Inhibition of biofouling by marine microorganisms and their metabolites

Development of microbial biofilms and the recruitment of propagules on the surfaces of man-made structures in the marine environment cause serious problems for the navies and for marine industries around the world. Current antifouling technology is based on the application of toxic substances that can be harmful to the natural environment. For this reason and the global ban of tributyl tin (TBT), there is a need for the development of "environmentally-friendly" antifoulants. Marine microbes are promising potential sources of non-toxic or less-toxic antifouling compounds as they can produce substances that inhibit not only the attachment and/or growth of microorganisms but also the settlement of invertebrate larvae and macroalgal spores. However, so far only few antilarval settlement compounds have been isolated and identified from bacteria. In this review knowledge about antifouling compounds produced by marine bacteria and diatoms are summarised and evaluated and future research directions are highlighted.     

Inhibition of phosphofructokinases by copper(II)

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.     

Replicate divergence between and within sounds in a marine fish: the copper rockfish (Sebastes caurinus)

Understanding the factors that influence larval dispersal and connectivity among marine populations is critical to the conservation and sustainable management of marine resources. We assessed genetic subdivision among ten populations of copper rockfish (Sebastes caurinus) representing paired samples of outer coast and the heads of inlets in five replicate sounds on the west coast of Vancouver Island, British Columbia, using 17 microsatellite DNA loci. Overall, subdivision (F_ST) was low (F_ST = 0.031, P < 0.001), but consistently higher between paired coast and head of inlet sites (mean F_ST = 0.047, P < 0.001) compared to among the five coast sites (mean F_ST = ?0.001, P > 0.5) or among the five head of inlet sites (mean F_ST = 0.026, P < 0.001). Heterozygosity, allelic richness and estimates of effective population size were also lower in head of inlet sites than in coast sites. Bayesian analysis identified two genetic groups across all samples, a single genetic group among only coast samples, two genetic groups among head of inlet samples and two genetic groups within each sound analysed separately. Head of inlet copper rockfish tended to be shorter with lower condition factors and grew more slowly than coast sites fish. Reduced physical connectivity and selection against immigrants in contrasting outer coast–head of inlet environments likely contribute to the evolution of population structure of copper rockfish. Based on genetic connectivity, coast sites appear to be better served by existing marine protected areas than are head of inlet sites.     

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis $\text{in vitro}$ with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. The inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.     

Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish

Homologous peptides belonging to the pancreatic polypeptide (PP) family were isolated from the pancreas of a teleostean fish, the American eel (Anguilla rostrata), an holostean fish, the bowfin (Amia calva) and an elasmobranch fish, the skate (Raja rhina), and their primary structures were determined. The peptides show stronger homology to neuropeptide Y, particularly in their COOH-terminal regions, than to peptide YY or pancreatic polypeptide and contain an alpha-amidated COOH-terminal tyrosine residue. The skate peptide Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Asp10-Asp-Ala-Ala-Pro-Glu-Glu- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2 represents the first member of the PP family to be isolated from a cartilaginous fish. The primary structure of the pancreatic PP family peptide has been more strongly conserved among the phylogenetically more ancient holostean and elasmobranch fishes than among the teleosts. A comparison of the primary structures of all PP family peptides supports the hypothesis and evolution has acted to conserve features of tertiiary structure in the molecules (e.g., the polyproline- and alpha-helices) rather than individual amino acid residues.     

Biology of parasite populations: population dynamics of bothriocephalids (Cestoda-Pseudophyllidea) in teleostean fish

Relationships between sympatric populations of four species of Cestoda belonging to the genus Bothriocephalus ( B. barbarus. B. gregarius, B. funiculus and B. scorpii ) and populations of their respective hosts Scophthalmus rhombus, Pserra maxima, Ciliata mustela and Myoxocephalus scorpius were investigated. The parasites were overdispersed within their host populations. The mean intensities of B. burbarus were 1.59 ( s.d. =0.83) in the Mediterranean (Gulf of Lions) and 1.25 ( s.d. =0.53) in the Atlantic (coasts of Brittany). The mean intensities of B. gregarius were 62.3 ( s.d. =49.7) in the Mediterranean and 14.06 ( s.d. =18.48) in the Atlantic, compared to 1.78 ( s.d. =0.3) for B. funiculus and 21.6 ( s.d. =17.3) for B. scorpii .
The distribution of prevalence of B. gregarius corresponded to a lognormal distribution while those of B. barbatus and B. fwticulus corresponded to negative binomials. No differences in infection could be related to host sex. The parasitization of B. gregariusdepended on host size. In brill, infection by E. barbatus was independent of host size and remained stable after the fish reached 180mm in length. Parasitization of turbot by E. gregarius was lower in young fish (≤110–150 mm) and increased with the host size. Compared to the Atlantic populations of B. gregarius and B. barbatus, the Mediterranean populations were more frequently and intensively infected. The differences in the population dynamics of B. gregarius and B. barbarus would appear to depend on whether or not the life cycle involves a paratenic host.     

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites.

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis in vitro with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. Th inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.     

Tissue damage by oxy-radicals: the possible involvement of iron and copper complexes

Increasing evidence points to a role for catalytic forms of iron and copper in mediating oxidative damage associated with degenerative processes.     

Double-mouth deformity in a trout (Salmo trutta) and its cause

Gudger (1930) ends an article on this deformity by stating that double-mouth deformity in fishes is always due to injury so far as our present knowledge goes. The feature common to all cases is displacement of the lower end of the hyoid arch downwards and backwards through a gap in the mouth floor. This occurs because the protractor muscles of the arch have been put out of action, usually by severe accidental injury. The retractor muscles, thus left unopposed, pull the lower end of the arch into the deformed position. It is difficult to imagine how accidental wound could produce the condition seen in the Shetland trout. Here is only a cleft like a clean cut in the midline of the mouth floor ending at the displaced arch. There is neither irregular scarring nor obvious tissue loss, the head retaining its bilateral symmetry. In it the protractors of the arch must also have been destroyed, but probably by some other kind of damage than wound from without. With this possibility in mind I wrote to the managers of several trout-farms, enclosing a sketch of the deformity. Each was asked if he had come across anything like it when grading his stock. One letter in the affirmative suggested that air trapped in the floor of the mouth might be the cause. Later the writer sent me two young trout, radiographs of which confirmed this statement and led me to think that obstruction of the pneumatic duct might account for the trapped air. The consequences of extirpation of the swimbladder in physostomes explains how the trapped air is permanently at increased pressure, and supports this hypothesis.     

Metabolic origins of 5-hydroxytryptamine in enteric neurons in a teleostean fish (Platycephalus bassensis), a toad (Bufo marinus) and the guinea-pig

1. 5-Hydroxytryptamine (5-HT) content and synthesis in mucosa-free intestine of guinea-pig, the teleost Platycephalus bassensis and the amphibian Bufo marinus was studied by HPLC with electrochemical detection or by TLC. 2. The 5-HT content of small intestine was: guinea-pig 0.58; Bufo: 1.23; Platycephalus: 26.88 nmol/g. 3. Intestine from each species synthesized 5-HT from exogenous 5-HTP. 4. Platycephalus preparations synthesized labelled 5-HT from 14C-tryptophan, but no labelled 5-HT was detected after similar incubation of guinea-pig or Bufo preparations. 5. Incubation of guinea-pig preparations with tryptophan did not increase tissue 5-HT or 5-HIAA content. 6. 5-HT in Platycephalus enteric neurons may be synthesized from tryptophan in situ; 5-HT in Bufo and guinea-pig neurons may be synthesized elsewhere, perhaps in enterochromaffin cells.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Inhibition of oestrone sulphatase activity by tibolone and its metabolites.

Tibolone is a 19-nortestosterone derivative commonly used in hormone replacement therapy. Although tibolone and its 3alpha/beta-hydroxy metabolites exert oestrogenic effects on bone and the vasomotor system, they do not appear to stimulate breast tissue proliferation. It has been proposed that the lack of an oestrogenic effect on breast tissues may result from the inhibition of oestrone sulphatase (E1-STS) in this tissue by tibolone and its metabolites. In this study we have examined the ability of tibolone and its metabolites to inhibit E1-STS activity in intact breast cancer cells, its effect on E1-STS activity in placental microsomes and also the expression of E1-STS mRNA in more detail. As the major proportion of hydroxytibolone metabolites circulate in a sulphated form, the ability of the 3alpha-sulphate and 3alpha,17beta-disulphate metabolites to inhibit E1-STS activity was also examined. In MCF-7 cells, tibolone and its 3beta-hydroxylated metabolite were relatively potent inhibitors; they inhibited activity by 48 % and 46 %, respectively. In these cells, the 3alpha-sulphate and 3alpha,17beta-disulphate metabolites of tibolone inhibited E1-STS activity by 95% and 79% at 10 microM, respectively. No effects of tibolone or its metabolites on the expression of E1-STS mRNA in MCF-7 cells were detected. Using T-47D breast cancer cells, evidence was obtained that the sulphated metabolites of tibolone could continue to inhibit E1-STS activity after removal of the drugs and extensive washing of cells. In placental microsomes, however, the 3beta-hydroxy metabolite was the most potent inhibitor with an IC50 of 20.5 microM; the sulphated metabolites were less potent. Neither tibolone nor its metabolites had any inhibitory effect on the conversion of oestrone to oestradiol in breast cancer cells. Results from this study have confirmed that tibolone and its metabolites can inhibit E1-STS activity. This may explain the absence of breast stimulation as observed in clinical studies.     

Inhibition of proteinase K activity by copper(II) ions

Disease-related prion protein, PrPSc, can be distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Several studies have suggested that copper(II) ions can convert PrPC to a proteinase K-resistant conformation; however, interpretation of these studies is complicated by potential inhibition of proteinase K (PK) by copper(II) ions. Here we have examined directly the kinetic and equilibrium effects of copper(II) ions on PK activity using a simple synthetic substrate, p-nitrophenyl acetate. We show that at equilibrium two to three copper(II) ions bind stoichiometrically to PK and destroy its activity (Kd < 1 microM). This inhibition has two components, an initial reversible and weak binding phase and a slower, irreversible abolition of activity with a half-time of 6 min at saturating copper(II) ion concentrations. Copper(II) ions produce a similar biphasic inhibition of PK activity in the presence of brain homogenate but only when the copper(II) ion concentration exceeds that of the chelating components present in brain tissue. Under these conditions, the apparent resistance of PrPC to proteolysis by PK appears to be directly attributable to the inhibition of PK activity by copper(II) ions.     

Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect of P. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth of V. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not. P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P. fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V. anguillarum at 10(4) to 10(5) CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 10(7) CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.     

Inhibition of acetylcholinesterase from electric eel by (-)-and (+)-physostigmine and related compounds

Unnatural (+)-physostigmine (2) inhibited acetylcholinesterase (AChE) from electric eel considerably less than natural (-)-physostigmine (1), but 2 may because of its possible lower toxicity still be an interesting anticholinesterase agent. (-)-Eseroline (3), a major metabolite of (-)-physostigmine (1) and a potent narcotic analgetic, and its unnatural (+)-antipode (4), were both poor inhibitors of the enzyme. (-)-Dihydrosecophysostigmine (5), a ring-open analog of (-)-physostigmine was less, but (-)-N-methylphysostigmine (6) much more potent than the natural alkaloid. The availability of compounds related to (-)- and (+)-physostigmine by improved chemical synthesis suggests that further structural variation may well lead to other biologically interesting AChE inhibitors.