Ionic Current Measurements in the Squid Giant Axon Membrane

The concepts, experiments, and interpretations of ionic current measurements after a step change of the squid axon membrane potential require the potential to be constant for the duration and the membrane area measured. An experimental approach to this ideal has been developed. Electrometer, operational, and control amplifiers produce the step potential between internal micropipette and external potential electrodes within 40 microseconds and a few millivolts. With an internal current electrode effective resistance of 2 ohm cm.², the membrane potential and current may be constant within a few millivolts and 10 per cent out to near the electrode ends. The maximum membrane current patterns of the best axons are several times larger but of the type described by Cole and analyzed by Hodgkin and Huxley when the change of potential is adequately controlled. The occasional obvious distortions are attributed to the marginal adequacy of potential control to be expected from the characteristics of the current electrodes and the axon. Improvements are expected only to increase stability and accuracy. No reason has been found either to question the qualitative characteristics of the early measurements or to so discredit the analyses made of them.     

Prevention of flowering and increasing sugar yield of sugarcane by application of ethephon (2-chloroethylphosphonic acid)

The inhibition of flowering in sugarcane by ethephon (2-chloroethylphosphonic acid) applied to experimental plots is well-documented; however, verification of its efficacy in large field trials is lacking. Large-scale field trials were established at Mauna Kea Agribusiness Company, Inc., a sugar and macadamia nut plantation located on the island of Hawaii, to determine whether flower inhibition attributed to ethephon would increase sugar yield. Summarization of results from 35 paired block experiments showed an 87% reduction in tasseling in the ethephon-treated blocks. The yield of sugarcane was increased by 7.5%, and the yield of sugar by 10%. The correlation (r ²) between the decrease in flowering and increase in cane and sugar yield was only 0.02 and 0.08%, respectively, indicating that the yield increase attributed to ethephon was not adequately explained by its effect on flowering. Paper No. 665 in the journal series of the Experiment Station, Hawaiian Sugar Planters' Association.     

N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Recombination associated with replication of malarial mitochondrial DNA.

Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.     

Multiple adenovirus serotypes use alpha v integrins for infection.

The nucleotide sequence encoding the penton base integrin-binding domains of several human adenoviruses was obtained by homology PCR. Each of the penton base proteins contains a conserved Arg-Gly-Asp (RGD) sequence that is predicted to lie at the apex of two extended alpha helices. The penton base RGD domain promotes efficient infection of host cells by multiple adenovirus serotypes via interaction with alpha v integrins, thus indicating that alpha v integrins play a central role in the entry of adenoviruses into host cells.