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A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microglia maintain brain homeostasis as the main immune cells in the central nervous system. Activation of sigma-1 receptor (Sig1R) plays neuroprotective and anti-inflammatory roles in microglia. Recent studies showed that Sig1R expression level has been reduced in the brain of the patients with neurodegenerative diseases including Alzheimer's disease. However, the mechanisms underlying the down regulation of the Sig1R has not been clear. Treatment of rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of Sig1R mRNA in a concentration and time-dependent manner. The effects of LPS were blocked by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) antagonist. Furthermore, inhibitors of transforming growth factor beta-activated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK) and histone deacetylase 6 (HDAC6) restored the LPS-induced downregulation of Sig1R. Thus, the current findings demonstrate that TLR4 activation leads to the downregulation of the Sig1R expression via TLR4-TAK1-p38 MAPK pathway and the inhibition of HDAC6 can increase Sig1R expression in microglia. The current findings suggest that downregulation of Sig1R may contribute to neuroinflammation-induced microglial dysfunction, regulation of microglial Sig1R may be novel therapeutic drug candidates for neurodegenerative and neuroinflammatory diseases., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest in this study., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Aging is a predominant risk factor for the development and progression of cardiovascular complications. Physiologically and anatomically, the heart undergoes numerous changes that result in poor cardiac function in the elderly population. Recently, several studies have provided promising results, confirming the ability of the senescence-accelerated mouse-prone 8 (SAMP8) model to accurately model age-related cardiovascular alterations. In this study, using a murine model of senescence, SAMP8, we aimed to investigate the effect of 3,4-dihydroxybenzalacetone (DBL), a catechol-containing phenylpropanoid derivative isolated from Inonotus obliquus (Chaga), on cardiac aging. DBL was administered at the doses of 10 mg/kg and 20 mg/kg by oral gavage to SAMP8 mice to examine aging-mediated cardiac changes, such as oxidative DNA damage, oxygen radical antioxidant capacity (ORAC) value, fibrosis, inflammation, and apoptosis. The treatment with DBL at both doses significantly reduced aging-mediated oxidative DNA damage, and simultaneously increased the ORAC value in the SAMP8 assay. Cardiac fibrosis was assessed with Azan-Mallory staining, and the number of cardiac remodeling markers was found to be significantly reduced after the treatment with DBL. We also observed a decrease in cardiomyocyte apoptosis as measured by the terminal transferase-mediated dUTP nick end labeling (TUNEL) staining method and the caspase-3 levels in SAMP8 mice compared with senescence-resistant control (SAMR1) mice. The findings from this study suggest that DBL has a potentially beneficial effect on aging-mediated myocardial alterations. Further studies are warranted to confirm the promising potential of this catechol compound against aging-associated myocardial dysfunction., Competing Interests: The authors declare no conflicts of interest.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by a sensitive photoabsorber following exposure to NIR light. Most studies of NIR-PIT have been performed in xenograft models of cancer. The purpose of this study was to evaluate the therapeutic effects of NIR-PIT in a transgenic model of spontaneous lung cancer expressing human EGFR (hEGFR-TL). Mice were separated into 3 groups for the following treatments: (1) no treatment (control); (2) 150 μg of photoabsorber, IR700, conjugated to panitumumab, an antibody targeting EGFR [antibody-photoabsorber conjugate (APC)] intravenously (i.v.) only; (3) 150 μg of APC i.v. with NIR light administration. Each treatment was performed every week up to three weeks. MRI was performed 1 day before and 3, 6, 13, 20, 27, and 34 days after first NIR-PIT. The relative volume of lung tumors was calculated from the tumor volume at each MRI time point divided by the initial volume. Steel test for multiple comparisons was used to compare the tumor volume ratio with that of control. Tumor volume ratio was inhibited significantly in the NIR-PIT group compared with control group (P < 0.01 at all time points). In conclusion, NIR-PIT effectively treated a spontaneous lung cancer in a hEGFR-TL transgenic mouse model. MRI successfully monitored the therapeutic effects of NIR-PIT. Mol Cancer Ther; 16(2); 408-14. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: The aim of this study was to identify the biomarkers associated with chemotherapeutic efficacy and long-term survival for patients with advanced squamous cell carcinoma of the esophagus (SCCE) who had received neoadjuvant chemotherapy with docetaxel and cisplatin plus 5-fluorouracil (NAC-DCF)., Methods: This study included 45 patients with advanced SCCE who received NAC-DCF between 2008 and 2012. The NAC-DCF was conducted as a phase II study (UMIN000007408). The expressions of excision repair cross-complementing-1 (ERCC1), class III beta-tubulin, breast cancer susceptibility gene I (BRCA1), and thymidylate synthase were investigated simultaneously in the pre-treatment endoscopic tumor biopsy samples., Results: A multivariate logistic regression analysis indicated that pathological responses were significantly associated with tumors with low ERCC1 expression (P = 0.016) and with tumors with high BRCA1 expression (P = 0.030). The multivariate Cox proportional hazard model analysis for relapse-free survival revealed high BRCA1 expression (P = 0.031, hazards ratio 4.39) as the factor associated with survival., Conclusions: Low ERCC1 expression and high BRCA1 expression in patients with SCCE were associative biomarkers for chemotherapeutic efficacy. High BRCA1 expression was considered the factor associated with survival. These findings may be helpful for tailoring chemotherapy.

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on β-globin expression. In contrast, transduction with both transcription factors stimulated β-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of β-globin equivalent to that of adult erythroid cells. Interestingly, this increase in β-globin was coincident with a decrease in ε- and ζ-, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of β-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin., (Copyright© Ferrata Storti Foundation.)

Aberrant expression of MIR9 predicts a poor prognosis in acute myelogenous leukemia. To evaluate its clinical significance in acute lymphoblastic leukemia, we analyzed expression levels of MIR9 in bone marrow samples from patients with acute lymphoblastic leukemia and compared them to those in normal bone marrow cells. Approximately 20% of them showed higher expression compared with controls. There was a tendency that patients who showed overexpression of MIR9 underwent worse clinical courses, but without statistical significance. However, when the analyses were restricted to patients who did not receive a stem cell transplant, overexpression of MIR9 was significantly associated with worse overall survival. Interestingly, exaggerated MIR9 expression and higher white blood cell count at presentation were independent unfavorable prognostic factors in all patients for overall survival by multivariate analysis. The presence of higher MIR9 expression could be a useful indicator for treatment stratification.

Objective of the study was to assess the breed effect on fatty acid (FA) composition of different adipose tissues and on mRNA expression of genes involved in adipogenesis and fat metabolism. Japanese Black (JB) and Holstein (HS) steers were kept under equivalent conditions with high energy intake resulting in large differences in intramuscular fat (IMF) accumulation in longissimus muscle (LM). The relative FA composition of muscle, intermuscular fat, visceral fat, and perirenal fat was comparable between JB and HS steers. Circulating fatty acids were also similar in both breeds. Most relevant breed effects were identified in IMF, underlining the uniqueness of this adipose tissue site. JB steers had more monounsaturated FA and less saturated FA. Perilipin 1 and adipose differentiation-related protein (ADFP) mRNA levels were higher in IMF of JB. The results suggest advanced maturity of IMF cells in JB and altered local conditions in muscle influencing IMF accumulation and composition., (© 2013.)

We analyzed the expression profiles of several steroidogenic enzymes in normal adrenals, aldosterone-producing adenomas (APA), cortisol-producing adenomas combined with Cushing's syndrome (CPA) or with subclinical Cushing's syndrome (SCPA), and nonfunctioning adrenal adenomas (NFA) to clarify the nature and characteristics of steroidogenesis in APA. Clinical data were collected for all subjects. In resected adrenal glands (normal adrenals, APA, CPA, SCPA, and NFA), the mRNA expression levels of the CYP17, HSD3B2, CYP11B1, and CYP11B2 genes were studied using real-time quantitative PCR and immunohistochemistry. The CYP11B2 mRNA level in APA was significantly higher than that in other groups. The CYP17/HSD3B2 ratio for mRNA in APA was significantly lower than those in the other groups. Low ratio of CYP17/HSD3B2 with high expression of CYP11B2 seems to explain steroidogenic characteristics of APA.

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the β-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).

The RUNX1 gene is frequently rearranged in acute leukemia. We cloned a novel RUNX1 chimeric gene generated by t(7;21)(q11.2;q22) in a patient with acute myeloid leukemia. 3'-rapid amplification of cDNA ends analysis showed a tail-to-tail fusion between RUNX1 on 21q22 and DTX2 on 7q11.2, with an insertion of short complementary sequence from UPK3B adjacent to DTX2. DTX2 encodes a putative E3-ubiquitin ligase with no known biological function. There are two possible functions of RUNX1-reversed UPK3B-DTX2: one from aberrant RUNX1 chimeric protein and the other from the reversed sequence of DTX2. The predicted aberrant protein expressed under the RUNX1 promoter was highly structurally similar to RUNX1a. In a reporter assay, the aberrant protein inhibited the trans-activation function of RUNX1 in a dominant-negative manner, similar to RUNX1a. In contrast, the DTX2 reversed sequence may degrade wild-type DTX2 transcript or suppress its translation. In conclusion, we identified a novel fusion RUNX1 partner, DTX2, which chimerize in a reverse direction. This is the first example of RUNX1 chimera in an opposing direction generated by chromosomal translocation in leukemia. In addition to the aberrantly truncated RUNX1 protein, the DTX2 antisense sequence may play some role in the development of leukemia carrying the t(7;21) translocation.

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca(2+) accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.

(-)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5-20 microM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure-activity relationship study revealed that the exogenously produced H(2)O(2) significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH(2)-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.

The gene encoding NADP(+)-dependent L-1-amino-2-propanol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 was cloned and sequenced. A 780-bp nucleotide fragment was confirmed to be the gene encoding AADH by agreement of the N-terminal and internal amino acid sequences of the purified AADH. The gene (aadh) codes a total of 259 amino acid residues, and the deduced amino acid sequence shows similarity to several short-chain dehydrogenase/reductase family proteins. An expression vector, pKKAADH, which contains the full length aadh was constructed. Escherichia coli cells possessing pKKAADH exhibited a 10.4-fold increase in specific activity as to catalysis of the reduction of (S)-1-phenyl-2-methylaminopropan-1-one (MAK), as compared with that of R. erythropolis MAK154 induced by 1-amino-2-propanol (1 mg/ml). Coexpression of aadh with a cofactor regeneration enzyme (glucose dehydrogenase) gene was also performed, and a system for sufficient production of d-pseudoephedrine from racemic MAK was constructed.

Butylparaben and benzylparaben, used as preservatives mainly in cosmetic products, have recently been found to be weakly estrogenic. Batch activated-sludge treatment and batch chlorination were carried out to roughly determine the removal efficiency of a wastewater treatment plant. Combining the removal efficiency with the estimated annual consumption and the unaltered excretion ratio, the maximum predicted environmental concentration (PEC) was estimated. Conventional acute/chronic toxicity tests were conducted using Japanese medaka (Oryzias latipes), daphnia (Daphnia magna), and green algae (Pseudokirchneriella subcapitata) for n-butylparaben, i-butylparaben, and benzylparaben. Medaka vitellogenin assays were also conducted for the three compounds and DNA microarray analysis was carried out to examine the effects of benzylparaben on gene expression. The plasma vitellogenin concentration of male medaka increased for concentrations of 200, 100, and 100 microg L(-1) n-butylparaben, i-butylparaben, and benzylparaben for 14 days, respectively, while the expression levels of genes encoding proteins such as p53, cytochrome P450 3A40, and choriogenin-L increased for concentrations higher than 4 microg L(-1) of benzylparaben. Furthermore, the predicted no-effect concentration (PNEC) was calculated using the lethal or effect concentration 50 (LC50 or EC50) values and no-effect concentrations (NOECs) obtained in the toxicity tests for these compounds. The maximum concentrations found in the aquatic environment or sewage effluent (MEC eff) were used to carry out preliminary environmental risk assessment. The calculated MEC/PNEC ratio suggests the necessity of further study such as a more detailed large-scale monitoring and chronic toxicity tests including reproduction inhibition and endocrine disruption.

Previously we demonstrated that gene transduction of the granulocyte-macrophage colony stimulating-factor (GM-CSF) gene into mouse tumor cells eliminated tumorigenicity in vivo. The rejection process of the subcutaneous tumor was as follows: transient tumor growth peaked around 10 days after tumor injection, then the tumors were rejected within a week. In this paper, we analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression method to identify molecules associated with the antitumor effect. We then screened those genes that were differentially expressed between the parental and the GM-CSF-transduced tumors and identified a group of genes that are suggested to have a relationship with tumor rejection, including a cytokine receptor, adhesion molecules, chemokines, cytotoxicity-related molecules, and others. Focusing on the chemokine genes TARC and RANTES, which were preferentially expressed in the GM-CSF-transduced tumors, their forced expression on mouse tumor cells showed moderate suppression of tumor formation. Transduction of GM-CSF in combination with either the TARC or the RANTES gene into tumor cells profoundly inhibited tumor establishment. Histological findings suggested the significant contribution of CD4+ T cells to tumor regression in both TARC/GM-CSF- and RANTES/GM-CSF-transduced tumor cells, in excess of that seen with GM-CSF transduction alone.

Purpose: To investigate the expression pattern of myocilin in the trabecular meshwork of normal and dexamethasone-induced ocular hypertensive rat eyes., Materials and Methods: An ocular hypertension model was generated by application of topical dexamethasone to rat eyes 4 times daily for 1, 2, and 4 weeks. Age-matched untreated eyes served as controls. The intraocular pressure (IOP) was monitored by electronic Tono-pen under anesthesia. The protein and mRNA levels of myocilin in the trabecular meshwork and endothelial lining of Schlemm's canal were investigated by immunohistochemistry and in situ hybridization, respectively. For semiquantitative evaluation, areas with positive staining were analyzed by a computer-assisted image-processing system with NIH software., Results: The IOP in rat eyes was elevated after 2 weeks of topical dexamethasone treatment. Despite the IOP elevation, the protein and mRNA levels of myocilin in the trabecular meshwork and around Schlemm's canal in the steroid-treated eyes were not different from those of controls., Conclusion: No discernible changes in myocilin expression in the chamber angle indicate that myocilin may not be directly linked to ocular hypertension, at least not in rat eyes after relatively short-term steroid application.

Intrahepatic cholangiocarcinoma is a neoplasm arising in the liver, and its incidence is increasing in Japan as well as in Western countries. Prognosis of patients with this type of tumor remains unsatisfactory because no effective chemotherapeutic drugs are available, we have no sensitive tumor markers to detect this tumor in its early stage, and it is difficult to identify a high-risk group for the disease. To clarify the molecular mechanism of tumorigenesis and identify molecular targets for diagnosis and treatment, we analyzed global gene-expression profiles of 25 intrahepatic cholangiocarcinomas using tumor cell populations purified by laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 52 genes that were commonly upregulated and 421 that were downregulated in intrahepatic cholangiocarcinomas compared with noncancerous biliary epithelial cells. From the 52 upregulated genes, we selected P-cadherin and survivin for further investigation and corroborated enhanced expression of their products in cancer tissues by immunohistochemical staining. Furthermore, comparison between tumors with lymph node metastasis and those without metastasis identified 30 genes that were associated with lymph node involvement. In conclusion, these data should be helpful for a better understanding of the tumorigenesis of intrahepatic cholangiocarcinoma and should contribute to the development of diagnostic and therapeutic strategies for this type of tumor. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.

We previously investigated the correlations between the expression of 9216 genes and various chemosensitivities in a panel of 39 human cancer cell lines(1)) and found that the expression levels of AKR1B1 and CTSH were correlated with sensitivity and resistance to multiple drugs, respectively. To validate these correlations, we investigated the expression of these two genes and the chemosensitivities in 12 additional gastric cancer cell lines. The expression of AKR1B1 in the additional cell lines exhibited significant correlations with sensitivities to 8 of the 23 drugs examined, while that of CTSH displayed a significant negative correlation with only one (MS-247) of the 27 drugs examined. Their expressions were weakly correlated with sensitivity and resistance, respectively, to the remainder of the drugs. Moreover, when the 12 cell lines were divided into high-expressing and low-expressing groups, a comparison of these groups using Mann-Whitney's U test revealed that high expression levels of AKR1B1 and CTSH were related to sensitivity to 21 of the drugs and resistance to 8 of the drugs, respectively. The present results suggest that AKR1B1 and CTSH may be good markers for prediction of sensitivity to certain drugs and that our panel of 39 cell lines has the potential to identify candidate predictive marker genes.

Background: Lack of androgen receptor (AR) expression or mutation on the AR gene creates the tendency for androgen independence and progression of prostate cancer. However, the association between the progression and AR expression or mutations is still controversial. In this study, we evaluated the prognostic significance of AR expression and mutations in prostate cancers., Methods: Forty-two prostate adenocarcinomas and three lymph node metastatic lesions sampled prior to hormonal therapy were included in this study; AR expression was analyzed immunohistochemically using an antibody against AR and the result was scored as the percentage of AR-positive tumor cells in the total tumor cells. Polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing were used to detect AR mutations., Results: Our study revealed the average AR expression in the prostate adenocarcinoma was 52.2 +/- 27.1%, which was significantly lower than that in the adjacent non-tumorous prostate tissue (68.3 +/- 18.3% in average) (P < 0.001). A significant correlation was obtained between progression-free survival and AR expression (P < 0.01). By SSCP analysis, three silent mutations (T649T, E709E and E711E) were detected in three separate prostate carcinomas., Conclusion: : We conclude that AR expression is a useful prognostic indicator for tumor progression. Androgen receptor mutation may be an uncommon molecular event in untreated prostate cancer in Japanese men.

To analyse the functions of genes that are expressed and potentially involved in the development of embryonic gonad cells, a method was developed by which foreign genes can be introduced and expressed in cultured mouse genital ridges. Genital ridges from mouse embryos at 12.5 days post coitus (dpc) were injected with plasmid DNA of a green fluorescence protein (GFP) gene construct and then placed between small electrodes. Rectangular pulses were charged to electroporate DNA into the cells. The treated genital ridges were cultured on a membrane of a culture insert, and GFP gene expression was observed under a fluorescence microscope. Green fluorescence protein expression in the genital ridges was found as early as 1 h after electroporation. Thereafter, the expression gradually increased, peaked after 1 day, and then decreased. A significant number of cells were, however, still positive for fluorescence even after 2 weeks in the culture, in which both gonads and germ cells had continued to develop. The GFP gene was expressed in 1-2% of cells in each genital ridge in a DNA concentration-dependent manner. In addition, we confirmed that an electroporated red fluorescent protein (DsRed) gene construct was expressed in GFP-expressing primordial germ cells in genital ridges of Oct-4/GFP transgenic embryos, although the DNA was mainly found in somatic cells in genital ridges. Finally, an expression vector containing the internal ribosome entry site-green fluorescent protein (IRES-GFP) gene was constructed. An inserted lacZ gene showed similar expression pattern to that of GFP Using this vector, we can easily monitor the expression of an inserted gene of interest by GFP expression. Therefore, this experimental system could be useful for quick assays of gene function in genital ridges.

Purpose: To investigate the gene expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and their high-affinity receptors (TrkA, TrkB and TrkC) in cultured human Müller cells., Methods: The polymerase chain reaction was performed using specific primers for NGF, BDNF, NT-3, TrkA, TrkB and TrkC with complementary DNAs as the template, which were generated from poly-A+ RNA of cultured human Müller cells., Results: We detected the precursor mRNA transcripts for NGF, BDNF, NT-3, TrkB and TrkC, but not TrkA., Conclusion: Human Müller cells can direct mRNA expressions of a number of neurotrophins which may have a neurotrophic function in the retina. An autocrine mode of action is suggested, since Müller cells not only synthesize neurotrophic factors, but also express their specific receptors., (Copyright 2002 S. Karger AG, Basel)

Although long-term treatment with low doses of 14-membered macrolides is widely applied in management of patients with chronic inflammatory diseases, e.g., diffuse panbronchiolitis, chronic bronchitis, or chronic lung damage in newborns, the physiological mechanisms underlying the action of macrolides in these conditions are unclear. To clarify the pathological basis of these diseases and also to aid in the design of novel drugs to treat them, we chose to investigate the molecular target(s) of macrolides. Our experiments involved long-term culture of human small airway epithelial cells (hSAEC) in media containing 14-membered macrolides erythromycin (EM) or clarithromycin (CAM), or a 16-membered macrolide, josamycin (JM), which lacks clinical anti-inflammatory effects. We then analyzed gene expression profiles in the treated cells using a cDNA microarray consisting of 18,432 genes. We identified nine genes whose expression was significantly altered during 22 days of culture with EM, and seven that were altered by CAM in that time. Four of those genes revealed similar behavior in cells treated with either of the 14-membered macrolides, but not JM. The products of these four genes may be candidates for mediating the ability of 14-membered macrolides to suppress chronic inflammation., (Copyright 2001 Academic Press.)

The expression of the ubiquitin (Ub) gene in dermatophytes was examined for its relation to resistance against the antifungal drug fluconazole. The nucleotide sequences and the deduced amino acid sequences of the Ub gene in Microsporum canis were proven to be 99% similar to those of the Ub gene in Trichophyton mentagrophytes. Expression of mRNA of Ub in M. canis and T. mentagrophytes was enhanced when the fungi were cultured with fluconazole. The antifungal activity of fluconazole against these dermatophytes was increased in the presence of Ub proteasome inhibitor.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

Erythropoietin (Epo) is known to be a lineage specific cytokine which regulates the number of circulating erythrocytes. Most of it is produced in the kidney. Recently, Epo has been reported to be synthesized in the normal brain, placenta, and capillary endothelium. We also have found that uterine endometrium expresses Epo signals in an estrogen-dependent manner, and that Epo contributes to angiogenesis in the endometrium in mice. To clarify the functional activity of Epo in human reproductive organs, we examined Epo signaling in these organs by Southern analysis of RT-PCR products and studied the distribution of substances relevant to Epo signal transduction by immunohistochemistry and Western blotting. Epo mRNA is expressed in the normal human cervix, endometrium and ovary, but it is not always detected in the specimens. Immunohistochemical analysis revealed Epo-receptor (EpoR) protein in: a) the endothelium of vessels, in glandular and surface epithelial cells, in decidual cells of the endometrium, and b) in follicles at various stages including oocytes, granulosa, theca interna cells and lutein cells of the ovary. Moreover, co-expression of JAK2 and phosphotyrosine, which reflects tyrosine phosphorylation via JAK2, and co-expression of EpoR and STAT5, which is a transcriptional factor relevant to mitogenic activity, were seen at these Epo-responsive sites. Western blotting analysis of these organs confirmed the immunohistochemical results. These findings imply that female reproductive organs can produce Epo, and that signal transduction of Epo contributes to the cyclic changes in the female reproductive organs.

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Prophet of Pit-1 (Prop-1), which is a paired-like homeodomain transcription factor, is capable of binding to sites in an early enhancer of the Pit-1 gene and regulating its expression. As human Pit-1 is expressed considerably in pituitary adenomas, we studied human Prop-1 gene expression in pituitary adenomas. We also sequenced the Prop-1 cDNAs in pituitary adenomas. Human Prop-1 transcript was detected in all pituitary adenomas examined by RT-PCR analysis. The expression of human Prop-1 in pituitary adenomas was confirmed by in situ hybridization in one of the GH-producing adenomas. The sequence analysis of human Prop-1 cDNAs in these pituitary adenomas revealed that there were no mutations except 5 silent nucleic acid substitutions, suggesting that mutations of Prop-1 gene do not represent a frequent mechanism of human pituitary tumorigenesis.

DA41 was previously identified as one of the DAN-binding proteins, via a yeast-based two-hybrid screening strategy. In the present study, we cloned a human homolog of DA41 cDNA. Structural analysis revealed that human DA41 cDNA consisted of 2,861 nucleotides in length and encoded a protein of 589 amino acids, with a predicted molecular mass of 62.4kDa. Human DA41 exhibited an 86% amino acid sequence identity to rat DA41, indicating the evolutionarily conserved structure and function of DA41. A database search for DA41-related protein(s) identified mouse PLIC-1, PLIC-2, frog XDRP1, and yeast DSK2. DA41 and each DA41-related protein contain a ubiquitin-like domain in their amino-terminal regions. DA41 was expressed ubiquitously in adult human tissues, with relatively higher levels in pituitary gland, adrenal gland, kidney, thymus, and placenta. Fluorescence in situ hybridization (FISH) revealed that DA41 was mapped to human chromosome 9q21.2-q21.3, a position overlapping the candidate tumor suppressor locus for bladder cancer.

Centrally administered thyrotropin-releasing hormone produces a number of physiological and behavioral changes, e.g., a general antidepressant effect, increasing body temperature, and elevated blood pressure. However, the specific brain sites of action responsible for the centrally activating property of thyrotropin-releasing hormone have not been precisely determined. Using chloral hydrate-anesthetized adult Sprague-Dawley rats, we compared the distribution of Fos-like immunoreactivity after intracerebroventricular administration of thyrotropin-releasing hormone with the results after intracerebroventricular injection of vehicle alone. Some rats were paralysed and artificially ventilated to avoid possible Fos expression secondarily induced by autonomic (e.g., respiratory) disturbances. In thyrotropin-releasing hormone administered rats, selective Fos-like immunoreactivity was observed in V/VI layers of the pre- and infralimbic areas of the medial prefrontal cortex, the ventral midline thalamus, and the nucleus of the solitary tract as well as in the adjacent reticular formation. Fos-like immunoreactivity was significantly reduced in most areas of the cerebral cortex (II/III layers), the shell of the nucleus accumbens, the medial amygdaloid nucleus, parts of the hypothalamus, and the periaqueductal gray. These data suggest that various behavioral and autonomic responses induced by centrally administered thyrotropin-releasing hormone might be produced through the complex neural circuitry comprising the above structures, which are presumed to be implicated in limbic and/or autonomic functions.

In this series of projects sequencing the entire genome of Arabidopsis thaliana chromosome 5, non-redundant P1 and TAC clones have been sequenced according to the fine physical map, and as of May 7, 1999, the sequences of 16.2 Mb representing approximately 60% of chromosome 5 have been accumulated and released at our web site. In parallel, structural features of the sequenced regions have been analyzed by applying a variety of computer programs, and to date we have predicted a total of 2380 potential protein-coding genes in the 10,154,580 bp regions, which are covered by 142 P1 and TAC clones. In this paper, we newly analyzed the structural features of the 1,011,550 bp regions covered by additional 17 P1 and TAC clones, and predicted 298 protein-coding genes. The average density of the genes identified was 1 gene per 3394 bp. Introns were observed in 67% of the genes, and the average number per gene and the average length of the introns were 3.2 and 159 bp, respectively. The gene density became higher than the value estimated in the previously analyzed regions (1 gene per 4,267 bp), as the data in this paper were compiled based on a new standard of gene assignment including the computer-predicted hypothetical genes. The regions also contained 8 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available on the database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.

Allelic imbalance of the insulin-like growth factor II (IGF II) gene expression is often seen in hepatocellular carcinoma (HCC). To investigate the role of allelic imbalance in hepatocarcinogenesis, we have studied allelic expression status of the IGF II gene in dysplastic nodules, which are precancerous lesions of HCC, as well as in HCCs of different histological grade, and the influence of the allelic imbalance on IGF II gene expression has also been examined. Allelic imbalance was observed in 3 of 7 dysplastic nodules, in 7 of 9 well-differentiated HCCs, and in 8 of 9 moderately differentiated HCCs. IGF II gene expression level, which was studied by a semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR), was significantly higher (3.6-fold) in the dysplastic nodules than the control livers, but a significant increase in the IGF II gene expression was not observed in well- and moderately differentiated HCCs as compared with the control livers. These results demonstrate that the allelic imbalance of the IGF II gene expression is seen in the early stage (precancerous lesions) of hepatocarcinogenesis. Association of the allelic imbalance with an increased expression of the IGF II gene in the precancerous lesions might suggest a possible involvement of an IGF II autocrine loop in the pathogenesis of these lesions.

Retinoic acid (RA) plays a major role in neuronal cell differentiation. Neuroblastoma cells differentiate in vitro by extending neurites and forming ganglion-like aggregates in response to RA. In the present study, we have examined a biological role(s) of DAN in the regulation of RA-mediated cellular differentiation in neuroblastoma cells. RTBM1 and SH-SY5Y cells undergo marked morphological changes associated with a remarkable induction of DAN gene expression when exposed to RA. By transfecting an expression vector harboring a rat DAN cDNA into SH-SY5Y cells, we have obtained two independent transfectants which express a large amount of DAN. The forced expression of DAN gene enhanced the neurite extension in the presence of RA, suggesting that DAN gene product might contain some regulatory role(s) in the RA-induced cellular differentiation in neuroblastoma cells.

To achieve a better understanding of the mechanism of intimal thickening, we used a rabbit model in which aorta was denuded mechanically by a balloon catheter. Total RNA was prepared from each aorta 1, 2, 7, 14, 23, or 30 days after denudation, and from intact aorta of non-denuded control rabbits. Subsequently, using the differential display method, we identified eight genes that were expressed differently during the time course after injury. One of them, RESP18 (encoding regulated endocrine secretory protein 18), was suppressed during the acute reaction. The other seven showed increase in expression during the acute phase: the genes for hTAFII68 (human TATA-binding protein associated factor), NPAT (nuclear protein mapped to the AT locus), OSF2 (osteoblast-specific factor 2), Pyst1, casein kinase 1 alpha, integrin alpha 1, and XP-C complementing protein. Although hTAFII68, NPAT, OSF2, and Pyst1 are thought to be related to transcription, not all four are positive regulators. Considering that none of these genes had previously been reported as being implicated in intimal hyperplasia, we conclude that many known or unknown genes play roles in this process. We believe that differential display is an effective method for screening genes whose variations in expression can provide clues toward understanding the molecular mechanism of intimal hyperplasia.

The present study was undertaken to investigate the expression of beta1 integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the beta 1, alpha 1, alpha 2, and alpha 5 subunits of the beta1 integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of beta1 integrins in vitro. The immunohistochemical distribution of beta1 integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and mesenchymal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of the beta 1, alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of beta 1 integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of beta 1, integrins expression. Immunohistochemistry confirmed the beta 1 integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. Autoradiography of immunoprecipitate subjects to SDS-PAGE revealed three major polypeptides with molecular weights of 130 kDa (beta 1 subunit), 165 kDa (alpha 2 subunit), and 210 kDa (alpha 1 subunit) under reducing conditions. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed beta1 integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, beta 1 integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation.

We isolated a human cDNA clone encoding a novel protein homologous to cyclophilins, specific cellular targets of cyclosporin A, which are conserved in species ranging from human to prokaryotes. This cDNA, designated hCyPX, contained an open reading frame of 498 nucleotides encoding 166 amino acids. Computer analysis indicated that its predicted amino acid sequence had 41.6%, 40.4%, and 39.2% homology to those of human, bovine, and Drosophila cyclophilins, respectively. Northern blot analysis indicated ubiquitous expression in adult human tissues, but most abundant expression in heart. Fluorescence in situ hybridization to human metaphase chromosomes localized this gene (PPIL1, peptidylprolyl isomerase [cyclophilin]-like 1) to chromosome bands 2p23.3-->p23.1.

From a human fetal-brain cDNA library we isolated a novel cDNA clone encoding a protein containing a CAP-GLY domain that is highly conserved among several cytoskeleton-associated proteins. The CAP-GLY domain is thought to be essential for their association with microtubules. The cDNA, designated CKAPI (for cytoskeleton-associated protein I, glycine motif) contained an open reading frame of 579 nucleotides encoding 193 amino acids. Northern-blot analysis revealed expression of three transcripts, 1.0, 3.4, and 4.6 kb in size, in all tissues examined. The 1.0-kb transcript was significantly higher in brain and heart than in other tissues. This gene was mapped by FISH to chromosome bands 19q13.11-->q13.12.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates both humoral and cellular immune responses. Accumulating evidence suggests that the infection of T cells and other cell types with human T-lymphotropic virus type 1 (HTLV-1) results in the constitutive expression of IL-6. However, the underlying molecular mechanisms are little understood. When a reporter plasmid, pIL6-CAT-E3, in which the human IL-6 enhancer/promoter region from -630 to +14 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, was transfected, HTLV-1-infected but not -uninfected T-cell lines activated the IL-6 promoter. This indicated the presence of a factor transactivating the IL-6 gene in the infected cells. To evaluate the involvement of the HTLV-1-encoded transacting factor (Tax) in this transactivation, we examined the effect of transient cotransfection with the Tax-expression plasmid, pMAX-Neo, on the transcription from the IL-6 promoter by use of COS1 cells. The cotransfected COS1 has about six-times greater the CAT activity than that transfected with pIL6-CAT-E3 alone. The analysis of a series of deletions of the IL-6 promoter suggested that the region (-105/-47) containing a NF kappa B site was crucial for the Tax responsiveness. We further examined the effect of Tax on endogenous IL-6 gene expression using the Jurkat clone, JPX-9, stably transfected with pMAX-Neo. JPX-9 accumulated steady state transcripts of the endogenous IL-6 gene in response to the induction of Tax expression. Our findings indicate an important role of the Tax protein in the expression of IL-6 in cells infected with HTLV-1.

The expression of pEL98 (also termed mts1), an S100-related calcium-binding protein, was induced during macrophagic or granulocytic differentiation of human promyelocytic leukemia HL-60 cells in response to phorbol 12-myristate 13-acetate or dimethylsulfoxide, respectively. However, pEL98 expression remained at the low level during granulocytic differentiation of the cells by the treatment with all-trans retinoic acid. The expression of cell motility, but not adhesiveness or phagocytic ability, was found to be coincided with that of pEL98 (mts1), suggesting that pEL98 (mts1) is involved in regulating cell motility. Immunocytochemical analysis demonstrated that pEL98 (mts1) was expressed in human monocytes, macrophages and polymorphonuclear leukocytes.

RUNX3, a runt-related transcription factor, has a crucial role in dorsal root ganglion neurogenesis. Recent studies have suggested that RUNX3 acts as a tumor suppressor in stomach, colon and breast cancer. However, the biological role of RUNX3 in neuroblastoma remains elusive. Here we report that high levels of RUNX3 expression contribute to the favorable outcome in patients with neuroblastoma, whereas low levels of RUNX3 expression result in poor outcome. Array-based analysis suggested that the allelic loss at chromosome 1p36 is one of the reasons why expression of RUNX3 is downregulated in advanced neuroblastomas. Interestingly, the several patients survived from neuroblastoma with both high mRNA expressions of MYCN and RUNX3, suggesting that RUNX3 high expression might overcome the aggressive behavior of MYCN. Exogenous expression of RUNX3 strongly inhibits cell proliferation and migration in neuroblastoma cell lines. Furthermore, RUNX3 reduces the stability of MYCN protein in MYCN-amplified neuroblastoma cell lines, and this RUNX3-mediated MYCN degradation may depend on the physical interaction between RUNX3 and MYCN. Thus, our findings provide a tumor-suppressing mechanism by which RUNX3 inhibits the MYCN activity in neuroblastoma. [ABSTRACT FROM AUTHOR]

Background:We and others previously reported the prognostic significance of PTEN mutational status on favourable survival in endometrial carcinomas. Here, we demonstrate that loss of PTEN expression in immunohistochemistry is an independent prognostic marker for favourable survival in endometrial carcinomas.Methods:We conducted immunohistochemical analyses of PTEN, PIK3CA, phosphorylated Akt (p-Akt), and p27 in primary endometrial carcinomas from 221 patients. Mutation of PTEN was analysed further.Results:Expression of PTEN was lost in 56 patients (25%), and PIK3CA was overexpressed in 159 patients (72%). Overexpression of PIK3CA was associated with p-Akt overexpression (P<0.001), which was in turn associated with loss of nuclear p27 expression (P=0.028). Loss of PTEN expression was found to be associated with endometrioid histology (P=0.03), and was inversely associated with the presence of lymphovascular space invasion (P=0.03). Univariate and multivariate survival analyses revealed that factors of PTEN loss, age <70, histological grade 1, early International Federation of Gynecology and Obstetrics (FIGO) stage, and absence of lymphovascular invasion were independent prognostic indicators for better overall survival (P=0.03, 0.04, 0.01, <0.001, and 0.03, respectively). The subset analysis showed a stronger tendency of PTEN loss towards favourable survival in advanced-stage (III and IV) disease than in early-stage (I and II) disease (P=0.05 vs 0.14). Moreover, our mutational analysis demonstrated that PTEN expression loss was associated with PTEN-truncating mutations (P=0.03).Conclusion:The current observations further support the prognostic significance of PTEN aberration on favourable outcome in endometrial carcinomas, providing useful implications for the individualised management of the disease. [ABSTRACT FROM AUTHOR]

Molecularly targeted drugs for cancer therapy represent a therapeutic advance, but the proportion of patients who receive clinical benefit is still very limited. We present here the rationale and initial results of our program to define molecules involved in lung carcinogenesis with the goal of identifying new therapeutic targets and/or predictive biomarkers for drug response. We have used gene expression analysis of 120 lung cancers followed by RNA interference, tumor-tissue microarray analysis, and functional analyses to systematically distinguish potential target molecules specifically expressed in cancer cells. Through this approach, we have identified oncoproteins that provide the starting point for the development of therapeutic antibodies, dominant negative peptides, small-molecule inhibitors, and therapeutic cancer vaccines. We believe that the approach we describe should result in new molecularly targeted therapies with minimal risk of adverse events. [ABSTRACT FROM AUTHOR]

We determined whether a potential probiotic bacterium, Bifidobacterium bifidum OLB6378 (BB6378), exerts beneficial effects on the mucosal immune system in a mouse intestinal explant model. The addition of heat-inactivated BB6378 to intestinal explants prepared from embryonic day 18 BALB/c mice increased the expression of polymeric immunoglobulin receptor (pIgR) mRNA by two- to fivefold. These effects were observed on ileal and colonic explants but not on jejunal explants, suggesting that the BB6378-induced pIgR upregulation is site-specific within the mouse intestine. The upregulation of pIgR protein expression in colonic explants was also detected after 24 h of culture. The results of DNA microarray analysis of ileal and colonic samples indicated that BB6378 increased the gene expression of interleukin (IL)-1α and IL-1β, and IL-1α content in colonic explants was significantly increased after 20 h of culture with BB6378. We then examined the involvement of endogenously induced IL-1α in pIgR mRNA upregulation by using IL-1α knockout (KO) mice. Contrary to our expectations, pIgR mRNA expression was equally upregulated by BB6378 in colonic explants from BALB/c and IL-1α KO mice. Conversely, we examined the involvement of Toll-like receptors in pIgR mRNA upregulation by using MyD88 KO mice. The upregulation of pIgR was completely suppressed in the explants derived from MyD88 KO mice. Taken together, we conclude that in a mouse intestinal explant model, the heat-inactivated potential probiotic BB6378 increases intestinal pIgR expression in a site-specific manner and that the upregulation of pIgR could be explained by a direct microbial effect on the epithelium via Toll-like receptors. [ABSTRACT FROM AUTHOR]