Antigenic similarity between cytoplasmic tetrodotoxin-sensitive protein and neuronal membrane proteins

Membrane proteins with a molecular weight of 290, 180, and 55 kDa were isolated using immunosorbent attached to sepharose and rabbit antibodies to cytoplasmic tetrodotoxin-sensitive protein from beef brain gray matter. A technique used for research into voltage-dependent sodium channels was applied to reconstruction of these proteins and investigation of toxin-dependent sodium flows through the lipoprotein membrane. Findings are interpreted as evidence of the similarity between cytoplasmic tetrodotoxin-sensitive protein and that of sodium channels at the cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev; A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 485–489, July–August, 1989.     

Spread of fast thermowaves through the cerebral cortex produced by visual stimulation

Thermowaves spreading through the cerebral cortex were observed and investigated during acute experiments on white rats using a new technique — that of thermoencephaloscopy (TES) through the intact skull. These waves were induced by regular visual stimulation (at intervals of 1.5–3 min) or by initiating one of the flashes and spread through the cortex along trajectories of five basic types; amplitude of thermowaves: 0.005–0.1°C; length: 10–15 mm; duration 1.2–11.4 sec; velocity: 1–33 mm/sec; extent of pathway: 2–56 mm. They appeared with a high degree of probability (of 0.92) during the interval between 15 sec before and 26 sec after the flash. Numbers of moving waves declined by the point of stimulus application and rose by 7–8 sec after the flash. Waves arose in 50% of cases in the contralateral visual cortex (areas 17 and 18a), spreading to the midline and crossing to the ipsilateral hemisphere (areas 17, 18a, and 7). Local waves moving along a circular trajectory were also discovered in the contralateral visual cortex. Several types of wave, differing in trajectory, also arose in the ipsilateral visual cortex. Mechanisms and possible significance of this effect are examined.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 467–475, July–August, 1989.     

Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.     

Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics

Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender with 4% glycerol. Spermatozoal quality was assessed at four different points: at less than 15 min after collecting and before processing (Step 1); after centrifugation and just before freezing (Step 2); immediately after thawing less than 3 h after freezing (Step 3); and immediately after thawing 10 to 20 d after freezing (Step 4). Acrosome-specific monoclonal antibody detected differences (P <0.05) among steps and ejaculates within stallions. All parameters of spermatozoal motion, including the percentage of motile sperm, percentage of progressively motile sperm, curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and radius of the average path for circularly swimming sperm, differed (P <0.05) among steps, and most of these parameters differed among ejaculates within a stallion and among stallions. For Steps 2 and 3, 62 and 37% of the sperm were motile, and 56 and 23% of all motile sperm had a curvilinear velocity of >100 mum/sec. Most damage to sperm occurred as a result of freezing-thawing, whereas centrifugation of sperm caused only minor damage.     

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPNDC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Can a simple function for the dielectric response model electrostatic effects in globular proteins?

The relationship between the effective dielectric constant that models the electrostatic effect from a charged side chain in a protein was evaluated both experimentally and theoretically. Experimental values were obtained from the shifts in pKa that resulted from point mutations of side chains in subtilisin. Theoretical values were obtained from an iterative solution to Poisson's equation that considers the dielectric response of the protein and the solvent together with charge positions. There is no simple relationship between the effective dielectric constant and the distance from the charge responsible for the interactions. For some charge positions a linear but not a direct proportional relationship of the effective dielectric with distance of separation was observed. Thus, simple models such as a linear distance-dependence for the dielectric response are not suitable to evaluate electrostatic effects in proteins.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.