Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.     

The fungicide methyl 2-benzimidazole carbamate causes infertility in male Sprague-Dawley rats

A serial breeding technique was used to evaluate the fertility of male Sprague-Dawley rats after exposure to the fungicide carbendazim (methyl 2-benzimidazole carbamate). Proven-fertile male rats (90 days old) received 10 daily doses of corn oil or carbendazim (400 mg/kg/day) peroral. Each male was bred with a new female each week; breeding began on the third day of treatment and continued for 32 wk after the last day of chemical exposure. Twelve days after each breeding period, the females were killed, their uteri were examined for resorptions, and the number of dead and viable fetuses was determined. All males were killed 35 wk post exposure, and testicular tissue was prepared for histopathological examination by vascular perfusion. Fertility (percent fertile as indicated by pregnant females) of males in the carbendazim-treated group was depressed the first post-exposure week; 10 of the 24 treated males failed to produce a pregnant female as compared with no failures in the control group. By the fifth post-exposure week, 16 of the 24 carbendazim-treated males were infertile. Of these 16 males, 4 recovered fertility after a failure to produce a pregnant female for 5-11 consecutive breeding periods. However, 12 of the males did not recover fertility during the remainder of the 32-wk post-exposure period. Histological examinations of testicular sections 245 days post exposure revealed that exposure to carbendazim caused severe seminiferous tubular atrophy (greater than 85% of tubules were atrophic) in those carbendazim-treated males that failed to recover fertility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional representation of chemical gradients

Perspective display techniques are applied to chemical and biochemical data sets. These represent spatially distributed gradients of reactive compounds that participate in pattern-formation processes due to reaction-diffusion or reaction-convection coupling. The patterns form in thin solution layers and are observed as chemical waves in the Belousov-Zhabotinskii reaction, as convection-induced stationary structures during oscillating glycolysis in yeast cytoplasm, and as the diffusive spreading of enzyme-catalyzed metabolic turnover in a substrate layer. The digital data are measured with a two-dimensional spectrophotometer based on a computerized video equipment with high spatial, temporal and intensity resolution. By application of three-dimensional procedures detailed structural properties of chemical and biochemical model systems will be presented yielding localization of reaction and transport events.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

Low intensity tetanic stimulation induces long-term potentiation in hippocampal neurons

A minimal intensity of the stimulation necessary for the induction of long-term potentiation of synaptic transmission (LTP) was investigated by intracellular recording in guinea pig in vitro hippocampal slices. High frequency stimulation of afferent fibres at intensities evoking in CA 1 neurons control excitatory postsynaptic potentials (EPSPs) of amplitudes 1-5 mV, resulted usually in a long-lasting increase in response amplitude. LTP was not observed at lower stimulus strength. The coactivation of a certain, though small number of synaptic contacts is thus necessary for the production of LTP.     

Modification of two peptides of bacteriorhodopsin with a pentaamminecobalt (III) complex

Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Acetylcholinesterase activity in antennal receptor neurons of the sphinx moth Manduca sexta

Summary We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the antenna of the sphinx moth Manduca sexta. High levels of echothiophate-insensitive (presumably intracellular) AChE activity were found in six different types of antennal receptors localized in specific regions of the three antennal segments of the adult moth. Mechanosensory organs in the scape and pedicel, the Böhm bristles and Johnston's organ, are innervated by AChE-positive neurons. In each annulus of the antennal flagellum, AChE-positive neurons are associated with six sensilla chaetica and a peg organ, probably a sensillum styloconicum. At least 112 receptor neurons (8–10 per annulus) innervating the intersegmental membranes between the 14 distalmost annuli also exhibit high levels of echothiophate-resistant AChE. In addition, each annulus has more than 30 AChE-positive somata in the epidermis of the scale-covered (back) side of the flagellum, and 4 AChE-positive somata reside within the first annulus of the flagellum. Since none of the olfactory receptor neurons show a high level of echothiophateresistant AChE activity, and all known mechanoreceptors are AChE-positive, apparently intracellular AChE activity in the antenna correlates well with mechanosensory functions and is consistent with the idea that these cells employ acetylcholine as a neurotransmitter.