Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish.

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.Expert Rev. Proteomics 3(4), 399-408 (2006) In photoaffinity labeling (PAL), a ligand possessing a UV-photoactivating group is incubated with its receptor to form, first, a noncovalently bound ligand-receptor complex in which the ligand binds specifically to the binding site or pocket in the receptor due to its affinity. Some ligands, such as oligonucleotides, contain intrinsic photoactivating groups; alternatively, these groups can be introduced by synthesis into ligands for peptides and small molecules, such as drugs. It is important to establish that the incorporated photoactivating groups do not significantly alter the binding affinity of the ligand to its receptor and its functionality, compared with the nonderivatized ligand. Subsequent to the complex formation, UV irradiation triggers the photoactivating group in the ligand and leads to the conversion of the noncovalent binding into a covalent link between the ligand and its receptor at the binding site. A covalent ligand-receptor complex makes the study of the complex easier, since it permits the use of more rigorous but harsher analysis tools, such as SDS-PAGE, HPLC and mass spectrometry (MS), which would typically lead to the dissociation of the complex with information regarding the binding sites no longer being obtainable.Photoaffinity labeling with radioactively labeled ligands is a widely used method to determine the competitive binding affinities of other molecules for the binding sites in the binding pocket occupied by the radioactively labeled ligand and, as such, is a valuable tool for drug screening. When photoaffinity techniques are combined with modern MS, this approach can provide additional structural and mechanistic information regarding the protein complex, such as the ligand-receptor stoichiometry, a map of the binding pocket, and even the exact identification of the amino acid residues, which are involved in the ligand-receptor interaction. The data obtained can be used to create a 3D model of the ligand-binding pocket that might provide better insight into the nature of the ligand-receptor interaction. This knowledge can be v...

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent inJection at the same site ofPseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria.

This paper describes the isolation of an approximately 3.7 kDa, basic, antibacterial peptide (designated callinectin), which represents the major antibiotic activity in blue crab, Callinectes sapidus, hemocytes. A single-step purification using low-pressure cation-exchange chromatology yielded a highly purified (>95%) peptide. Purity was confirmed by C4 reverse-phase high-performance liquid chromatography (RP-HPLC), native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electophoresis, and mass spectral analysis. The partial amino acid sequence obtained via Edman degradation revealed no significant homology to other reported peptides in the Basic Local Alignment Search Tool (BLAST) program database.

ABSTRACT:We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health.KEY WORDS: Endobiotics · Non-specific immunity · Diagnostic stress test · ELISA Resale or republication not permitted without written consent of the publisherDis Aquat Org 44: [97][98][99][100][101][102][103][104][105][106][107] 2001 Barton 1997). Correlations between stress-associated immunosuppression and susceptibility of fish to infectious disease have been demonstrated in various fish species, such as rainbow trout Oncorhynchus mykiss (Angelidis et al. 1987), chinook salmon Oncorhynchus tshawytscha (Maule et al. 1989), and carp Cyprinus carpio (Yin et al. 1995).A number of indicators have been used to study the effects of stress on the immune responses of fish. These methods can generally be divided into 3 categories (Anderson 1990): (1) non-specific assays that do not involve antigenic stimulation (e.g., hematocrit and leukocrit) (Ellsaesser & Clem 1986, Pickering & Pottinger 1987; (2) non-specific or specific functional indicators, which may or may not use antigenic stimulation (e.g., chemiluminescent assay of phagocytic oxidative burst potential; T and B cell mitogenesis assays) (Stave & Roberson 1985, Ellsaesser & Clem 1986 and (3) indicators that involve immunization of the fish and subsequent assay of the specific immune response (e.g., antibody assays and pathogen challenge) (Hetrick et al. 1979, Cipriano et al. 1985, Smith et al. 1992, Cobb et al. 1998.The importance of non-specific immune responses as a first-line of protection against infectious di...