Artificial neural network analysis of data from multiple in vitro assays for prediction of skin sensitization potency of chemicals.

In order to develop in vitro risk assessment systems for skin sensitization, it is important to predict a threshold from the murine local lymph node assay (LLNA). We first confirmed that the combination of the human Cell Line Activation Test (h-CLAT) and the SH test improved the accuracy and sensitivity of prediction of LLNA data compared with each individual test. Next, we assessed the mutual correlations among maximum amount of change of cell-surface thiols (MAC value) in the SH test, CV75 value (concentration giving 75% cell viability) in a cytotoxicity assay, EC150 and EC200 values (thresholds concentrations of CD86 and CD54 expression, respectively) in h-CLAT and published LLNA thresholds of 64 chemicals. Based on the results, we selected MAC value and the minimum of CV75, EC150 (CD86) and EC200 (CD54) as descriptors for the input layer of an artificial neural network (ANN) system. The ANN-predicted values were well correlated with reported LLNA thresholds. We also found a correlation between the SH test and the peptide-binding assay used to evaluate hapten-protein complex formation. Thus, this model, which we designate as the "iSENS ver. 1", may be useful for risk assessment of skin sensitization potential of chemicals from in vitro test data.

Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc gamma and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells.

The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as FcgammaRII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in FcgammaRII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of FcgammaRII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fcgamma and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.

RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway.

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.