Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis

Focal biliary cirrhosis causes significant morbidity and mortality in cystic fibrosis (CF). Although the mechanisms of pathogenesis remain unclear, bile acids have been proposed as potential mediators of liver injury. This study examined bile acid composition in CF and assessed altered bile acid profiles to determine if they are associated with incidence and progression of liver injury in CF-associated liver disease (CFLD). Bile acid composition was determined by gas-liquid chromatography/mass spectrometry in bile, urine, and serum samples from 30 children with CFLD, 15 children with CF but without liver disease (CFnoLD)), and 43 controls. Liver biopsies from 29 CFLD subjects were assessed histologically by grading for fibrosis stage, inflammation, and disruption of the limiting plate. A significantly greater proportion of endogenous biliary ursodeoxycholic acid (UDCA) was demonstrated in CFnoLD subjects vs. both CFLD subjects and controls (2.4- and 2.2-fold, respectively; ANOVA, P = .04), and a 3-4 fold elevation in endogenous serum UDCA concentration was observed in both CFLD subjects and CFnoLD subjects vs. controls (ANOVA, P < .05). In CFLD, there were significant correlations between serum cholic acid and hepatic fibrosis, inflammation, and limiting plate disruption as well as the ratio of serum cholic acid/chenodeoxycholic acid to hepatic fibrosis, inflammation, and limiting plate disruption. In conclusion, elevated endogenous UDCA in CFnoLD suggests a possible protective role against liver injury in these patients. The correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in CFLD.

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, T-m, Was unchanged, but additional phase transitions appeared above T-m. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small-and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTP-1 above T-m, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Solution structure of the cyclotide palicourein: Implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

A nearly idealized 6 '-O-methylated t-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and C-13, H-1, and C-13/H-1 heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6-O-methylated iota-earrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. (C) 2004 Elsevier Ltd. All rights reserved.

The role of the cyclic peptide backbone in the anti-HIV activity of the cyclotide kalata B1

The plant cyclotides, the largest known family of circular proteins, have tightly folded structures and a range of biological activities that lend themselves to potential pharmaceutical and agricultural applications. Based on sequence homology, they are classified into the bracelet and Mobius subfamilies. The bracelet subfamily has previously been shown to display anti-HIV activity. We show here that a member of the Mobius subfamily, kalata B1, also exhibits anti-HIV activity despite extensive sequence differences between the subfamilies. In addition, acyclic permutants of kalata B1 displayed no anti-HIV activity, suggesting that this activity is critically dependent on an intact circular backbone. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and G termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Increased adjuvant activity of minimal CD8 T cell peptides incorporated into lipid-core-peptides

A problem facing the use of subunit peptide and protein vaccines is their inability to stimulate protective immune responses. Many different approaches have been utilized to overcome this inefficient immune activation. The approach we have taken is to modify the vaccine antigen so that it now has adjuvant properties. To do this, multiple copies of minimal CD8 T cell epitopes were attached to a poly lysine lipid core. These constructs are known as lipid-core-peptides (LCP). The research presented here examines the adjuvant activity of LCP. Using mouse models, we were able to show that LCP were indeed able to activate antigen-presenting cells in vitro and to activate cytotoxic T-cell responses in vivo. More importantly, LCP were able to stimulate the development of a protective antitumour immune response.

Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel ( GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

Mutational analysis of the large periplasmic loop 7-8 of the putrescine transporter PotE in Escherichia coli

The PotE protein is a putrescine-ornithine antiporter found in many gram-negative bacteria. It is a member of the APA family of transporters and has 12 predicted alpha-helical transmembrane spanning segments (TMS). While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function. We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake. Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity. Glycine-scanning mutagenesis of this region showed that Val(249) and Leu(254) were required for optimal transporter function. The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity. In contrast, the L254G mutant had a higher substrate affinity. A series of Val(249) mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function. Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic alpha-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space. The study shows that loop 7-8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter. (C) 2003 Elsevier Ltd. All rights reserved.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation

Overexpression of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 has been observed in a variety of tumour types, however, it is unknown whether this dysregulation is a consequence of, or a driving force for, unregulated cell proliferation. We have shown that the levels of hnRNPs A1, A2 and B1, but not A3, are modulated during the cell cycle of Colo16 squamous carcinoma cells and HaCaT immortalized keratinocytes, suggesting that A1, A2 and B1 are needed at particular cell cycle stages. However, the levels of hnRNP A1, A2 and B1 mRNAs were constant, indicating that regulation of protein levels was controlled at the level of translation. RNAi suppression of hnRNP At or A3 alone did not affect the proliferation of Colo16 cells but the proliferation rate was significantly reduced when both were suppressed simultaneously, or when either was suppressed together with hnRNP A2. Reducing hnRNP A2 expression in Colo16 and HaCaT cells by RNAi led to a non-apoptotic-related decrease in cell proliferation, reinforcing the view that this protein is required for cell proliferation. Suppression of hnRNP A2 in Colo16 cells was associated with increased p21 levels but p53 levels remained unchanged. In addition, expression of BRCA1 was downregulated, at both mRNA and protein levels. The observed effects of hnRNP A2 and its isoforms on cell proliferation and their correlation with BRCA1 and p21 expression suggest that these hnRNP proteins play a role in cell proliferation.

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.

Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A

Cyclotides are a family of plant proteins that have the unusual combination of head-to-tail backbone cyclization and a cystine knot motif. They are exceptionally stable and show resistance to most chemical, physical, and enzymatic treatments. The structure of tricyclon A, a previously unreported cyclotide, is described here. In this structure, a loop that is disordered in other cyclotides forms a beta sheet that protrudes from the globular core. This study indicates that the cyclotide fold is amenable to the introduction of a range of structural elements without affecting the cystine knot core of the protein, which is essential for the stability of the cyclotides. Tricyclon A does not possess a hydrophobic patch, typical of other cyclotides, and has minimal hemolytic activity, making it suitable for pharmaceutical applications. The 22 kDa precursor protein of tricyclon A was identified and provides clues to the processing of these fascinating miniproteins.