Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Chemical synthesis, characterization and activity of RK-1, a novel alpha-defensin-related peptide

The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial alpha-defensin family, was synthesized by the continuous now Fmoc-solid phase method. The crude, cleaved and S-reduced Linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by H-1-NMR spectroscopy and confirmed to be a Cys(1)-Cys(6), Cys(2)-Cys(4), Cys(3)-Cys(5) arrangement similar to other mammalian alpha-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418, Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (h beta c) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3R alpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type h beta c exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type h beta c, despite failing to undergo IL-3-stimulated h beta e tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and h beta c tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-I DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP. (C) 2000 Elsevier Science B.V. All rights reserved.

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wild-type and beta(3)-adrenoceptor knockout mice

1 The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wild-type (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. 2 (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. 3 In oesophagi precontracted with 1 mu M carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 mu M) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. 4 In ureter, (-)-CGP 12177 (2 mu M) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7.5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. 5 Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)- adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of(-)-CGP 12177 in colon, oesophagus and ureter.

Effects of promoters on catalytic activity and carbon deposition of Ni/gamma-Al2O3 catalysts in CO2 reforming of CH4

Catalytic reforming of methane with carbon dioxide was studied in a fixed-bed reactor using unpromoted and promoted Ni/gamma-Al2O3 catalysts. The effects of promoters, such as alkali metal oxide (Na2O), alkaline-earth metal oxides (MgO, CaO) and rare-earth metal oxides (La2O3, CeO2), on the catalytic activity and stability in terms of coking resistance and coke reactivity were systematically examined. CaO-, La2O3- and CeO2-promoted Ni/gamma-Al2O3 catalysts exhibited higher stability whereas MgO- and Na2O-promoted catalysts demonstrated lower activity and significant deactivation. Metal-oxide promoters (Na2O, MgO, La2O3, and CeO2) suppressed the carbon deposition, primarily due to the enhanced basicities of the supports and highly reactive carbon species formed during the reaction. In contrast, CaO increased the carbon deposition; however, it promoted the carbon reactivity. (C) 2000 Society of Chemical Industry.

Social-cognitive and perceived environment influences associated with physical activity in older Australians

Background, Regular physical activity in older adults can facilitate healthy aging, improve functional capacity, and prevent disease. However, factors associated with physical inactivity in older populations are poorly understood. This study attempts to identify social-cognitive and perceived environmental influences associated with physical activity participation in older populations. Methods. In a randomly selected sample of 449 Australian adults age 60 and older, we assessed self-reported physical activity and a range of social-cognitive and perceived environmental factors. Respondents were classified as sufficiently active and inactive based on energy expenditure estimates (kcal/week) derived from self-reported physical activity. Two logistic regression models, with and without self-efficacy included, were conducted to identify modifiable independent predictors of physical activity. Results. Significantly more males than females were physically active. Physical activity participation was related to age with a greater proportion of those age 65-69 being active than those age 60-64 or 70 or older. High self-efficacy, regular participation of friends and family, finding footpaths safe for walking, and access to local facilities were significantly associated with being active. Conclusion. Identifying predictors of physical activity in older populations, particularly social support, facility access, and neighbourhood safety, can inform the development of policy and intervention strategies to promote the health of older people. (C) 2000 American Health Foundation and Academic Press.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the immune-privileged nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully mature iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Effects of an oral vasopressin receptor antagonist (OPC-31260) in a dog with syndrome of inappropriate secretion of antidiuretic hormone

Objective The syndrome of inappropriate secretion of antidiuretic hormone is a rare disorder in dogs characterised by hypo-osmolality and persistent arginine vasopressin production in the absence of hypovolaemia and/or hypotension. The study describes the efficacy and safety of the nonpeptide selective arginine vasopressin V-2 receptor antagonist OPC-31260 in a dog with the naturally occurring syndrome. Design The detailed case history of a dog with spontaneous syndrome of inappropriate secretion of antidiuretic hormone that received long-term therapy with oral OPC-31260 is presented. Effects of the first dose of OPC-31260 and of a dose administered after a continuous dosing period of 12 days are reported. Procedure Packed cell volume, plasma sodium, total protein, arginine vasopressin, renin activity, atrial natriuretic peptide, urine specific gravity, urine output, heart rate and body weight were monitored for 2 h before, and for 4 h after, the first dose of OPC-31260. The same parameters plus plasma osmolality and urine osmolality were monitored when an identical dose was administered after 12 days of therapy. Results Oral administration of OPC-31260 at 3 mg/kg body weight resulted in marked aquaresis with increased urine output and decline in urine specific gravity within 1 h. Corresponding increases in concentrations of plasma sodium, plasma osmolality and plasma renin activity were recorded over a 4 h period. Arginine vasopressin concentration remained inappropriately elevated throughout the study. Results were similar when the trial procedure was repeated after a stabilisation period of 12 days. Long-term therapy with OPC-31260 at a dose of 3 mg/kg body weight orally every 12 h resulted in good control of clinical signs with no deleterious effects detected during a 3-year follow-up period. Despite sustained clinical benefits observed in this case, plasma sodium did not normalise with continued administration of the drug. Conclusions Treatment of a dog with naturally occurring syndrome of inappropriate secretion of antidiuretic hormone with OPC-31260 at 3 mg/kg body weight orally every 12 h resulted in marked aquaresis and significant palliation of clinical signs with no discernible side-effects detected over a 3-year period. Thus, OPC-31260 appears to offer a feasible medical alternative to water restriction for treatment of dogs with syndrome of inappropriate secretion of antidiuretic hormone. Higher doses of OPC-31260 may be required to achieve and maintain normal plasma sodium in dogs with this syndrome.

Occupation, hours worked, and leisure-time physical activity

Background. International research indicates that blue-collar employees typically exhibit lower rates of leisure-time physical activity. While lack of time and work demands are commonly reported barriers to activity, the extent to which time-at-work mediates the relationship between occupation and leisure-time physical activity is unclear. This study investigated the association between occupation, time spent in paid employment, and participation in leisure-time physical activity. Methods. This was a secondary analysis of cross-sectional data from the 1995 Australian Health Survey, focusing on employed persons ages 18-64 years (n = 24,454), Occupation was coded as per the Australian Standard Classification of Occupations and collapsed into three categories (professional, white-collar, blue-collar). Hours worked was categorized into eight levels, ranging from 1-14 to more than 50 h per week. Participation in leisure-time physical activity was categorized as either insufficient or sufficient for health, consistent with recommended levels of energy expenditure (1600 METS-min/fortnight). The relationship between occupation, hours worked, and leisure-time physical activity was examined using logistic regression. Analyses were conducted separately for male and female, and the results are presented as a series of models that successively adjust for a range of potential covariates: age, living arrangement, smoking status, body mass index, and self-reported health. Results. Individuals in blue-collar occupations were approximately 50% more likely to be classified as insufficiently active. This occupational variability in leisure-time physical activity was not explained by hours worked. There was a suggested relationship between hours worked and leisure-time physical activity; however, this differed between men and women, and was difficult to interpret. Conclusions. Occupational variability in leisure-time physical activity cannot be explained by hours worked. Therefore, reports that work constitutes a barrier to participation should be explored further. Identification of the factors contributing to occupational variability in leisure-time physical activity will add to our understanding of why population subgroups differ in their health risk profiles, and assist in the development of health promotion strategies to reduce rates of sedentariness and health inequalities. (C) 2000 American Health Foundation and Academic Press.

The anti-tumor activity of IL-12: Mechanisms of innate immunity that are model and dose dependent

IL-12 has been demonstrated to have potent anti-tumor activities in a variety of mouse tumor models, but the relative roles of NK, NKT, and T cells and their effector mechanisms in these responses have not been fully addressed. Using a spectrum of gene-targeted or Ab-treated mice we have shown that for any particular tumor model the effector mechanisms downstream of IL-12 often mimic the natural immune response to that tumor. For example, metastasis of the MHC class I-deficient lymphoma, EL4-S3, was strictly controlled by NK cells using perforin either naturally or following therapy with high-dose IL-12. Intriguingly, in B16F10 and RM-1 tumor models both NK and NKT cells contribute to natural protection from tumor metastasis, In these models, a lower dose of IL-12 or delayed administration of IL-12 dictated a greater relative role of NKT cells in immune protection from tumor metastasis. Overall, both NK and NKT cells can contribute to natural and IL-12-induced immunity against tumors, and the relative role of each population is turner and therapy dependent.