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The cellular repressor of adenovirus early region 1A-stimulated gene 1 (CREG1) is a secreted glycoprotein involved in cell differentiation and energy metabolism. It also binds to insulin-like growth factor 2 receptor (IGF2R), a protein implicated in muscle regeneration. However, whether CREG1 regulates the regeneration and metabolism of skeletal muscles via IGF2R remains unclear. This study investigates the role of CREG1 in skeletal muscle regeneration and glucose uptake in C2C12 myotubes and a cardiotoxin (CTX)-induced mouse skeletal muscle regeneration model. CTX-treated skeletal muscle showed significantly higher levels of IGF2R, CREG1, phospho-AMPKα Thr 172 , and GLUT4 proteins. Similarly, treatment of myotubes with CREG1 also stimulated AMPKα phosphorylation and GLUT4 expression. CREG1-induced AMPKα phosphorylation and 2DG uptake in myotubes were suppressed by IGF2R knockdown and Compound C, an AMPK inhibitor. These results suggest that CREG1 stimulates glucose uptake in skeletal muscles partially through AMPK activation. Hence, CREG1 plays an essential role in muscle regeneration by affecting glucose metabolism in skeletal muscles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2022 Elsevier Inc. All rights reserved.)

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (K A ) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The K A of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of K A , and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high K A by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high K A by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and K A of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of K A with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takashi Masuko reports financial support was provided by Healios KK., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We designed and synthesized a celecoxib derivative UTX-121 to enhance its anti-tumor activity. Similar to celecoxib, this compound could also inhibit matrix metalloproteinase (MMP)-9 activity. In addition, UTX-121 suppressed membrane-type 1 MMP (MT1-MMP)-mediated pro-MMP-2 activation by disturbing the cell surface expression of MT1-MMP. UTX-121 also impeded the glycosylation of cell surface proteins, resulting in the suppression of cell attachment to fibronectin. This inhibition by UTX-121 caused the reduction of fibronectin-stimulated focal adhesion kinase activation, Akt activation, and cell migration. Consequently, UTX-121 treatment significantly inhibited fibronectin-induced HT1080 cell invasion into the Matrigel. UTX-121 may be a potent lead compound that can be used to develop a novel anti-tumor drug., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (p < 0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21 Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (p < 0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21 Cip1 and p27 Kip1 . Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ubiquitin-conjugating enzyme E2S (UBE2S), a family of E2 protein in the ubiquitin-proteasome system, is highly expressed in several types of cancers; however, its roles in oral squamous cell carcinoma (OSCC) have not yet been well elucidated. The purpose of this study was to clarify the functional activities of UBE2S in OSCCs. We analyzed the expression levels of UBE2S in nine OSCC cell lines and primary OSCC tissues by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry (IHC). The correlations between UBE2S expression and clinical classifications of OSCCs were analyzed using the IHC scoring system. We also used UBE2S knockdown OSCC cells for functional assays (proliferation assay, flow cytometry, and Western blotting). UBE2S was overexpressed in OSCCs in vitro and in vivo and was correlated significantly (P < 0.05) with the primary tumoral size. The cellular growth was decreased and the cell-cycle was arrested in the G2/M phase in the UBE2S knockdown (shUBE2S) cells. The expression level of P21, a target of the ubiquitin-proteasome system, was increased in the shUBE2S cells because of lower anaphase activity that promotes complex subunit 3 (APC3), an E3 ubiquitin ligase, compared with shMock cells. These findings might promote the understanding of the relationship between UBE2S overexpression and oral cancer proliferation, indicating that UBE2S would be a potential biomarker of and therapeutic target in OSCCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zrt-Irt-like protein 4 (ZIP4) is critical molecule for proper mammalian development and releasing zinc from vesicular compartments. Recent studies suggested that ZIP4 plays an important role of tumor progression in pancreatic, prostate, and hepatocellular cancers, however, little is known about the detail mechanism of ZIP4 in their cancers. In the present study, we examined the possibility of ZIP4 as a new molecular target for oral squamous cell carcinoma (OSCC). We evaluated ZIP4 expression in OSCC-derived cell lines and primary OSCC samples by quantitative RT-PCR, immunoblotting, and immunohistochemistry (IHC). We also analyzed the clinical correlation between ZIP4 status and clinical behaviors in patients with OSCC. In addition, ZIP4 knockdown cells (shZIP4 cells) and ZnCl 2 treatment were used for functional experiments, including cellular proliferation assay, zinc uptake assay, and cell-cycle analysis. ZIP4 mRNA and protein were up-regulated significantly in OSCCs compared with normal counterparts in vitro and in vivo. IHC showed that ZIP4 expression in the primary OSCC was positively correlated with primary tumoral size. The shZIP4 cells showed decrease accumulation of intercellular zinc and decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. Since cellular growth of OSCC cells after treatment with zinc was significantly greater than control cells, we speculated that intercellular ZnCl 2 accumulation is an important factor for cellular growth. Consistent with our hypothesis, not only decreased zinc uptake by ZIP4 knockdown but also chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), showed inhibitory effects of cellular proliferation. Therefore, our data provide evidence for an essential role of ZIP4 and intracellular zinc for tumoral growth in OSCC, suggesting that zinc uptake might be a potential therapeutic targeting event for OSCCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We reported previously that decorin (DCN) is significantly up-regulated in chemoresistant cancer cell lines. DCN is a small leucine-rich proteoglycan that exists and functions in stromal and epithelial cells. Accumulating evidence suggests that DCN affects the biology of several types of cancer by directly/indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis, however, the molecular mechanisms of DCN in chemoresistance and its clinical relevance are still unknown. Here we assumed that DCN silencing cells increase chemosusceptibility to S-1, consisted of tegafur, prodrug of 5-fluorouracil. We first established DCN knockdown transfectants derived from oral cancer cells for following experiments including chemosusceptibility assay to S-1. In addition to the in vitro data, DCN knockdown zenografting tumors in nude mice demonstrate decreasing cell proliferation and increasing apoptosis with dephosphorylation of AKT after S-1 chemotherapy. We also investigated whether DCN expression predicts the clinical responses of neoadjuvant chemotherapy (NAC) using S-1 (S-1 NAC) for oral cancer patients. Immunohistochemistry data in the preoperative biopsy samples was analyzed to determine the cut-off point for status of DCN expression by receiver operating curve analysis. Interestingly, low DCN expression was observed in five (83%) of six cases with complete responses to S-1 NAC, and in one (10%) case of 10 cases with stable/progressive disease, indicating that S-1 chemosensitivity is dramatically effective in oral cancer patients with low DCN expression compared with high DCN expression. Our findings suggest that DCN is a key regulator for chemoresistant mechanisms, and is a predictive immunomarker of the response to S-1 NAC and patient prognosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function., Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O2 for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC)., Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p<0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p<0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p<0.01)., Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cellobiose triggers the production of two endoglucanases, EglA and EglB, in Aspergillus nidulans. The cellulose responsive element (CeRE) cis-element that is essential for induction has been identified on the eglA promoter, but transcription factors that bind to CeRE have not yet been identified. CeRE contained a consensus sequence CC(A/T)6GG for binding of the SRF-type MADS box proteins. Introduction of a missense mutation into mcmA, encoding for the sole SRF-MADS protein in A. nidulans, caused a significant reduction in cellulase induction. Real-time RT-PCR analysis revealed that inductive expression of not only eglA but also eglB and cbhA by cellobiose were under control of McmA. The McmA protein expressed in Escherichia coli specifically bound to two regions of the eglA promoter: CeRE and its upstream proximal region. These results, together with our previous study on the eglA promoter structure, imply that McmA regulates eglA expression by binding directly to its promoter. This is the first evidence for participation of an SRF-MADS protein in cellulase regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5'-kinase, and 2',3'-cyclic phosphate 3'-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5'-tri/diphosphate RNAs is dependent on ATP, a 2'-phosphate-3'-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5' terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5'-phosphate and 2'-phosphate-3'-hydroxyl ends. Two RNA molecules having 5'-hydroxyl and 2',3'-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications., (Copyright © 2010. Published by Elsevier Inc.)

Wheat RNA ligase contains 5'-hydroxyl kinase, 2',3'-cyclic phosphate 3'-phosphodiesterase, and 5'-phosphate 2'-phosphate-3'-hydroxyl RNA ligase activities in a 110-kDa polypeptide. Taking advantage of a wheat cell-free protein production system, we prepared various fragments containing a part of the enzyme. The method allowed us to check the activities of the fragments rapidly, eliminating the time-consuming cloning and sequencing steps for the expression of the fragment proteins. The results showed that each of the three activities can be assigned to a non-overlapping domain that does not require the presence of the other part(s) of the enzyme for its activity. This contrasts to the case of yeast tRNA ligase, in which the central kinase domain has been suggested to require to be tethered to one of the flanking domains for its activity., (Copyright 2010 Elsevier Inc. All rights reserved.)

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERalpha and ERbeta. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.

Type-I ribosome-inactivating protein (RIP), which is found in many plants, catalyzes depurination of a specific adenine in the sarcin/ricin domain (SRD) of the large rRNA causing loss of ribosomal activity. Previously, we found a RNA apurinic site-specific lyase (RALyase) that catalytically cleaved the phosphodiester bond at the RIP-dependent depurination site by beta-elimination reaction. Here we show that both the RIP activity and RIP-RALyase-mediated cleavage of SRD in the cytoplasmic ribosome were induced at the late stage of senescence of wheat coleoptiles. Following this process, tissue death was observed. Furthermore, transgenic tobacco plants expressing glucocorticoid-induced RIP developed senescence-like phenotype. Our results suggest that ribosome inactivation due to the cleavage of SRD by the inducible RIP and constitutively expressed RALyase may be a unique plant system that mediates programmed cell death at the late senescent stage.

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 to be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.

Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.

DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique.

Apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) is a potent inhibitor of infection by a wide range of retroviruses. Although recent reports have suggested that human APOBEC3G exerts antiviral activity against hepatitis B virus, APOBEC3G expression is normally low in the human liver. To clarify the role of APOBEC3G in cellular defenses against hepatitis viruses, the regulation of the APOBEC3G expression was investigated in human hepatocytes. Endogenous transcripts of nine APOBEC family members were barely detectable in quiescent liver cells. However, APOBEC3G was significantly up-regulated in response to interferon-alpha (IFN-alpha) stimulation in HepG2, Huh-7, and primary human hepatocytes. IFN regulatory factor elements that are important for IFN-inducible promoter activity were identified 5' upstream from the human APOBEC3G gene. Our findings provided the first evidence showing that APOBEC3G is induced by IFN stimulation in human hepatocytes and thus could be involved in host defense mechanisms directed against hepatitis viruses.

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.

CYP3A41 is a female-specific major CYP3A in mouse livers. Adrenalectomy decreased expression of CYP3A41 as well as CYP3A11, another major CYP3A, and dexamethasone (DEX) restored the decreased expression. Hypophysectomy completely abolished CYP3A41 expression and growth hormone (GH) replacement only slightly restored the expression. Treatment with DEX alone did not induce expression of either CYP3A41 or CYP3A11 in hypophysectomized mice. However, combined treatment with GH and DEX strongly induced expression of CYP3A41 but not CYP3A11. In primary cultured mouse hepatocytes, DEX induced expression of both CYP3A41 and CYP3A11, and DEX-inducible expression of CYP3A41 was suppressed by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself enhanced basal expression of CYP3A11 mRNA, while it showed no inhibitory effect on DEX-inducible expression. These observations indicate that glucocorticoids may participate in the GH-dependent control of the Cyp3a41 gene expression, probably mediated via the glucocorticoid receptor, which may be different from that of the Cyp3a11 gene expression.

Chromafenozide (ANS-118) is a non-steroidal ecdysone mimic and its insecticidal effect is highly specific to lepidoptera. In order to evaluate the transcription-inducing activity via nuclear ecdysone receptor (EcR) and the mode of action of chromafenozide, ecdysone-responsive reporter gene assay systems were developed in Sf9 and Kc cells. Ponasterone A, a full EcR agonist, induced reporter transcription in a dose-dependent manner in both Sf9 and Kc cells. In contrast, chromafenozide activated reporter transcription with comparable potency to ponasterone A only in Sf9 cells, although its maximum activity was 4-fold lower than that of ponasterone A. When chromafenozide was applied together with ponasterone A to Sf9 cells, it antagonized ponasterone A at nanomolar concentrations. These results suggest that chromafenozide is a potent partial EcR agonist specific to lepidoptera; it appears to bind lepidopteran EcR with comparable affinity to ponasterone A, but may activate the EcR in a different manner., ((c)2002 Elsevier Science (USA).)

The effect of peroxisome proliferator-activated receptor (PPAR) gamma activators, thiazolidinediones, on plasminogen activator type 1 (PAI-1) was examined in cultured human umbilical vein endothelial cells (HUVEC). Tumor necrosis factor alpha (TNF-alpha) enhanced PAI-1 secretion and mRNA expression by approximately 2-fold. The thiazolidinediones, troglitazone and pioglitazone, decreased basal and TNF-alpha-stimulated PAI-1 secretion and mRNA expression in HUVEC in a dose-dependent fashion. PPARgamma mRNA in HUVEC could be detected by reverse transcriptase-polymerase chain reaction using specific primers. These results suggest that PPARgamma may regulate PAI-1 expression in HUVEC and that thiazolidinediones have a therapeutic potential for improving endothelial dysfunction observed in insulin resistance., (Copyright 1999 Academic Press.)

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

We report here the comparative analysis of human Mcm/P1 proteins (HsMcm2, -3, -5 and -7), including a characterization of their mutual interactions, cell cycle dependent expression and nuclear localization during the cell cycle and the quiescent state. The mRNA levels of these genes, which undergo cell cycle dependent oscillations with a peak at G1/S phase, may be regulated by E2F motifs, two of which were detected in the 5' upstream region of the HsMCM5 gene. In contrast, the protein levels of these Mcm proteins were found to remain rather constant during the HeLa cell cycle. However, their levels gradually increased in a variable manner as KD cells progressed from GO into the G1/S phase. In the GO stage, the amounts of HsMcm2 and -5 proteins were much lower than those of HsMcm7 and -3 proteins, suggesting that they are not present in stoichiometric amounts, and that only a proportion of these molecules actively participate in cell cycle regulation as part of Mcm/P1 complexes.

The implication of reactive oxygen species for the Ca2+ ionophore ionomycin-induced apoptosis was investigated in cultured cortical neurons from embryonic rats. Ionomycin increased the production of intracellular peroxides as measured by flow cytometric analysis with 6-carboxy-2'7'-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester). Low doses of ionomycin increased the level of manganese-superoxide dismutase (Mn-SOD). In addition, N-acetyl-L-cysteine prevented apoptotic neuronal death induced by ionomycin in a dose-dependent manner. Buthionine sulfoximine suppressed the effect of N-acetyl-L-cysteine. These results suggest that peroxides and redox-regulation play an important role in the apoptosis of neurons induced by elevation of intracellular Ca2+ concentration.

We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.

The RNR3 gene encodes the large subunit of ribonucleotide reductase. Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker. To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region. An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3. This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide. The other repressive element homologue to DRE1 was located at -263 to -254. One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found. These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage.

Several oxygenated cholesterols and their derivatives (yakkasteroids) were prepared as candidate ligands for cytosolic-nuclear tumor promoter binding protein (CN-TPBP). Among the compounds, a possible natural substance, 3 beta,5 alpha-dihydroxycholestan-6-one (yakkasterone), showed the highest binding affinity to CN-TPBP. It also showed high specificity for binding to CN-TPBP: it does not bind to protein kinase C. Investigation of the structure-activity relationships of yakkasteroids revealed that the structure of the side chain at the 20-position is important for the binding activity to CN-TPBP.

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.

Active human thyroid-stimulating hormone (hTSH) was produced by Xenopus laevis oocytes following injection of an mRNA mixture of hTSH beta and alpha subunits synthesized by T3 RNA polymerase. Some of the hTSH molecules were secreted into the medium, while others remained in the cells. The active molecules consisted of alpha and beta subunits and were in highly glycosylated form. The Xenopus laevis oocyte-produced hTSH stimulated the rat thyroid cell line FRTL-5 to produce and secrete the cyclic AMP as does authentic hTSH.

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.