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Background Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.

The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. Sphingosine 1-phosphate (S1P) induces peripheral T cell depletion. Therefore, we hypothesized that S1P1receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective sphingosine analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼73 and ∼69% with doses of 240 and 48 μg/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44116, a selective S1P1receptor antagonist. Furthermore, SEW-2871, a selective S1P1agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼70% with a dose of 10 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-2871 treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S1P1mRNA expression following IRI that begins after 2 h with the maximum expression at ∼4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S1P1receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S1P1receptor.

Molecules such as benzene, that have six pi electrons cyclically delocalized, have the special stability that chemists call aromaticity. When electricity passes through a molecular wire containing a benzene ring a quinone-like structure is induced, and there is some loss of aromaticity. The conductance of such systems correlates with the magnitude of this effect. A molecule such as cyclobutadiene with only four cyclically delocalized pi electrons has special instability, called antiaromaticity. Evidence that such antiaromatic systems could convey high conductance in appropriate systems is derived from electrochemical studies, which also detect geometric changes in some thiophene-derived nanolength wires.