Protein responses in blue mussels (Mytilus edulis) exposed to organic pollutants: a combined CYP-antibody/proteomic approach.

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.

Effect of treatment of hypothyroidism on the plasma concentrations of neuroactive steroids and homocysteine.

Autoimmune thyroiditis with hypothyroidism is frequently accompanied by symptoms of psychiatric disorders and atherogenic changes in lipid metabolism. Recent studies suggest that some neuroactive steroids and homocysteine are involved in the pathophysiology of both disorders. Homocysteine metabolism may be affected by some steroids. We were interested if the treatment of hypothyroidism would affect the above factors. We studied plasma concentrations of allopregnanolone, pregnenolone sulfate, dehydroepiandosterone and its sulfate, progesterone, estradiol and homocysteine in 14 patients (12 women, 2 men) during the 3-month treatment with levothyroxine. Steroids and thyroid function were monitored by measuring thyrotropin, free triiodothyronine, free thyroxine and levels of thyroid antimicrosomal antibodies and antibodies to thyroglobulin. We have found that with the restoration of the thyrotropin level, free triiodothyronine, free thyroxine and homocysteine levels decreased, but the levels of steroids were not significantly altered. Steroid concentrations correlated negatively with the level of thyroid antimicrosomal antibodies.

[The prevalence of antithyroid antibodies and long term follow-up in patients with lipiodol iodine-induced goiter].

We have carried out a follow-up study on iodine-induced goiter to clarify whether or not iodine could be a factor in the progression as well as the promotion of thyroid autoimmunity. We selected 143 women of child-bearing age without any previous thyroid disorders who had received hysterosalpingography (H.S.G.). 45 Sex and age-matched healthy subjects were chosen as controls. Serum nonhormonal iodine (S.N.I.) levels, frequency of goiter and antimicrosomal antibody (MCHA) in all the Lipiodol-cases were significantly higher than those in the controls (p < 0.001, < 0.01 and < 0.01), respectively. When the subjects were divided into 9 groups according to the duration of each 5 months after H.S.G., serum TSH and S.N.I. levels, incidence of goiter and MCHA in the initial group were significantly higher than those in the other groups (p < 0.05). The S.N.I. levels became normalized in 30 months after H.S.G. and the goiters disappeared in almost the same duration, while the incidence of higher MCHA titers declined gradually but significantly around 40 months after H.S.G. compared with that in the first 5 months after H.S.G. (p < 0.05). The frequency of goiter and MCHA in 44 cases after a 6-39 month follow-up decreased significantly compared to that in the initial group (p < 0.05). Therefore, we tried an individual longitudinal follow-up study on MCHA titer in 12 cases for 35-103 months, resulting in a significant reduction or negativeness of the titer in 6 cases. Likewise, MCHA titers in all cases decreased significantly (p < 0.05) on later evaluation. The present data suggest that iodine in Lipiodol administered via the vagina will act not only as the promoting factor, but as an aggravating agent for thyroid autoimmunity.

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Antibodies that inhibit Ca2+-pump systems of cardiac and skeletal muscles.

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.

Iodized oil treatment for endemic goiter does not induce the surge of positive serum concentrations of anti-thyroglobulin or anti-microsomal autoantibodies.

Forty-three goitrous patients (grade II and III, WHO classification), living in areas of chronic iodine deficiency, were treated with an injection of iodized oil (470 mg iodine). Serial measurements of serum thyroid hormone levels after the therapy revealed increasing concentrations of both hormones, with a significantly lower serum T3/T4 ratio, and progressively significantly lower serum TSH mean values. Serum Tg mean value, initially elevated (58 +/- 9 ng/ml), decreased after 6 months and returned to the normal range at 36 months of therapy. In none of the examined patients (except for one subject with positive autoantibodies before therapy), it was observed the surge of positive anti-thyroglobulin or anti-microsomal autoantibodies after the iodized oil. We conclude that iodized oil therapy does not induce an abnormal autoimmune reaction in endemic goiter patients.

Significance of a common epitope of plant and animal endomembranes.

A rat monoclonal antibody that was raised against a common epitope on bean (Phaseolus vulgaris) endomembranes has been shown to cross-react with microsomal polypeptides from a number of plant and animal species. Immunoblotting has shown that the epitope is present on a large subset of polypeptides on microsomes of five animal species. The antigenic site appears to be accessible on intact bean membranes since it is readily digested by trypsin. The epitope is probably not derived post-translationally since the same Mr range is immunoprecipitated from polypeptides newly synthesized in vivo and in vitro. The polypeptides in bean appear to be regulated independently, one of Mr 58,000, in particular, was highly induced by treatment of suspension cultures with fungal elicitor. Preincubation of membranes enriched with endoplasmic reticulum and Golgi apparatus with the antibody blocks transfer of radioactivity from one compartment to the other in vitro. The common antigenic site could possibly be concerned in recognition or some fusion event during membrane trafficking within the cell.

Antigen-antibody reactions in rat brain sites induce transient changes in drinking behavior.

The septum or hypothalamus of rat brain was injected through implanted cannulas with antibody against membrane antigens in the rat brain or with antibody against exogenous soluble antigens (such as ovalbumin) followed by the specific antigen. Both immunological systems produced a moderate but highly significant decrease in drinking by thirsty rats. This phenomenon is suggested as an experimental model for behavioral disorders resulting from nondegenerative, immunological processes in the brain.

Subcellular fractions from dermis and epidermis in contact sensitization of guinea pigs to 1-chloro-2,4-dinitrobenzene.

Subcellular fractions were prepared from the epidermis of guinea pigs which had been painted with 1-chloro-2,4 dinitrobenzene 14 h previously. The microsomal fraction showed less activity than other fractions in the MIF test. Similarly, the microsomal fraction from the dermis of skin irritated with croton oil before DNCB painting was relatively inactive. However, the microsomal fractions from the epidermis and more especially from the dermis of skin painted with DNCB while undergoing allergic contact dermatitis from pentadecylcatechol showed significant MIF activities.