The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages

Mycobacterium tuberculosis is an important pathogen of mammals that relies on 2-hydroxyphenyloxazoline-containing siderophore molecules called mycobactins for the acquisition of iron in the restrictive environment of the mammalian macrophage, These compounds have been proposed to be biosynthesized through the action of a cluster of genes that include both nonribosomal peptide synthase and polyketide synthase components. One of these genes encodes a protein, MbtB, that putatively couples activated salicylic acid with serine or threonine and then cyclizes this precursor to the phenyloxazoline ring system. We have used gene replacement through homologous recombination to delete the mbtB gene and replace this with a hygromycin-resistance cassette in the virulent strain of M. tuberculosis H37Rv, The resulting mutant is restricted for growth in iron-limited media but grows normally in iron-replete media. Analysis of siderophore production by this organism revealed that the biosynthesis of all salicylate-derived siderophores was interrupted. The mutant was found to be impaired for growth in macrophage-like THP-1 cells, suggesting that siderophore production is required for virulence of M. tuberculosis, These results provide conclusive evidence linking this genetic locus to siderophore production.

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D H-1 NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the H-1 NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Her chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and IID-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin), Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma, These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin, Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-IO, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-IO, Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production, Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma, (C) 2001 Wiley-Liss. Inc.

Cervical screening: population-based comparisons between self-reported survey and registry-derived Pap test rates

Objective: To compare self-reported cervical screening rates, as recorded in the 1998 NSW Health Survey, with registry-based cervical screening rates for NSW for the same period; and to examine factors associated with over-estimates of cervical screening rates by self-report. Methods: Self-reported cervical screening data was extracted from the 1998 NSW Health Survey, biennial screening rates estimated and compared with biennial cervical screening rates for 1997-98, as recorded on the NSW Pap Test Register (PTR). Rates and differences were related to socio-demographic characteristics of the 17 Area Health Services of NSW. Results: According to the 1998 NSW Health Survey, 74% of women reported having a Pap test during the previous two years. The equivalent rate recorded on the NSW PTR for 1997-98 was 62% (p

Aspergillicins A-E: five novel depsipeptides from the marine-derived fungus Aspergillus carneus

A search for new antiparasitic agents from a strain of the fungus Aspergillus carneus isolated from an estuarine sediment collected in Tasmania, Australia, yielded the known terrestrial fungal metabolite marcfortine A ( 1) as an exceptionally potent antiparasitic agent. This study also yielded a series of new depsipeptides, aspergillicins A - E ( 2 - 6) and the known terrestrial fungal metabolite acyl aszonalenin ( 7). Marcfortine A ( 1) and acyl aszonalenin ( 7) were identified by spectroscopic analysis, with comparison to literature data. Complete stereostructures were assigned to aspergillicins A - E ( 2 - 6) on the basis of detailed spectroscopic analysis, together with ESIMS analysis of the free amino acids generated by acid hydrolysis, and HPLC analysis of Marfey derivatives prepared from the acid hydrolysate. The peptide amino acid sequence for all aspergillicins was unambiguously assigned by MSn ion-trap ESI mass spectrometry.

Molecular phylogenetics of Chaetodon and the Chaetodontidae (Teleostei : Perciformes) with reference to morphology

Butterflyfish are colourful, pan-tropical coastal fish that are important and distinctive members of coral reef communities. A successful systematic scheme and a robust phylogeny is considered essential in understanding further their biogeography and ecology, although recent cladistic treatments of butterflyfish phylogeny, based on soft tissue and bone morphology and coded at the generic and subgeneric levels, differ in character coding and subsequently tree topology. This study provides an independent test of the morphologically based hypotheses, using molecular systematic data from two partial mitochondrial gene fragments, cytochrome b (cytb) and small subunit rRNA (rrnS), for 52 ingroup chaetodontids and seven pomacanthids used to root the molecular trees. Individual gene trees were largely compatible and a combined molecular phylogeny, inferred from Bayesian analysis, was used to test alternative hypotheses suggested by morphological analyses. The tree was also used to map the latest morphological matrix in order to evaluate potential synapomorphies for various nodes defining butterflyfish interrelationships. A clade comprised of Chelmon and Coradion was sister group to other chaetodontids. Heniochus and Hemitaurichthys were each resolved as monophyletic groups, and as sister taxa Of the taxa sampled, Prognothodes was resolved as the sister genus to Chaeotodon. Of the ten Chaetodon subgenera sampled, all were monophyletic but their interrelationships differed significantly from that inferred from morphological characters. Lepidochaetodon was the most basal subgenus followed by Exornator and the remaining subgenera. Molecular data support the sister group relationship between Corallochaetodon and Citharoedus suggested by morphology, but major differences occur among the remaining more derived taxa. Chaetodon trifascialis and C. oligacanthus were resolved as sister taxa adding weight to the inclusion of the latter in C. Megaprotodon. Of those pairs of taxa known to hybridize and sampled with molecular data, all were closely related phylogenetically, except those hybrids known to occur in the Rabdophorus subgenus. Two base changes separated C. pelewensis from C. paucifasciatus which have been regarded previously as a single species. Cytb provided greater resolution than rrnS and will likely provide additional resolution with greater taxon sampling.

Experience-induced preference for oviposition repellents derived from a non-host plant by a specialist herbivore

Foraging adults of phytophagous insects are attracted by host-plant volatiles and supposedly repelled by volatiles from non-host plants. In behavioural control of pest insects, chemicals derived from non-host plants applied to crops are expected to repel searching adults and thereby reduce egg laying. How experience by searching adults of non-host volatiles affects their subsequent searching and oviposition behaviour has been rarely tested. In laboratory experiments, we examined the effect of experience of a non-host-plant extract on the oviposition behaviour of the diamondback moth (DBM), Plutella xylostella, a specialist herbivore of cruciferous plants. Naive ovipositing DBM females were repelled by an extract of dried leaves of Chrysanthemum morifolium, a non-host plant of DBM, but experienced females were not repelled. Instead they were attracted by host plants treated with the non-host-plant extract and laid a higher proportion of eggs on treated than on untreated host plants. Such behavioural changes induced by experience could lead to host-plant range expansion in phytophagous insects and play an important role in determining outcome for pest management of some behavioural manipulation methods.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Antibacterial activity of bovine lactoferrin-derived peptides

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified. one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf. one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond, These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ton-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC), They were characterized by. N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination, Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques, This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 mu M or less, Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC, Subfragment 1 (residues I to 10) was active against most of the test microorganisms at concentrations of 10 to 50 mu M. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 mu M. These antibacterial studies indicate that the activity of lactoferricin Is mainly, but not wholly, due to its N-terminal region.

Conformational analysis of LYS(11-36), a peptide derived from the beta-sheet region of T4 lysozyme, in TFE and SDS

The solution conformation of a peptide LYS(11-36), which corresponds to the beta-sheet region in T4 lysozyme, has been examined in aqueous solution, TFE, and SDS micelles by CD and H-1 NMR spectroscopy. Secondary structure predictions suggest some beta-sheet and turn character in aqueous solution but predict a helical conformation in a more hydrophobic environment. The predictions were supported by the CD and NMR studies which showed the peptide to be relatively unstructured in aqueous solution, although there was some evidence of a beta-turn conformer which was maintained in 200 mM SDS and, to a lesser extent, in 50% TFE. The peptide was significantly helical in the presence of either 50% TFE or 200 mM SDS. TFE and SDS titrations showed that the peptide could form helical, sheet, or extended structure depending on the TFE or SDS concentration. The studies indicate that peptide environment is the determining factor in secondary structure adopted by LYS(11-36).

Petrodentine in derived dipnoan tooth plates

Most lungfish tooth plates, that are arranged in radiating ridges derived from the fusion of separate cusps in young juveniles, are based on a framework of enamel, mantle dentine and bone that encloses a mass of specialized dentines forming the occlusal surface. In most taxa, the specialized dentines are interdenteonal and circumdenteonal dentine, but a few derived genera have petrodentine as well. Petrodentine, as originally defined, describes a specific form of hypermineralized dentine in adult tooth plates of the Recent African lungfish Protopterus. The ontogeny of fossil and Recent lungfish tooth plates demonstrates that petrodentine is derived by continuous enhancement of the hard tissue of the primary core of the initially isolated cusps of the tooth plate, and that interdenteonal dentine with denteons of circumdenteonal dentine is a secondary development in the tooth plate around and below the first formed cusps of the ridges. In dipnoans that lack petrodentine in adults the primary core of the cusps is not enhanced, but is removed by wear. The hard tissues of the dipnoan tooth plate provide useful characters for defining dipnoan taxa, as do the differing arrangements of the tissues in each species. Details of the arrangement of the enclosed specialized dentines are surprisingly variable among genera, and are significant for the structure and function of the tooth plate. Little regularity of structure is discernible in the histology of tooth plates of early dipnoans, but derived genera have more predictable structure. Consistent with other uniquely dipnoan characters, like the composition of the dermal skull, an evolutionary progression is evident within the group in the fine structure of the dentition, and, as with the bones of the dermal skull, little similarity is demonstrable between the dentines of dipnoans and tetrapods.