Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.     

Reciprocal syndromes caused by deficiency duplication resulting from maternal t(10;18)(p12;q22) translocation

The detection of a familial translocation, t(10;18)(p12;q22), has made possible the observation in type and countertype of two related persons with opposite chromosomal imbalance: trisomy 18q22----18qter with monosomy 10p12----10pter in one of the two and monosomy 18q22----10pter in the other. In each case the abnormalities attributable to monosomy overrule those attributable to monosomy overrule those attributable to the associated trisomy.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Blockade of a motor response by cholinergic stimulation of the toad midbrain tegmentum

Application of an acid solution to the dorsal skin of conscious toads having intact nervous system induces a scratching reflex and escape movements, as well as autonomic alterations (hypertension and tachycardia) that are part of the defense response. The motor components of this response are abolished or reduced by microinjection of 60, 30, 15 or 7.5 ng carbachol into the midbrain tegmentum. The cardiovascular components, however, continue to be present, although their amplitude is reduced. The depression of the motor response is statistically significant up to 15 minutes for the 60 ng dose, up to 10 minutes for the 15 and 30 ng doses, and only up to 5 minutes for the 7.5 ng dose. The data suggest that the midbrain tegmentum may modulate the reflex motor response triggered by a noxious stimulus and also participate in the organization of the escape movements. The importance of cholinergic agents in this modulation is discussed. The persistence of the cardiovascular component of the response shows the importance of this parameter as an indicator of alert situations.     

A new dimension to observations in minirhizotrons: A stereoscopic view on root photographs

Summary With a stereoscope, as used for the inspection of aerial photographs, sequential photographs of roots obtained by the endoscope method from minirhizotrons can yield much more information than hitherto. A series of photographs shows that most of the roots seen in a minirhizotron in grassland grew on the surface of the lexan tube, while there was a gap between the roots and the soil. Decay of the extensive root hair zones around the roots may make new root growth in the gap between rhizotron wall and soil invisible. Some consequences of these observations for the endoscope method are discussed.     

Beneficial effect of S-adenosyl-L-methionine in lead intoxication. Another approach to clinical therapy

Five patients with chronic lead intoxication were treated with S-adenosyl-L-methionine (12 mg/kg body weight, daily), given intravenously, over a period of 22 days. A significant recovery of erythrocytic ALA-D was observed in all cases, after therapy. Blood lead content significantly pathologic at the beginning of SAM administration, rapidly decreased within 24-48 h of initiating treatment, reaching nearly control values at the end of the trial. A good correlation between recovery of ALA-D activity and decreased concentration of lead in RBC was found. GSH content in blood was diminished in lead poisoned patients, increasing to normal levels after SAM administration. Other biochemical parameters such as Deaminase activity in RBC, ALA, PBG, porphyrins and lead in urine and serum gamma-GT were measured, showing no important deviations from control values before, during or after treatment. Both biochemical and clinical improvement was observed, indicating that SAM therapy is beneficial in the treatment of lead intoxication. No untoward signs were observed. The mechanism of action of SAM is not yet clear; however, a chelating effect could be excluded, and very likely its action can be attributed to glutathione availability.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.