior as a function of hydration. Although the glycogen behaves as a typical polymer in the dry state, addition of water makes a significant difference in the spectral appearance. Water addition decreases the carbon spin-lattice relaxation times by 2 orders of magnitude over the range from 7% to 70% water by weight. The proton-carbon dipole-dipole coupling, which broadens the carbon spectrum and permits cross-polarization spectroscopy, is lost with increasing hydration over this range. By 60% water by weight, scalar decoupling methods are sufficient to achieve a reasonably high-resolution spectrum. Further, at this concentration, the carbon spin-lattice relaxation times are near their minimum values at a resonance frequency of 50.3 MHz, making acquisition of carbon spectra relatively insensitive to intensity distortions associated with saturation effects. Though motional averaging places the spectrum in the solution phase limit, the static spectrum shows a residual broader component that would not necessarily be detected readily by using high-resolution liquid-state experiments.     

The fluorescence induction kinetics as a non-destructive tool for investigating spruce treated with ozone

Summary The scattering coefficient of yellow spruce needles exceeds that of green needles by a factor of 2, whereas the fluorescence efficiency is approximately equal for both needle colours. As shown by the angular distribution the fluorescence light is diffusely emitted. However, the scattered light consists of a diffuse and a reflecting portion below 20° with a ratio of the intensities of 1 : 2 at perpendicular observation (0°). Control measurements show that in the rejection region the effective transmission of cut-off-filters commonly used to separate fluorescence light and excitation light exceeds the value calculated from the filter specifications by a factor of 100. Therefore, the portion of the scattered light in the measuring signal must be controlled if the fluorescence induction kinetics is measured from specimen of different colour. A device for the determination of the fluorescence induction kinetics is described which employs a He-Ne laser, a mechanically working shutter with an opening time of 4 ms for the excitation, and a computer for data storage and device control. Two filters select the fluorescence components at 685 nm and 730 nm and they reduce the portion of the scattered light in the measuring signal to 0.18% and 0.55%, respectively. In order to consider the temporal development of the fluorescence kinetics the sampling rate is reduced from 2 kHz to 1 Hz. From the data stored in the computer maximum valueF _P, and steady-state-valueF _S are determined for both fluorescence components. Measurements on 4-year-old spruce exposed to ozone-concentrations of 0, 300 ppb, 600 ppb, and 1000 ppb were repeated every week. With increasing concentration and duration of treatmentR _fd =(F _P-F_s)/F _S was decreased for both fluorescence components. With the highest ozone concentration a reduction ofR _fd of 23% and 24%, respectively, was obtained for the two fluorescence components after three weeks.     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)