Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans.

Crustins are antibacterial proteins of ca. 7-14 kDa with a characteristic four-disulphide core-containing whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans. Over 50 crustin sequences have been now reported from a variety of decapods, including crabs, lobsters, shrimp and crayfish. Three main types seem to occur but all possess a signal sequence at the amino terminus and a WAP domain at the carboxyl end. Differences between types lie in the structure of the central region. Those crustins purified as the native protein or expressed recombinantly all kill Gram-positive bacteria, and gene studies have shown that they are constitutively expressed, often at high levels, but show no consistent patterns of change in expression following injection of bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recovery from wounding, trauma or physiological stress.

Purification of antibodies and preparation of antibody fragments.

Antibodies may be purified from serum, ascitic fluid, and tissue culture supernatant by a number of methods. Purified antibodies can be treated to produce fragments that may have enhanced properties for use in immunochemistry.

Nuclear import and activity of histone deacetylase in Xenopus oocytes is regulated by phosphorylation.

Most of the histone deacetylase (HDAC) activity detected in oocytes and early embryos of Xenopus can be accounted for by the presence of a protein complex that contains the maternal HDACm protein. This complex appears to fulfil the conditions required of a 'deposition' histone deacetylase, its primary function being to deacetylate the core histones incorporated into newly-synthesized chromatin during the rapid cell cycles leading up to blastula. A major event in the assembly and accumulation of the HDAC complex is the translocation of the HDACm protein into the germinal vesicle during oogenesis. Here we examine the features of HDACm that are responsible for its nuclear uptake and enzyme activity, identifying the charged C-terminal domain as a target for modification by phosphorylation. Whereas, one phosphorylation site lying within the putative nuclear localization signal, T445, is required for efficient nuclear import of a GST-carboxy-tail fusion, two others, S421 and S423, appear to effect release from the import receptors. Although overexpression of recombinant HDACm in oocytes leads to premature condensation of endogenous chromatin, this effect is abrogated in vivo by mutation of S421A and S423A. Thus, both translocation and activity of HDACm appear to be regulated by specific phosphorylation events. These results have implications for techniques involving the transfer of somatic nuclei into enucleated oocytes.

Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss.

A potent antimicrobial peptide, tentatively named oncorhyncin II, was isolated from an acid extract of rainbow trout skin secretions. Amino acid sequencing showed that the first 17 residues of oncorhyncin II are identical to residues 138-154 of histone H1 from rainbow trout. Matrix-assisted laser desorption ionization mass spectrometry revealed that the purified peptide has a molecular mass of 7195.3Da. Taken together, these data indicate that oncorhyncin II is a 69-residue C-terminal fragment of histone H1, probably phosphorylated at two residues. Oncorhyncin II has minimal inhibitory concentrations in the submicromolar range against Gram-(+) as well as Gram-(-) bacteria and it does not display significant haemolytic activity towards trout erythrocytes. The purified peptide was found to induce a marked destabilisation of planar lipid bilayers without the formation of stable ion channels. Oncorhyncin II is possibly a cleavage product of histone H1 with a potentially important role in mucosal defence of rainbow trout.

Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss.

The partial N-terminal amino acid sequence of the antimicrobial peptide reported in the present paper has been submitted to the TrEMBL database under the accession number P83338. A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography, (t)C(18) solid-phase extraction, and C(18) reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1-66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 microM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.

Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss.

Skin exudates of rainbow trout contain a potent 13.6 kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3 microM all test bacteria lose viability after 30 min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3 microM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.

Isolation of two cytochrome P450 cDNAs, CYP1A1 and CYP1A2, from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus).

Two cytochrome P450 (CYP), CYP1A1 and CYP1A2, cDNA sequences have been isolated and cloned from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus). EROD, a model substrate for CYP1A, and heterologous antibodies have been employed as a biomarker in marine mammals, however the CYP1A sequences have not been characterised in these two seal species. mRNA was used as the template in RT-PCR, rather than DNA as this indicates transcription of the CYP1A gene in these seal species exposed to environmental contaminants. Harp and grey seal CYP1A1 amino acid sequences exhibited >99% identity and the CYP1A2 sequences were >98% identical. Phylogenetic analyses of the two seal species with other mammalian, and avian CYP1A sequences, showed the CYP1A1 and CYP1A2 sequences clustered with corresponding sequences in other mammalian species. The closest sequences to the seal CYP1As was dog CYP1A. The CYP1A sequence information presented in this study has provided the necessary data for the future production of species-specific probes for the use as biomarkers of environmental contaminant exposure.