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Reproduced from New J. Chem., 2023, 47, 231-237 with permission from the Royal Society of Chemistry., https://doi.org/10.1039/d2nj04361k, Ionic clathrate hydrates (ICHs) have been applied to thermal storage materials, which can be utilized as unusual thermal energy sources. In the present study, we developed a novel ICH including tri-n-butyl(cyclobutylmethyl)phosphonium bromide (P444(1c4)-Br) as an unprecedented guest species. The highest equilibrium temperature and the dissociation enthalpy of P444(1c4)-Br ICH were 279.5 ± 0.1 K and 202 ± 2 J g−1, respectively, at the mole fraction of 0.0255 ± 0.0008. The highest equilibrium temperature of P444(1c4)-Br ICH was slightly lower than that of the tetra-n-butylphosphonium bromide (P4444-Br) ICH. The dissociation enthalpy of P444(1c4)-Br ICH was larger than that of other ICHs with similar highest equilibrium temperatures. Cyclic hydrocarbon groups, together with normal, branched, saturated, and unsaturated hydrocarbon chains, can be one of the options for tuning the equilibrium temperature of ICHs.

Azuma S., Shimada J., Tsunashima K., et al. Equilibrium Phase Relations and Dissociation Enthalpies of Tri- n-butylalkenylphosphonium Bromide Semiclathrate Hydrates. Journal of Chemical and Engineering Data, 67, 6, 1415 - 1420, May 25, 2022, © 2022 American Chemical Society. https://doi.org/10.1021/acs.jced.2c00146., Semiclathrate hydrates (SCHs) have received attention as thermal storage materials because of their equilibrium temperature and relatively large dissociation enthalpy. The alkyl length of quaternary onium cations significantly affects the equilibrium temperature and dissociation enthalpy of quaternary onium salt SCHs. To investigate the effects of the double bond introduced in onium cations, in the present study, the thermodynamic stability of tri-n-butylalkenylphosphonium bromide (P444(alkenyl)-Br: alkenyl = 3-butenyl (3═1) or 4-pentenyl (4═1)) SCH was investigated. The equilibrium temperatures and the dissociation enthalpies were determined to be (277.5 ± 0.1) K and (199 ± 2) J/g for P444(3═1)-Br SCH, and (274.8 ± 0.1) K and (188 ± 2) J/g for P444(4═1)-Br SCH. The equilibrium temperatures of P444(3═1)-Br SCH and P444(4═1)-Br SCH were lower than those of the corresponding normal alkyl SCHs (P4444-Br and P4445-Br), although the equilibrium temperature of tri-n-butylallylphosphonium bromide SCH was higher than that of the P4443-Br SCH.

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by γ-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (

Supplementary Figure 2 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Figure 1 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Figure 3 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Figure Legends 1-6 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Figure 4 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Figure 5 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Supplementary Table 1 from NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

Phase equilibrium (temperature–composition) relations of the tetra-n-butylphosphonium trifluoroacetate (TBP-TFA) + water binary system including solid (semiclathrate hydrate, SCH)-liquid equilibria and liquid-liquid equilibria were investigated. Phase diagram of TBP-TFA SCH exhibited peritectic behavior. The maximum decomposition temperature (peritectic temperature) of TBP-TFA SCH at atmospheric pressure were 275.9 ± 0.1 K. The stoichiometric composition of TBP-TFA SCH was located at the mole fraction of 0.027 ± 0.002. The decomposition enthalpy of TBP-TFA SCH was 199 ± 2 J/g. At temperatures above the lower critical solution temperature (LCST), the liquid-liquid phase separation appeared. Interestingly, the composition at the LCST was 0.026 ± 0.002, which is very similar to the stoichiometric composition of TBP-TFA SCH. It implies that both cluster-based solution structures are similar.

Semiclathrate hydrates (SCHs) are ionic crystalline compounds consisting of an organic guest cation in several cages formed by hydrogen bonding of water1. SCHs have recently been considered for application to air conditioning systems and refrigeration systems since desirable equilibrium temperatures and the considerable decomposition enthalpies are observed in the solid-liquid equillibria2. Ionic guest substances of SCHs include quaternary onium compounds such as tetrabutylammonium2 and tetrabutylphosphonium based salts3. We report here the preparation and thermodynamic properties of SCHs based on quaternary phosphonium bromides containing an unsaturated carbon-carbon double bond, as shown in Fig. 1, in comparison with the conventional saturated phosphonium salt, such as tetrabutylphosphonium bromide (P4444-Br). The unsaturated phosphonium bromides (P444(1All)-Br and P444(2All)-Br) were synthesized by quaternarization reaction of tributylphosphine with alkenyl bromides in a nitrogen atmosphere. The aqueous solutions were prepared from 5 to 50 w/w %. After thorough stirring, the aqueous solutions were cooled in a freezer to form SCHs. The SCHs were immersed into a thermostatic bath at 268 K for at least 1 day. The temperature in the thermostatic bath was raised from 268 K at a rate of 0.1 K/step (1 step: more than 5 h). The equilibrium temperatures were defined as the temperature at which SCHs were completely dissolved. The ionic crystal structure was analyzed by a powder X-ray diffraction (PXRD) technique, the dissociation enthalpy was measured by a micro differential scanning calorimeter (μDSC). Fig. 2 depicts the solid-liquid phase equilibrium diagram representing the phase equilibrium temperatures of SCHs. The equilibrium temperature of P444(1All)-Br SCH was higher than P4444-Br SCH. These results might be due to the steric effect of alkenyl group. As shown in Fig. 3, the ionic crystal structure of both P444(1All)-Br and P444(All)-Br SCHs were orthorhombic. The dissociation enthalpy of P444(1All)-Br SCHs and P444(2All)-Br SCHs were 188 J/g and 199 J/g, respectively. The unsaturated phosphonium bromides would be regarded as potential guest substances used for latent heat storage materials in air conditioning systems. References M. Oshima, et al, J. Chem. Thermodynamics, 90, 277 (2015). H. Oyama, et al, Fluid Phase Equilibria, 234, 131 (2005). J. Shimada, et al, Chem. Eng. Sci., 236, 116514 (2021). Figure 1

DRG1 and DRG2 comprise a highly conserved subfamily of GTP-binding proteins and are thought to act as critical regulators of cell growth. Their abnormal expressions may trigger cell transformation or cell cycle arrest. Our aim is to clarify their physiological functions and regulatory mechanisms. Here we report identification of novel proteins, DRG family regulatory protein (DFRP) 1 and DFRP2, which regulate expression of DRG proteins through specific binding. In transient transfection experiments, DFRP1 specifically binds DRG1, and DFRP2 preferentially binds DRG2. DFRPs provide stability to the target DRG proteins through physical association, possibly by blocking the poly-ubiquitination that would precede proteolysis of DRG proteins. DFRPs are highly conserved in eucaryotes, and the expression patterns of dfrp1 and drg1 transcripts in Xenopus embryos and tissues are similar, indicating that these genes work cooperatively in various types of eukaryotic cells. Immunofluorescence experiments have revealed that the interaction between DRG1 and DFRP1 may occur in the cytoplasm. We generated dfrp1- knockout cells and found that endogenous expression of DRG1 is regulated by DFRP1, confirming that DFRP1 is a specific up-regulator of DRG1 in vivo. On the basis of these results, we propose that DRG1 and DRG2 are regulated differently despite their structural similarities.

The developmentally regulated GTP-binding protein (DRG) subfamily is an uncharacterized member of the Obg family, an evolutional branch of GTPase superfamily proteins. GTPases act as molecular switches regulating diverse cellular processes. DRG2 and DRG1 comprise the DRG subfamily in eucaryotes. Although drg1 was first identified as a gene predominantly expressed during early development of the mouse central nervous system, comparative analysis of drg2 and drg1 expression during embryogenesis has never been reported, and the biochemical properties of the DRG family proteins remain to be elucidated. Thus, we first cloned Xenopus drg2 (Xdrg2) and examined the temporal and spatial expression patterns of Xdrg2 mRNA in comparison to those of Xdrg1. Both Xdrg2 and Xdrg1 are induced at late gastrula and subsequently increased during later stages of embryos (stage 13-41). Whole-mount in situ hybridization showed that Xdrg2 and Xdrg1 expression patterns are almost identical except that only Xdrg2 expression is detected in the stage 22 pronephric anlage. Strong transcripts of both genes are also observed at this stage in neural crest cells, blood islands, and developing eyes, and in brain, eyes, otic vesicle, branchial arches, pronephroses, spinal cord, notochord, head mesenchyme, and somites at stages 27 and 32. Northern blot analysis of adult tissues revealed that both genes are expressed highly in ovary and testis and rather moderately in other organs, except that Xdrg1 transcripts are scarcely detected in heart, lung, and liver. Accordingly, transcription or stability of Xdrg2 and Xdrg1 mRNAs may be regulated by different mechanisms. In addition, by generating recombinant XDRG2 and XDRG1 proteins, we found the RNA binding activity of these proteins in vitro. Our results suggest that the DRG proteins may play their physiological roles via RNA binding.

Background ERA (Escherichia coliRas-like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA-like proteins is not known. Results We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. Conclusion Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.

Background TRAF6, a member of the tumour necrosis factor receptor-associated factor family, was first identified as a transducer of CD40 and interleukin-1 receptor (IL-1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL-1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified. Results We show that TRAF6−/− mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6−/− mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6−/− mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6−/− mice are defective in lymph node organogenesis and IL-1 signalling in thymocytes. Conclusions The results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.

CD40 signaling modulates the immune response at least in part by activation of nuclear factor kappaB (NFkappaB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFkappaB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFkappaB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFkappaB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFkappaB activation by cyt-C is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFkappaB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFkappaB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.

CD40 signalings play crucial roles in B-cell function. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-beta receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required. Overexpression of TRAF6 activates transcription factor NFkappaB, and its TRAF-C domain suppresses NFkappaB activation triggered by CD40 lacking residues 246-277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain “driver” genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12–21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.

Constitutive nuclear factor (NF)-kappaB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kappaB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kappaB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kappaB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-kappaB activation by expression of IkappaBalpha super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-kappaB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-kappaB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-kappaB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer.

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by γ-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (

The forkhead transcription factor FoxA1 is thought to be involved in mammary tumorigenesis. However, the precise role of FoxA1 in breast cancer development is controversial. We examined expression of FoxA1 in 35 human breast cancer cell lines and compared it with that of ErbB2, a marker of poor prognosis in breast cancer. We found that FoxA1 is expressed at high levels in all ErbB2-positive cell lines and a subset of ErbB2-negative cell lines. Down-regulation of FoxA1 by RNA interference significantly suppressed proliferation of ErbB2-negative and FoxA1-positive breast cancer cell lines. Down-regulation of FoxA1 also enhanced the toxic effect of Herceptin on ErbB2-positive cell lines through induction of apoptosis. Taken together with previous data that FoxA1 is a marker of luminal cells in mammary gland, our present results suggest that FoxA1 plays an important role as a lineage-specific oncogene in proliferation of cancer cells derived from mammary luminal cells.

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

DRG1 and DRG2 comprise a highly conserved subfamily of GTP-binding proteins and are thought to act as critical regulators of cell growth. Their abnormal expressions may trigger cell transformation or cell cycle arrest. Our aim is to clarify their physiological functions and regulatory mechanisms. Here we report identification of novel proteins, DRG family regulatory protein (DFRP) 1 and DFRP2, which regulate expression of DRG proteins through specific binding. In transient transfection experiments, DFRP1 specifically binds DRG1, and DFRP2 preferentially binds DRG2. DFRPs provide stability to the target DRG proteins through physical association, possibly by blocking the poly-ubiquitination that would precede proteolysis of DRG proteins. DFRPs are highly conserved in eucaryotes, and the expression patterns of dfrp1 and drg1 transcripts in Xenopus embryos and tissues are similar, indicating that these genes work cooperatively in various types of eukaryotic cells. Immunofluorescence experiments have revealed that the interaction between DRG1 and DFRP1 may occur in the cytoplasm. We generated dfrp1- knockout cells and found that endogenous expression of DRG1 is regulated by DFRP1, confirming that DFRP1 is a specific up-regulator of DRG1 in vivo. On the basis of these results, we propose that DRG1 and DRG2 are regulated differently despite their structural similarities.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals that lead to activation of NFkappaB and AP-1, which is essential for cell differentiation and establishment of the immune and inflammatory systems. TRAF-interacting protein with a forkhead-associated domain (TIFA) was identified as a TRAF6-binding protein that could link IRAK-1 to TRAF6 and then activate TRAF6 upon stimulation. We report identification of a TIFA-related protein, TIFAB, that inhibits TIFA-mediated activation of NFkappaB. TIFAB does not associate with members of the TRAF family but does bind TIFA. We analyzed the effect of TIFAB expression on the TRAF6/TIFA interaction by immunoprecipitation of TRAF6 and found that TIFA coprecipitated with TRAF6 was not changed. However, when we analyzed this interaction by immunoprecipitation of TIFA, we found that TIFAB significantly increased the amount of TRAF6 coprecipitated with TIFA. These findings suggest that TIFAB inhibits the TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA.

Recurrent chromosomal translocations in neoplasms often generate hybrid genes that play critical roles in tumorigenesis. Desmoplastic small round-cell tumor (DSRCT) is an aggressive malignancy associated with the chromosomal translocation t(11;22)(p13;q12). This translocation generates a chimeric transcription factor, EWS-WT1, which consists of the transcriptional activation domain of the Ewing's sarcoma (EWS) protein and the DNA binding domain of the Wilms' tumor 1 (WT1) protein. One of the splice variants, EWS-WT1(-KTS) lacks three amino acid residues (Lys-Thr-Ser) in the DNA binding domain and transforms NIH3T3 cells. Therefore, it is likely that aberrant gene expression caused by EWS-WT1(-KTS) is involved in the malignant phenotype of DSRCT. Microarray analysis of 9600 human genes revealed that a gene encoding a tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1 (TALLA-1), was induced in EWS-WT1(-KTS)-expressing cell clones. This induction was EWS-WT1(-KTS)-specific, and more importantly, TALLA-1 protein was expressed in the three independent cases of DSRCT. Tetraspanin-family genes encode transmembrane proteins that regulate various cell processes such as cell adhesion, migration and metastasis. Our findings provide a novel insight into the malignant phenotype of DSRCT, suggesting that TALLA-1 is a useful marker for diagnosis and a potential target for the therapy of DSRCT.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that links signals from members of the TNFR superfamily and Toll/IL-1 receptor family to activation of transcription factors NFκB and AP-1. Analysis of TRAF6 -deficient mice revealed that TRAF6 is essential for normal bone formation and establishment of immune and inflammatory systems. Here we report that TRAF6 deficiency results in defective development of epidermal appendixes, including guard hair follicles, sweat glands, sebaceous glands of back skin, and modified sebaceous glands such as meibomian glands, anal glands, and preputial glands. Except the sebaceous gland impairment, these abnormal phenotypes are identical to those observed in Tabby ( Ta ), downless ( dl ), and crinkled ( cr ) mice, which are models of hypohidrotic (anhidrotic) ectodermal dysplasia in human. β-catenin and mucosal addressin cell adhesion molecule-1, an early marker of developing guard-hair follicles is absent in the skin of TRAF6 -deficient embryos. Thus, TRAF6 is essential for development of epidermal appendixes. TRAF6 does not associate with the cytoplasmic tail of the dl protein (DL)/ectodysplasin receptor (EDAR) receptor, which, when mutated, results in hypohidrotic (anhidrotic) ectodermal dysplasia. However, TRAF6 associates with X-linked ectodysplasin-A2 receptor (XEDAR) and TNFR super family expressed on the mouse embryo (TROY/toxicity and JNK inducer (TAJ), which are EDAR-related members of the TNFR superfamily that are expressed at high level in epidermal appendixes. Furthermore, TRAF6 is essential for the XEDAR-mediated NFκB activation. Our results suggest that TRAF6 may transduce signals emanating from XEDAR or TROY/TAJ that are associated with development of epidermal appendixes.

The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.

A cDNA encoding the human homolog of the tumor necrosis factor receptor-associated factor 5 (TRAF5) protein has been molecularly cloned from a cDNA library of Human Daudi B cell line. The sequence analysis revealed that the cDNA encoded a protein of 557 aa residues with a calculated molecular weight of 64,236. The encoded protein has typical structural characteristics shown in the TRAF family of proteins and binds to the cytoplasmic region of lymphotoxin-beta receptor more efficiently than to that of CD40 and CD30. The TRAF5 gene was mapped to the human chromosome 1q32.3-q41.1. Overexpression of human TRAF5 activates NF kappa B transcription factor in human 293T kidney cells. These results suggest that the human TRAF5 protein could be involved in the signal triggered by various members of the tumor necrosis factor receptor (TNFR) superfamily including CD40, CD30 and lymphotoxin-beta receptor.