Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

Lipochondria and the light response of Aplysia giant neurons

The ultrastructure, absorbance, and elemental content of lipochondria present in the cytoplasm of Aplysia giant neurons have been investigated before and after 30–1,200 sec doses of white light at intensities which produce saturated light responses. The effects of exposure to the calcium ionophore A-23187 and to EGTA were also examined. The lipochondria of nonilluminated neurons are membrane-bound, and contain lipids, protein, Na, K, Mg, Ca, Si, Cl, Br, P, and a pigment which is probably β-carotene. The cytoplasm appeared to have little pigment. When neurons were illuminated for 20 min, 60–70% of the lipochondria showed marked ultrastructural alterations, the most notable being the appearance of membranous material. Earlier changes which occur after 30 sec of illumination include the appearance of paracrystalline arrays and mottling. Less than 10% of lipochondria in nonilluminated neurons have a similar appearance. These effects were greatly enhanced in illuminated neurons exposed to the calcium ionophore or EGTA. In nonilluminated neurons, the ionophore also produced ultrastructural changes. In frozen specimens, the calcium content of the most electron dense lipochondria of illuminated neurons was reduced. Other elements which were counted were also reduced. The lipochondria are the main intracellular site of photopigment. They may also act as an intracellular source for calcium which, as the accompanying paper indicated, may mediate phototransduction in Aplysia neurons.     

The flight mechanism of the pigeon Columbia livia during take-off

High-speed film analysis of the mechanism of take-off of a pigeon ascending nearly vertically reveals the pattern of movements of the wing segments and the bones within them during each of the five phases of the wingbeat cycle. Differences in the type and extent of wing movements between the upstroke and downstroke portions of the first and successive wingbeat cycles are explained with reference to the upward vertical jump made during the first wingbeat cycle. The presence during pigeon take-off of a non-steady state pattern of airfoil action similar to that seen in some insects at the beginning of the downstroke was verified.     

Re-appraisal of the nature of the pigment produced by Aeromonas salmonicida

Abstract Aeromonas salmonicida grown aerobically in the presence of tyrosine produces a dark-brown pigment, previously considered to be of a melanin type. However, this pigment is very water-soluble in contrast to melanin's solubility. In addition, no evidence for the presence of key early reactions and intermediates of the melanogensis pathway has been obtained. Radioisotopic labelling experiments, spectral data and chromatographic analyses support the conclusion that pigment biosynthesis, from tyrosine by Aeromonas salmonicida differs substantially from that of melanogenesis, and strongly suggest that the first intermediate is 2,4-dihydroxyphenylalanine and not 3,4-dihydroxyphenylalanine.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.