Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian Go and is influenced by guanyl nucleotide binding. The 39 kDa Go-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.     

A biophysical model for buzz pollination in angiosperms

The stamens of most of the world's flowering plants are longitudinally dehiscent, releasing their pollen passively, whereupon floral visitors may collect it. In nearly 400 genera in 65 plant families, the anthers dehisce by means of short apical slits or true pores. In these forms, the small light pollen can only be efficiently released by native bees capable of vibrating these stamens. This intrafloral behavior propels pollen out of the pores striking the bees on their venters. It is then collected for use in larval cell provisions. Aspects of the historical development of this novel pollination syndrome, known as “buzz” or vibratile (equals vibrational) pollination, are presented including a discussion and figures of a poricidal anther, a buzzing bee and the model system.A biophysical model for the pollen/locule wall interactions resulting in pollen expulsion upon bee or artificial vibration is developed. The model was created with the morphology of anthers of Solanum (Solanaceae) in mind, but the results obtained are generally applicable to any apically dehiscent flower which is vibrated by bees to release pollen.The anthers were modeled as a tall rectangular box with an apical pore and containing numerous small particles. As the box vibrates, particles striking the walls rebound elastically. If a pollen grain strikes a receding wall, it loses energy. If a grain strikes an advancing wall, it gains energy in the collision. In each oscillation, there is a net gain in the energy of the particles. As the anther (box) is shaken, vibrational energy is transmitted from the pterothorax of the bees to the flower, the pollen grains gaining significant energy. As the energy increases and the particles begin to move about more and more vigorously, they will begin to escape through the hole in the box (or stamina] pore). The rate at which particles leave the box and time required to empty the box are calculated as functions of the geometry of the model system and the frequency of vibration.In order to test the influence of air currents, Bernolli effects and viscous drag, the flowers were mecahnically vibrated in vacuum. The pollen cloud thus produced was virtually unchanged ans so it seems unlikely that air plays any significant role in the phenomenon of vibrational pollen release.Finally, variables such as: inelastic interactions, electrostatic forces, slightly sticky pollen due to presence of “pollenkitt”, duration and types of bee buzzes are discussed in relation to the mathematical model presented.     

Rubella Epidemic in a Maternity Unit

Four cases of rubella, the last confirmed by laboratory tests, occurred among the nurses of a large obstetric unit. Immediate measures were taken to prevent the spread of the infection to pregnant patients and staff, and none was in fact infected.Persons susceptible to rubella and who become infected may pose a real danger to women in early stages of gestation during the incubation period. We recommend that the immune status of medical and nursing personnel working in obstetric departments should be ascertained serologically, and that rubella vaccine should be offered to those who are susceptible.     

The structure of sodium beta-fluoropyruvate: a gem-diol.

Sodium β-fluoropyruvate is shown by X-ray diffraction and infrared spectroscopy to exist as a gem-diol when crystallized from water in the monoclinic space group $\text{P2}{}_1\text{a}$. The unit cell dimensions are a = 8.693(1), b = 11.556(1), c = 5.252(1) Å, β = 94.85(8) °. Molecular dimensions of the gem-diol are presented. The carbon-carbon bond to the carboxyl group is long [1.551(2) Å], indicative of a bond that can break easily. The hydration of the carbonyl group is attributed to the high electronegativity of the fluorine atom. While reports exist in the literature of a crystalline form of β-fluoropyruvic acid that contains a carbonyl, rather than a gem-diol group, ¹³C nmr studies presented here demonstrate that, in aqueous solution, the gem-diol is the predominant (≥95%) form. The significance of these results in the study of the mechanisms of enzymes utilizing pyruvate as a substrate is discussed. If the carbonyl oxygen activity is important for the action of an enzyme, e.g., in the formation of a Schiff base, it is possible that fluoropyruvate may not be a substitute for pyruvate. In other cases the fluoropyruvate can behave as a substrate analog, although its greater bulk may slow down or prevent its entry into the active site.     

Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.     

Glycogen and its metabolism: some new developments and old themes

Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.