The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.     

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C₁₈ column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10–500 pM (3.9–195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of ‘trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.     

Using the eye-movement system to control the head.

We tested the hypothesis that A.I., a subject who has total ophthalmoplegia, resulting in a lack of eye movements, used her head to orientate in a qualitatively similar way to eye-based orientating of control subjects. We used four classic eye-movement paradigms and measured A.I.''s head movements while she performed the tasks. These paradigms were (i) the gap paradigm, (ii) the remote-distractor effect, (iii) the anti-saccade paradigm, and (iv) tests of saccadic suppression. In all cases, A.I.''s head saccades were qualitatively similar to previously reported eye-movement data. We conclude that A.I.''s head movements are probably controlled by the same neural mechanisms that control eye movements in unimpaired subjects.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

Met-204 and Tyr-205 are together important for binding GLP-1 receptor agonists but not their N-terminally truncated analogues

A mutagenesis study to systematically analyse residues spanning the first extracellular loop of the GLP-1 receptor identified a double mutant, Met-204/Tyr-205-Ala/Ala, which displayed: markedly reduced affinity for the natural agonist GLP-1; slightly reduced affinity for its analogue exendin-4; and unaltered affinity for several N-terminally truncated analogues of GLP-1 and exendin-4. This suggests that the locus is important for the formation of the binding site for the N-terminal residues of peptide agonists.     

Guanidylation and tail effects in cationic antimicrobial lipopeptoids

Background

Cationic antimicrobial peptides (CAMPs) are attractive scaffolds for the next generation of antimicrobial compounds, due to their broad spectrum of activity against multi-drug resistant bacteria and the reduced fitness of CAMP-insensitive mutants. Unfortunately, they are limited by poor in vivo performance, including ready cleavage by endogenous serum proteases.

Methodology/Principal Findings

To explore the potential for peptoid residues to replace well studied CAMP scaffolds we have produced a series of antimicrobial lipopeptoids, with sequences similar to previously reported lipopeptides. The activity of the peptoids was assessed against a panel of clinically relevant and laboratory reference bacteria, and the potential for non-specific binding was determined through hemolytic testing and repeating the antimicrobial testing in the presence of added bovine serum albumin (BSA). The most active peptoids displayed good to moderate activity against most of the Gram positive strains tested and moderate to limited activity against the Gram negatives. Antimicrobial activity was positively correlated with toxicity towards eukaryotic cells, but was almost completely eliminated by adding BSA.

Conclusion/Significance

The lipopeptoids had similar activities to the previously reported lipopeptides, confirming their potential to act as replacement, proteolytically stable scaffolds for CAMPs.