Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N-and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillinsusceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 mM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity AEHae, mean hydrophobic moment AEm H ae, and a-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity AEHae and mean hydrophobic moment AEm H ae. Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.Keywords: alanine scan; antibacterial activity; coiled-coil conformation; fallaxin; solid-phase peptide synthesis Reprint requests to: Paul Robert Hansen, Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark; e-mail: prh@life.ku.dk; fax: 45-35332398.Abbreviations: ACTH, adrenocorticotropic hormone; ATCC, American type culture collection; Boc, tert-butyloxycarbonyl; BSA: bovine serum albumin; CFU, colony forming unit; CPD, citrate-phosphatedextrose; DIEA, diisopropylethylamine; DMPC, 1,2-dimyristoyl-snglycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DTT: dithiothrethiol; Fmoc: 9-fluorenylmethoxy; AEHae, mean hydrophobicity; HC 50 : the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37°C; AEm H ae, mean hydrophobic moment; HATU, (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate); HOBt, 1-hydroxybenzotriazole; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; MIC, minimum inhibitory concentration; NMP, N-methyl-2-pyrrolidone; SUV, small unilamellar vesicles; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TIS, triisopropylsilane; Trt, triphenylmethyl.Article and publication are at

The splanchnic-hepatic metabolism of glucose, lactate, pyruvate, alanine, glycerol, non-esterified fatty acids (NEFA), ketone bodies and oxygen were investigated in five normal men and six juvenile diabetic subjects at rest and during exercise after an overnight fast. A linear relationship was found between load (arterial concentration multiplied by hepatic blood flow) and splanchnic-hepatic uptake of lactate, pyruvate, glycerol and NEFA. The uptake of alanine was highly sensitive to load, but was also regulated by the concentration of hepatic venous glucagon. The uptake of pyruvate was high in exercising diabetic subjects, who had a high lactate/pyruvate concentration ratio in hepatic venous blood. The rate of uptake of the total measured gluconeogenic precursors was significantly higher in the diabetic group at a given load. The rate of ketogenesis was linearly related to the NEFA load in both groups; however, the rate of ketogenesis was twofold at a given load in the diabetic group. The highest rates of ketogenesis were found coincident with the highest concentrations of glucagon in hepatic venous blood. The observed antiketogenic effect of exercise was due to a decreased load of NEFA, mainly caused by a decrease in the hepatic blood flow.

The role of the autonomic nervous system in the glucagon response to hypoglycemia has not been fully clarified. We have studied the effect of total pharmacological blockade of the autonomic nervous system (concomitant \g=a\-and \ g=b\ -adrenergic blockade with simultaneous atropine injection) and of isolated \g=a\-adrenergic blockade on hormonal responses to hypoglycemia and on blood glucose recovery after hypoglycemia in healthy subjetcs. Neither of the pharmacological blockades had any significant effects on plasma glucagon responses to hypoglycemia nor had they any effect on the rate of blood glucose recovery after hypoglycemia. We conclude that the autonomic nervous system has no major influence on the glucagon response to hypoglycemia in healthy man. Changes in autonomic nervous activity are not essential for blood glucose recovery after hypoglycemia in healthy man.Despite a major research effort, insulin-induced hypoglycemia remains a major clinical problem in insulin-treated patients, resulting in a considerable morbidity and mortality (1). The recovery of blood glucose after insulin-induced hypoglycemia in man is due to the combined actions of glucagon and adrenaline (2). In insulin-dependent diabetic pa¬ tients, the secretion of both hormones in response to hypoglycemia seems to diminish with increasing duration of diabetes (3). The cause of this abnor¬ mality in hormone secretion in unknown. The pos¬ sible role of the autonomie nervous system in glu¬ cagon secretion during hypoglycemia has been in¬ vestigated previously, the conclusion being that the autonomie nervous system plays no major role in glucagon secretion during hypoglycemia in man.This conclusion was primarily based on pharmaco¬ logical studies using either combined a-and ß-adrenergic blockade or isolated ß-adrenergic blockade during hypoglycemia. However, since it has been argued that increased vagal activity during hypoglycemia could be responsible for glu¬ cagon secretion (4), we have performed a study in which total autonomie blockade was achieved during hypoglycemia with concomitant a-and ß-adrenergic blockade as well as parasympathetic blockade with atropine. Furthermore, the effect of isolated a-adrenergic blockade on hormonal re¬ sponses to hypoglycemia was investigated. We as¬ sessed the effect of these blockades on glucagon, adrenaline, cyclic AMP and glucose recovery in healthy subjects. Subjects and Methods Study populationSix healthy male subjects (age 24±2 years) (mean ± sem), height 180±3 cm, weight 71 ±3 kg) participated in the study after giving informed consent. The subjects partici¬ pated in 3 hypoglycemia experiments on separate days at an interval of at least 2 weeks within a 4-month period.