Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

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Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.Human SUMO1 (small ubiquitin-related modifier) (also named UBL1, PIC1, GMP1, SMT3C, and sentrin in the literature) is a ubiquitin-like protein (1-3). It shares about 18% identity to ubiquitin (4). Human SUMO1 and its yeast homolog, Smt3p, also share a similar activation and conjugation pathway with ubiquitin but employ distinct sets of E1 2 and E2 enzymes (5-9). The E1 enzymes for activating human SUMO1 and yeast Smt3p are the heterodimeric proteins SAE1/SAE2 and Aos1p/Uba2p, respectively (6, 9). Ubc9 is the only E2 identified for SUMO1/Smt3p, although a dozen of E2 enzymes have been identified for ubiquitin in yeast (10 -14). Recently, proteases that specifically activate SUMO1/Smt3p precursors and cleave SUMO1/Smt3p from their protein conjugates have been identified in yeast and mammalian cells (15)(16)(17)(18). Many proteins such as RanGAP1 (19), promyelocytic leukemia protein (20), IB␣ (21), RAD51, RAD52 (22, 23), p53 (24), and centromere proteins (25, 26), which have diverse functions, have been shown to interact with Ubc9/SUMO1 or be covalently modified by SUMO1. SUMO1 appears to primarily target nuclear proteins (23). Although the function of SUMO1 is still elusive, there are a few interesting observations that may shed light on the function of SUMO1. First, Pods (promyelocytic leukemia protein oncogenic domains, also called nuclear bodies or ND10) (27) and the nuclear envelope appear to be the major site of localization of SUMO1 conjugates in the nucleus (28). Second, SUMO1 and ubiquitin appear to share the same conjugation site(s) on some target proteins (e.g. IB␣ and MDM2) (21). Third, stress-caused protein unfolding (e.g. heat s...

These results suggest that overexpression of the resistin gene in adipocytes may be a local determinant factor in the pathogenesis of PCOS.

Thyroid hormone induces tumor cell and blood vessel cell proliferation via a cell surface receptor on heterodimeric integrin αvβ3. We investigated the role of thyroid hormone-induced internalization of nuclear integrin αv monomer. Physiological concentration of thyroxine (free T4, 10(-10) M), but not 3,5,3'-triiodo-l-thyronine (T3), induced cellular internalization and nuclear translocation of integrin αv monomer in human non-small-cell lung cancer (H522) and ovarian carcinoma (OVCAR-3) cells. T4 did not complex with integrin αv monomer during its internalization. The αv monomer was phosphorylated by activated ERK1/2 when it heterodimerized with integrin β3 in vitro. Nuclear αv complexed with transcriptional coactivator proteins, p300 and STAT1, and with corepressor proteins, NCoR and SMRT. Nuclear αv monomer in T4-exposed cells, but not integrin β3, bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor-α, cyclooxygenase-2, hypoxia-inducible factor-1α, and thyroid hormone receptor β1 in chromatin immunoprecipitation assay. In summary, monomeric αv is a novel coactivator regulated from the cell surface by thyroid hormone for the expression of genes involved in tumorigenesis and angiogenesis. This study also offers a mechanism for modulation of gene expression by thyroid hormone that is adjunctive to the nuclear hormone receptor (TR)-T3 pathway.

The results of this study provide a baseline QoL in infertile couples in Taiwan, and could potentially be used as a guide for clinical counseling and future works.

Hybrid carbon nanotube composites with two different types of fillers have attracted considerable attention for various advantages. The incorporation of micro-scale secondary fillers creates an excluded volume that leads to the increase in the electrical conductivity. By contrast, nano-scale secondary fillers shows a conflicting behavior of the decreased electrical conductivity with micro-scale secondary fillers. Although several attempts have been made in theoretical modeling of secondary-filler composites, the knowledge about how the electrical conductivity depends on the dimension of secondary fillers was not fully understood. This work aims at comprehensive understanding of the size effect of secondary particulate fillers on the electrical conductivity, via the combination of Voronoi geometry induced from Swiss cheese models and the underlying percolation theory. This indicates a transition in the impact of the excluded volume, i.e., the adjustment of the electrical conductivity was measured in cooperation with loading of second fillers with different sizes.

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