1, INTRODUCTION MetallotlCxxins(MTs), low molecular-weight, heavy-metal-binding, cysteinc-rich cyrosolic proteins were first isolated from horse kidney and characterized by Vallee and co-workers [1,2]. Since then, MTs have been found in a number of species [3]. The function of MT has been debated ever since its discovery. A role in metal metabolism or detoxification is strongly suggested by the ability of MTs to bind to and be induced by heavy metal ions [4], Kelley et al, reported that overexpression of MT represents one mechanism of resistance to anticancer agents [S]. In recent years, immunohistochemical methods have provided new interesting information on MT intracellular distribution in the hepatocytes. MT is mainly localized in the nuclei of both fetal and neonatal rat hepatocytes, and the intranuclear 10calization of MT decreases with age [6], In human liver, the localization of MT is very similar to that in rat hepatocytes [7]. However, the biological significance of the presence of MT in nuclei is not yet clearly understood.We observed that MT suppressed the formation of gastric ulcer in rats [S]. Moreover, immuno-

Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences. We now report the identification and verification of an electrically-active form of modified proteins recognized by a group of small molecules commonly used to interact with DNA. This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators. Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified Nε-pyrrolelysine as a key adduct. Unexpectedly, the pyrrolation conferred an electronegativity and electronic properties on the proteins that potentially constitute an electrical mimic to the DNA. In addition, we found that the pyrrolated proteins indeed triggered an autoimmune response and that the production of specific antibodies against the pyrrolated proteins was accelerated in human systemic lupus erythematosus. These findings and the apparent high abundance of Nε-pyrrolelysine in vivo suggest that protein pyrrolation could be an endogenous source of DNA mimic proteins, providing a possible link connecting protein turnover and immune disorders.

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRLlpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of 6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.

Abstract.In an attempt to clarify the influence of marine bacteria on the settling of fouling invertebrate larvae, we screened for inhibitors, produced by marine bacteria, of settling by cyprids of the barnacle, Balanus amphitrite. We found that the culture broth of Alteromonas sp. strain number KK10304, which was associated with the marine sponge, Halichondria okadai, effectively inhibited settling of the cyprids. Bioassay-guided isolation indicated ubiquinone-8 (1) as an effective inhibitor of cyprid settling. As ubiquinones are widely distributed in bacteria, several related compounds were also tested.Key words. Settlement inhibitor; barnacle; ubiquinone; sponge; Halichondria okadai; sponge associated bacteria; Alteromonas sp.Marine fouling invertebrates, such as barnacles and blue mussels, cause serious problems on ship hulls, in cooling systems of power plants, and in aquaculture systems. Organotin compounds such as TBTO [bis-(n-tributyltin)-oxide] have been used as effective antifouling agents against those organisms. However, stern warnings have been issued regarding the toxic effects of such heavymetal compounds on marine environments and wildlife, including fish and shellfish. Therefore, antifouling substances with no or reduced toxicity must be found and developed. In our screening for antifouling substances, using laboratory-reared larvae of the barnacle, Balanus amphitrite, we have reported fatty acids ~, steroids 1, a sesquiterpene hydrocarbon 2, a betaine 3, a furanoterpenoic acid 4, a pukalide derivative 4, a gramine derivative s and others as settling inhibitors in marine invertebrates. Crisp 7 reported that the larvae of fouling invertebrates were recruited to the settling surface in response to surface associated stimuli. He also revealed 7 that the stimuli played an important role not only in attracting larvae but also in inducing their metamorphosis. Mitchell and Kirchman s proposed that the stimuli were produced by bacteria growing on the solid surface. Maki et al. 9 reported that films of bacteria on solid substrate could positively or negatively influence the settling of marine invertebrate larvae. They 9 tested the effects of culture media of 18 different strains of marine bacteria on the settling of cyprids of the barnacle, B.amphitrite. They found that 8 out of 18 culture media showed inhibition of the settling and that the culture medium of Deleya (Pseudomonas) marina showed the strongest activity. These papers have suggested the existence of settling inhibitors produced by marine bacteria, although many of these compounds have remained unknown. In the present paper, we report the isolation and identification of the settling inhibitors from bacteria associated with a marine sponge. Materials and methods Isolation of bacteria.A common marine sponge, Ha[ichondria okadai, was collected from Numazu area in Suruga Bay of Shizuoka Prefecture, in 1990. This specimen was cut into small pieces (5 g), homogenized with 10 ml of sterilized seawater and diluted in a series of 10-fold dilutions. Each...

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