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Some typical immune proteins are expressed in the nervous system, among which the paired-immunoglobulin-like receptor B (PirB) is a receptor for major histocompatibility complex class I antigen (MHC-I), but may play a physiological role in the brain for neuronal circuitry stability by inhibiting synaptic plasticity. Chronic neuroinflammation is common to many neurodegenerative diseases and is often associated with neuronal/synaptic damage and dysfunction. Here we examined the expression of PirB in the rat brain following intracerebral application of lipopolysaccharide (LPS), which has been shown to induce proinflammatory changes and cognitive deficits in rodents. One month after unilateral intrahippocampal LPS injection (10 μg in 4 μl phosphate-buffered saline, PBS), increased protein levels and immunoreactivity of PirB were detected in the ipsilateral hippocampal formation and cortex of the experimental group relative to vehicle (PBS) control. The increased PirB labeling was localized to astrocytes and neurons. Reduced synaptophysin protein levels and immunoreactivity were also found in the ipsilateral hippocampal formation and cortex in LPS-treated rats relative to controls. Morris water maze tests indicated that hippocampus-dependent spatial learning and memory were impaired in LPS-treated animals. Our findings add new experimental data for an upregulation of immune proteins in neuronal and glial cells in the brain in a model of endotoxin-induced neuroinflammation, synaptic alteration, and cognitive decline. The results suggest that PirB modulation may be involved in the pathological process under neurodegenerative conditions., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

We report a green and efficient method for chemoselective deoxidization of graphene oxide via the ultraviolet irradiation catalyzed with 2,2,6,6-tetramethyl-4-piperidinol. While the sp(2)-hybridized oxygen functional groups are removed after the reduction, the epoxy and hydroxyl groups are retained in the chemoselectively reduced graphene oxide (CrGO). The obtained CrGO nanosheets exhibit the high solubility and excellent electronic stability, which allows for the fabrication of thin film devices through a solution processing. As a proof of concept, a CrGO-based write-once-read-many-times memory device with the desirable stability and long-time operation is fabricated.

Two highly selective two-photon fluorescent probes for cysteine over homocysteine, N-acetyl-L-cysteine, dithiothreitol, glutathione and other amino acids, and their fluorescent imaging in living cells have been shown., (This journal is © The Royal Society of Chemistry 2012)

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K(m) values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V(max) values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min(-1) mg protein(-1). This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn(2+)and Fe(2+) ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC(50) = 14.36 ± 0.15 μg protein/ml).

Background: Histone deacetylase (HDAC) is an enzyme that regulates chromatin structure and inflammatory gene expression. In patients with chronic obstructive pulmonary disease (COPD), while accumulating evidence indicates that the activity of HDAC is decreased in lung tissue alveolar macrophages, HDAC activity in peripheral inflammatory cells has not yet been evaluated in detail., Methods: HDAC activities in peripheral blood mononuclear cells (PBMC) were investigated in patients with stable COPD (n = 26), non-smoking controls (n = 13), and smoking controls (n = 10), respectively. HDAC activity was measured using an HDAC Activity/Inhibitor Screening Assay Kit. Serum interleukine-8 (CXCL8) levels were determined by ELISA techniques. Lung function test was carried out according to the ATS/ERS guidelines., Results: Compared with healthy non-smokers, HDAC activity in the PBMCs of COPD patients was decreased by 40% (13.06 ± 5.95 vs. 21.39 ± 4.92 (μM/μg), p < 0.001). In patients with COPD, HDAC activity was negatively correlated to smoke intensity (r = -0.867, p < 0.001). In COPD patients who had smoked for more than 40 pack-years, HDAC activity in PBMC was 40% lower than that in COPD patients who had smoked fewer than 40 pack-years.Moreover, serum CXCL8 levels in patients with COPD were significantly higher than that in controls and were negatively correlated to HDAC activities., Conclusion: In patients with COPD, HDAC activity in the PBMCs is lower than that in healthy controls. The reduction of HDAC activity may be associated with smoking exposure through inflammatory pathways.

The aryne [3 + 2] cycloaddition process with pyridinium imides breaks the aromaticity of the pyridine ring. By equipping the imide nitrogen with a sulfonyl group, the intermediate readily eliminates a sulfinate anion to restore the aromaticity, leading to the formation of pyrido[1,2-b]indazoles. The scope and limitation of this reaction are discussed. As an extension of this chemistry, N-tosylisoquinolinium imides, generated in situ from N'-(2-alkynylbenzylidene)-tosylhydrazides via an AgOTf-catalyzed 6-endo-dig electrophilic cyclization, readily undergo aryne [3 + 2] cycloaddition to afford indazolo[3,2-a]-isoquinolines in the same pot, offering a highly efficient route to these potential anticancer agents.

Background: Kruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated., Methodology/principal Findings: Quantitative real time polymerase chain reaction (RT-PCR) analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP) transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/CreER™) transgenic mice with KLF4(flox) mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreER™ and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreER™ and KLF4(flox) double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised cell migration., Conclusions/significance: KLF4 expression in mouse hair bulge stem cells plays an important role in cutaneous wound healing. These findings may enable future development of KLF4-based therapeutic strategies aimed at accelerating cutaneous wound closure.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.

Time-resolved fluorescence measurements of graphene oxide in water show multiexponential decay kinetics ranging from 1 ps to 2 ns. Electron-hole recombination from the bottom of the conduction band and nearby localized states to wide-range valance band is suggested as origin of the fluorescence. Excitation wavelength dependence of the fluorescence was caused by relative intensity changes of few emission species. By introducing the molecular orbital concept, the dominant fluorescence was found to originate from the electronic transitions among/between the non-oxidized carbon regions and the boundary of oxidized carbon atom regions, where all three kinds of functionalized groups C-O, C = O and O = C-OH were participating. In the visible spectral range, the ultrafast fluorescence of graphene oxide was observed for the first time.

In professional immune cells, Toll-like receptor 4 (TLR4) induces tightly regulated inflammatory response to avoid tissue damage via the induction of "endotoxin tolerance", which is a transient state of cell desensitization in response to lipopolysaccharide (LPS) restimulation after a prior LPS exposure. However, in endothelial cells, the regulation of TLR4-induced inflammation is not fully understood. In this study, we found that the gene transcripts for a lot of Toll-like receptors were expressed in various endothelial cells, including human umbilical vein endothelial cells (HUVEC), human aortic endothelial cell (HAEC), and mouse microvascular endothelial cells (bEND.3). Proteins of TLR4 and its coreceptor CD14 were also detected in HUVEC. LPS treatment significantly upregulated the expression of proinflammation cytokines such as IL-1β, IL-6, and IL-8 only in HUVEC, but not in HAEC and bEND.3, suggesting that vein endothelial cells are important source of proinflammatory cytokines in response to LPS. Unexpectedly, "endotoxin tolerance" was not induced in endothelial cell, but was induced in control glial cells, as LPS pretreatment downregulated the cytokine expression in control glial cells, but did not in endothelial cells, when the cells were restimulated with LPS. The upregulation of cytokine gene expression was dependent on NF-κB signaling, and NF-κB inhibitor repressed the induction of cytokines. Two important signal molecules MyD88 and TRIF, which are TLR4 downstream and NF-κB upstream, were upregulated in vein endothelial cells but were downregulated in control glial cells. These results suggested that vein endothelial cells may play important roles in the pathophysiology of systemic inflammation-associated diseases such as sepsis and septic cardiomyopathy.

Objective: To compare the expression of sex hormone binding globulin (SHBG) in normal placental tissues with placental tissues from patients with gestational diabetes mellitus (GDM) and to deduce the mechanism affecting placental SHBG in GDM., Methods: We detected SHBG localization and measured SHBG mRNA and protein using immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blotting, respectively, in normal and GDM placental tissues. The distribution of SHBG in placental cells was examined using immune electron microscopy., Results: Compared to controls, placental tissues from patients in the GDM group displayed disordered cell surface microvilli that were decreased in quantity, swollen, and had narrowed and broken gap junctions. Intracellular abnormalities included expanded rough endoplasmic reticula, swollen mitochondria, and irregular nuclear morphologies with non-uniform chromatin. SHBG localized primarily to trophoblast cell membranes and cytoplasm. SHBG was strongly expressed on the microvilli side and weakly expressed on the basement membrane with uneven staining. SHBG also was expressed in villous stromal cells and vascular endothelial cells. Compared to the controls, placental tissues from the GDM group displayed significantly decreased immunostaining rates for SHBG, as well as significantly lower levels of SHBG mRNA and protein expression (P<0.05)., Conclusion: SHBG was detected in placental trophoblast cells from patients with GDM, and the synthesis and secretion of SHBG were reduced when trophoblast cells were irregular. A decrease in SHBG could affect placental function or aggravate GDM. Our results suggest that placental SHBG plays an important role in the pathogenesis of GDM.

The [3 + 2] cycloaddition of arynes with N-tosylpyridinium imides, followed by an elimination of Ts(-), affords pyrido[1,2-b]indazoles under mild reaction conditions in good yields.

Cardiovascular disease prevalence and mortality are both increased by insulin resistance, hypertension, and atherosclerosis. The large-conductance Ca(2+)-activated K(+) channel (BK(Ca)) plays a pivotal role in the diastolic function of vascular smooth muscle cells. However, the role of this channel in insulin resistance remains unknown. Male Sprague-Dawley rats were randomly divided into an insulin resistant group and control group. We investigated the BK(Ca) current and subunit expression in myocytes from aortas and mesenteric arteries by Western blot, real-time PCR and the whole-cell patch-clamp methods. BK(Ca) current was decreased in smooth muscle cells in insulin resistant rats, compared with that in control group. Peak BK(Ca) current at + 60 mV was significantly decreased after iberiotoxin (IBTX) perfusion at 100 nmol/L (64.2 ± 4.7 versus 20.3 ± 3.5% in thoracic aortas and 65.6 ± 6.2 versus 29.3 ± 3.9% in mesenteric arteries, both p < 0.01). However, there was no significant difference in BK(Ca) alpha subunit between the two groups, both at the level of mRNA and protein. BK(Ca) beta 1 subunit expression in aortas and mesenteric arteries from the insulin resistant group was lower than in those from control group. The plasma level of nitric oxide was higher in the insulin resistant group than in the control group. Our results demonstrated that the BK(Ca) channel is decreased both in macrovessels and microvessels in insulin resistant rats. These impairments may be related to the down-regulation of β1 subunit expression and compensatory increase in plasma nitric oxide levels., (Copyright © 2011 John Wiley & Sons, Ltd.)

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1, side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.

Background: Myocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated., Methodology/principal Findings: The depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4(+)/CD8(+)IFN-γ(+) T cell percentages and reduced myocardial Th1 cytokine levels., Conclusion/significance: Our findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to GnRH receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, but GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHR-expressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show that luteinizing hormone-expressing gonadotropes, but not follicle-stimulating hormone-expressing gonadotropes, express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in luteinizing hormone secretion is needed to promote development of follicle-stimulating hormone-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling.

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR-dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF-kappaB and TGF-beta1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR-dependent different intracellular signaling pathways., (Published 2010 Wiley-Liss, Inc.)

We aimed to examine the physical interaction between CtBPs and KLF4 and the potential importance of this interaction. Co-immunoprecipitation indicated that CtBP1 indeed interacted with KLF4. This was supported by the co-localization of both KLF4 and CtBP1 in the promoter regions of KLF4 downstream target genes. In addition, overexpression of CtBP1 significantly decreased KLF4-mediated transcriptional activation in both an artificial (pGL5) and genuine (IAP and Keratin-4) reporter system. Mutations in the potential CtBP binding motif in KLF4 were accompanied by loss of the inhibitory effect of CtBP1 in the reporter assay and of the physical interaction with CtBP1. Overall, our results suggest that CtBPs attenuate KLF4-mediated transcriptional activation through the physical interaction with KLF4.

The genomic sequence of a strain of duck enteritis virus (DEV) was determined and analyzed in this study. The size of its genome is 158,091 bp in length and the genome is predicted to encode 78 putative proteins and resembles the members of the Alphaherpesvirinae in genomic organization and gene composition. The genome of the virus is composed of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region. Its genomic arrangement pattern (UL-IRS-US-TRS) corresponds to D-type herpesvirus and is consistent with the members of Varicellovirus and Iltovirus genera. Sequence analysis reveals that the genome of the virus contains 67 genes having homologs in most members of the Alphaherpesvirinae. Out of these genes, one gene has a homolog in cercopithecine herpesvirus 8 which is a virus of Betaherpesvirinae, and 5 genes have homologs in avian herpesviruses. Furthermore, the genome possesses three unique genes without homologs in any other herpesviruses. Like most members of the Alphaherpesvirinae, the genes in the UL region of its genome are well conserved, whereas the gene arrangement of IRS-US is similar to that of Marek's disease virus and equine herpesviruses 1. Therefore, our data based on the genomic analysis suggest that DEV represents an osculant taxonomic entity within the Alphaherpesvirinae.

Overexpression of trefoil factor 2 (TFF2) is associated with increased cell migration, resistance to apoptosis, and possibly increased gastric cancer invasion. Dysregulation of p53 is frequently observed in preneoplastic conditions of the stomach. Here, we investigated the effect of p53 on the expression and function of TFF2 in gastric cancer cell lines. Gene expression was determined by reverse transcription-polymerase chain reaction, and promoter activity was assessed by dual luciferase reporter assays. Apoptosis was detected by flow cytometry, and cell migration was evaluated by the Boyden chamber assay. Exogenous expression of p53 dose dependently inhibited endogenous TFF2 mRNA, protein, and promoter activity and resulted in induction of cell apoptosis and inhibition of cell migration. Downregulation of TFF2 by small interfering RNA sensitized gastric cancer cells to drug-induced p53-dependent apoptosis. Addition of human TFF2 peptide reversed p53-dependent apoptosis and inhibition of cell migration. The p53-responsive element was mapped to an AP-1-like cis-element at -182 bp upstream of the TFF2 transcription start site. Mutation of this AP-1-like element abrogated p53-mediated inhibition of TFF2 promoter activity. Gel shift and chromatin immunoprecipitation assays demonstrated that c-Jun and c-Fos bind to this AP-1-like element. Ectopic expression of c-Jun/c-Fos or p300 or treatment of cells with phorbol 12-myristate 13-acetate (PMA) stimulated endogenous TFF2 mRNA expression and promoter activity, and p53 inhibited the effects of AP-1 and PMA on TFF2. p53 induces cell apoptosis and inhibits cell migration in part by downregulating TFF2 expression through an AP-1-like site, suggesting that TFF2 may be an important downstream target of p53.

Objective: To analyze the clinical characteristics and treatment of bilateral and unilateral sudden sensorineural hearing loss (SSHL) and figure out the differences between bi-and unilateral sudden sensorineural hearing loss, and further improve the diagnosis and treatment of SSHL., Method: The retrospective study was based on 334 patients with SSHL (315 with uni-SSHL versus 18 with bi-SSHL) and all of the patients were in-patients., Result: The incidence of bi-SSHL was 5.4% of patients with SSHL. There was no significant difference on the clinical features such as age, gender, interval from onset of symptoms to seeing doctors and associated symptoms between bi-and unilateral SSHL. 27.8% of the patients with bi-SSHL had preexisting diabetes mellitus, which was significantly higher than the rate of the uni-SSHL (9.8%, P<0.05). Hearing loss was more profound in the uni-SSHL ones, however, the hearing improvements were better than their counterparts. The overall effective rate of the two groups was 58.4% and 13.9% respectively. Earlier diagnosis and treatment with steroids would have a good prognosis., Conclusion: Unilateral SSHL is more common than bilateral SSHL. Bilateral SSHL occurs more common with preexisting diabetes mellitus and has worse prognosis compared to unilateral SSHL. The earlier steroid treatment the better prognosis.

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.

Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2-like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

This is the first report of cryptosporidiosis in a bactrian camel (Camelus bactrianus) in China. Two Cryptosporidium isolates derived from the same bactrian camel (3-year-old) in November 2005 and April 2006 were characterized using sequence and phylogenetic analysis of the small-subunit rRNA (18S rRNA), 70-kDa heat shock protein (HSP70), actin and Cryptosporidium oocyst wall protein (COWP) genes. The sequences of the 18S rRNA and COWP were identical to all other Cryptosporidium andersoni isolates although minor differences were noticed between the isolates and the USA isolate at the actin locus (99.2% of similarity). The sequence of the HSP70 was identical to the Japanese C. andersoni isolate, with a minor difference from the Australian C. andersoni isolate (99.7% of similarity). Cross-transmission studies demonstrated that the C. andersoni isolates did not infect immunosuppressed or immunocompetent Kun-ming mice, severe combined immunodeficiency mice, and immunosuppressed or immunocompetent calves. Among the C. andersoni isolates reported so far, only isolates from Japan could infect SCID mice. Thus, the C. andersoni isolates from the bactrian camel were biologically similar to most bovine C. andersoni isolates characterized so far, but are different from bovine isolates from Japan.

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCK(B) receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.

Objective: To investigate the clinical characteristics, therapeutic efficacy and misdiagnosis status of sphenoid sinus malignant tumor in order to improve the diagnosis rate., Method: Analysing and summarizing 18 patients with sphenoid sinus malignant tumor in our department from 1996 to 2005., Result: The metastatic nasopharyngeal carcinoma is the most of sphenoid sinus malignant tumor,the second is chordocarcinoma and non-Hodgkin lymphoma, its clinical manifestation was headache (78%), sight alteration (50%), cranial nerve palsy (39%), nose bleed (11%); the clinical diagnosis rate was 56%., Conclusion: The nasal endoscopy is the convention examination for these patients with sphenoid sinus malignant tumor whose imageology diagnosis is of limitation.

KLF4 is a transcription factor that is highly expressed in the gastrointestinal tract. Previously we have demonstrated that KLF4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1 binding GC box and downstream gastrin responsive elements. However, the mechanism by which KLF4 inhibits HDC promoter is not well defined. In the current study, by using yeast two-hybrid screening, Tip60 was identified as a KLF4 interacting protein. Further coimmunoprecipitation and functional reporter assays support the interaction between these two proteins. In addition, Tip60 and HDAC7, previously shown to interact with each other and repress transcription, inhibited HDC promoter activity in a dose-dependent fashion. Consistently, knock down of Tip60 or HDAC7 gene expression by specific shRNA increased endogenous HDC mRNA level. Co-immunoprecipitation assays showed that HDAC7 was pulled down by KLF4 and Tip60, suggesting that these three proteins form a repressive complex. Further chromatin immuno-precipitation indicated that all three proteins associated with HDC promoter. Two-hour gastrin treatment, known to activate HDC gene expression, significantly decreased the association of KLF4, Tip60 and HDAC7 with HDC promoter, suggesting that gastrin activates HDC gene expression at least partly by decreasing the formation of KLF4/Tip60/HDAC7 repressive complexes at the HDC promoter.

Hypergastrinemia in INS-GAS mice leads to accelerated carcinogenesis of the stomach, but the mechanisms have not been well defined. We investigated the possible role of gastrin-induced gastric cell apoptosis in the development of gastric cancer. We examined apoptosis and the expression of Bcl-2 family proteins in INS-GAS mice of different ages, as well as in gastrin-deficient (GAS-KO) mice after gastrin-17 (G-17) infusion. In addition, we studied the effects of the gastrin/cholecystokinin-2 (CCK-2) receptor antagonist YF476 and/or histamine H2 (H-2) receptor antagonist loxtidine on apoptosis and atrophy in INS-GAS mice with or without Helicobacter felis (H. felis) infection. INS-GAS mice had age-associated increases in Bax protein expression and decreases in Bcl-2 protein expression, along with increased glandular and epithelial cell apoptosis. At 8-week gastrin infusions in GAS-KO mice resulted in a similar pattern of altered Bax and Bcl-2 expression, followed by gastric cell apoptosis. H. felis infection of INS-GAS mice led to increased apoptosis and the development of atrophy, whereas treatment with either YF476 and/or loxtidine strongly inhibited both apoptosis and atrophy. In vitro studies with Fas-expressing RGM1 cells showed that gastrin stimulation alone directly induced apoptosis via gastrin/CCK-2 receptor and synergized with FasL stimulation. These results indicate that gastrin can induce apoptosis in gastric epithelial cells and contribute to the development of gastric carcinogenesis.

Histidine decarboxylase (HDC) is the enzyme that converts histidine to histamine, a bioamine that plays an important role in many physiological aspects including allergic responses, inflammation, neurotransmission, and gastric acid secretion. In previous studies, we demonstrated that Kruppel-like factor 4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1-binding GC box (GGGCGG sequence) and downstream gastrin-responsive elements. In the current study, Yin Yang 1 (YY1), a pleiotropic transcriptional factor, was also shown in cotransfection assays to repress HDC promoter activity through the upstream GC box. DNA affinity purification assay demonstrated that YY1 was pulled down specifically by the upstream GC box. In addition, sterol-responsive element-binding protein 1a (SREBP-1a), a transcriptional factor that binds YY1, represses the HDC promoter. Interestingly, deletion analysis and cotransfection assays indicated that mutation of the upstream GC box or truncation of downstream gastrin-responsive elements in the HDC promoter disrupted the inhibitory effect of YY1 and SREBP-1a in an identical fashion. Furthermore, quantitative real-time PCR analysis indicated that gastrin treatment downregulated SREBP-1a gene expression and reduced the DNA binding activity of SREBP in EMSAs. Taken together, these results suggest that YY1 and SREBP-1a form a complex to inhibit HDC gene expression through both the upstream GC box and downstream gastrin-responsive elements and gastrin-induced activation of HDC gene expression is mediated at least partly through downregulation of transcriptional repressors such as SREBPs.

The effects of feeding CLA to hens on newly hatched chick hepatic and carcass lipid content, liver TAG accumulation, and FA incorporation in chick tissues such as liver, heart, brain, and adipose were studied. These tissues were selected owing to their respective roles in lipid assimilation (liver), as a major oxidation site (heart), as a site enriched with long-chain polyunsaturates for function (brain), and as a storage depot (adipose). Eggs with no, low, or high levels of CLA were produced by feeding hens a corn-soybean meal-basal diet containing 3% (w/w) corn oil (Control), 2.5% corn oil + 0.5% CLA oil (CLA1), or 2% corn oil + 1.0% CLA oil (CLA2). The egg yolk content of total CLA was 0.0, 1.0, and 2.6% for Control, CLA1, and CLA2, respectively (P< 0.05). Maternal dietary CLA resulted in a decrease in chick carcass total fat (P < 0.05). Liver tissue of CLA2 chicks had the lowest fat content (P < 0.05). The liver TAG content was 8.2, 5.8, and 5.1 mg/g for Control, CLA1, and CLA2 chicks, respectively(P < 0.05). The chicks hatched from CLA1 and CLA2 incorporated higher levels of cis-9,trans-11 CLA in the liver, plasma, adipose, and brain than Control (P< 0.05). The content of 18:0 was higher in the liver, plasma, adipose, and brain of CLA1 and CLA2 than Control (P< 0.05), but no difference was observed in the 18:0 content of heart tissue. A significant reduction in 18:1 was observed in the liver, plasma, adipose, heart, and brain of CLA1 and CLA2 chicks (P< 0.05). DHA (22:6n-3) was reduced in the heart and brain of CLA1 and CLA2 chicks (P < 0.05). No difference was observed in carcass weight, dry matter, or ash content of chicks (P> 0.05). The hatchabilities of fertile eggs were 78, 34, and 38% for Control, CLA1, and CLA2, respectively (P< 0.05). The early dead chicks were higher in CLA1 and CLA2 than Control (18 and 32% compared with 9% for Control), and alive but not hatched chicks were 15 and 19% for CLAl and CLA2, compared with 8% for Control (P < 0.05). Maternal supplementation with CLA leads to a reduction in hatchability, liver TAG, and carcass total fat in newly hatched chicks.

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.

The enterochromaffin-like (ECL) cell controls gastric acid secretion via histamine, generated by l-histidine decarboxylase (HDC). HDC expression is regulated by gastrin. However, gastrin is not alone in controlling ECL cell function. For example, the neural peptide pituitary adenylate cyclase-activating polypeptide (PACAP) also increases ECL cell proliferation. To investigate a potential role of PACAP in regulating HDC expression, we generated a series of HDC promoter-luciferase reporter constructs and transiently transfected them into PC12 cells (stably expressing the gastrin-CCK-2 receptor). We found that PACAP regulates HDC promoter activity. This is temporally biphasic, involving both adenyl cyclase and phospholipase C-dependent pathways. Deletional analysis, block mutation, and EMSA demonstrated a PACAP-response element at -177 to -170, wholly necessary for the effects of PACAP and discrete from known gastrin-responsive elements. Discrete neural and endocrine pathways regulate ECL cells through different patterns of postreceptor signaling and promoter activation, which may be appropriate to their functions in vivo.

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Sir proteins play a critical role in silent chromatin domains. While mutations can cause derepression of heterochromatin, it remains unclear whether silencing is actively involved in transcriptional control under changing environmental conditions. We find that TOR inhibits Sir3 phosphorylation. Rapamycin or stress induced by chlorpromazine leads to activation of MAP kinase Mpk1/Slt2, which phosphorylates Sir3. Sir3 hyperphosphorylation is correlated with reduced subtelomeric silencing, increased subtelomeric cell wall gene expression, and stress resistance to chlorpromazine, but does not affect the silent HML and rDNA loci. Based on these observations, we propose that regulation of silencing may be used to control gene expression at specific silent chromatin domains in response to stress and possibly other environmental changes.

Carbon and nitrogen are two basic nutrient sources for cellular organisms. They supply precursors for energy metabolism and metabolic biosynthesis. In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively. Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes. Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein. In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor. Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses.

Objective. To demonstrate that the recycled liquid, which originated from lettuce inedible biomass degraded by fixed microorganism (correction of microorgannism) and enzyme, can be used as a nutrient solution for lettuce hydroponic cultivation. Method. After biologically degrading the weighted, oven-dried and milled leaves and roots of lettuce in a biological reactor under aerobic condition, the original effluent and its supplemented effluent were used as nutrients for lettuce hydroponic cultivation. Result. The average dried weight (ADW) of lettuce from the original effluent group was approximately half of that from the control group, and the ADW from supplemented effluent group was about equal to that from the control group; some qualities of the lettuce such as a relatively lower content of NO3- from both the original effluent group and the supplemented effluent one improved, and some of those such as a relatively higher content of NO2- dropped. Conclusion. The biologically-degraded effluent was able to be used as nutrient solution for lettuce hydroponic cultivation, although the effects of the inorganic ion-supplemented effluent were much better; the plants of lettuce from the biologically-degraded effluent were safely edible.

Objective. To develop a nutrient delivery system for space vegetable planting prototype facility to be used in future space station, and to preliminarily testify its feasibility through ground-based demonstration experiments. Method. A nutrient delivery system in a space vegetable planting prototype facility was designed and fabricated, and ground based demonstration experiments of plant cultivation were conducted. Result. Nutrient could be steadily delivered to plant cultivation matrixes through capillary action, water content of planting matrixes could be controlled automatically and maintained constant, and the planted material lettuce showed basically normal morphology and color. Conclusion. The nutrient delivery system in a space vegetable planting prototype facility could basically meet the requirements for plant nutrient delivery under space microgravity environmental condition.

The Breadboard Project at Kennedy Space Center in NASA of USA was focused on the development of the bioregenerative life support components, crop plants for water, air, and food production and bioreactors for recycling of wastes. The keystone of the Breadboard Project was the Biomass Production Chamber (BPC), which was supported by 15 environmentally controlled chambers and several laboratory facilities holding a total area of 2150 m2. In supporting the Advanced Life Support Program (ALS Program), the Project utilizes these facilities for large-scale testing of components and development of required technologies for human-rated test-beds at Johnson Space Center in NASA, in order to enable a Lunar and a Mars mission finally.

Gln3p is a nitrogen catabolite repression-sensitive GATA-type transcription factor. Its nuclear accumulation was recently shown to be under the control of TOR signaling. Gln3p normally resides in the cytoplasm. When cells are starved from nitrogen nutrients or treated with rapamycin, however, Gln3p becomes translocated into the nucleus, thereby activating the expression of genes involved in nitrogen utilization and transport. To identify other genes under the control of Gln3p, we searched for the Gln3p-binding GATAA motifs within 500 base pairs of the promoter sequences upstream of the yeast open reading frames in the Saccharomyces Genome Database. APG14, a gene essential for autophagy, was found to have the most GATAA motifs. We show that nitrogen starvation or rapamycin treatment rapidly causes a more than 20-fold induction of APG14. The expression of APG14 is dependent on Gln3p; deletion of Gln3p severely reduced its induction by rapamycin, whereas depletion of Ure2p caused its constitutive expression. However, overexpression of APG14 led to only a slight increase in autophagy in nitrogen-rich medium. Therefore, these results define a signaling cascade leading to the expression of APG14 in response to the availability of nitrogen nutrients and suggest that the regulated expression of APG14 contributes to but is not sufficient for the control of autophagy.

Gln3p is a GATA-type transcription factor responsive to different nitrogen nutrients and starvation in yeast Saccharomyces cerevisiae. Recent evidence has linked TOR signaling to Gln3p. Rapamycin causes dephosphorylation and nuclear translocation of Gln3p, thereby activating nitrogen catabolite repressible-sensitive genes. However, a detailed mechanistic understanding of this process is lacking. In this study, we show that Tor1p physically interacts with Gln3p. An intact TOR kinase domain is essential for the phosphorylation of Gln3p, inhibition of Gln3p nuclear entry and repression of Gln3p-dependent transcription. In contrast, at least two distinct protein phosphatases, Pph3p and the Tap42p-dependent phosphatases, are involved in the activation of Gln3p. The yeast pro-prion protein Ure2p binds to both hyper- and hypo-phosphorylated Gln3p. In contrast to the free Gln3p, the Ure2p-bound Gln3p is signifcantly resistant to dephosphorylation. Taken together, these results reveal a tripartite regulatory mechanism by which the phosphorylation of Gln3p is regulated.

Objective. To construct an experimental facility for microorganism waste processing, which will be used to recover plant nutrient liquids from plant inedible biomass essential for growth and development of plants. Method. After technical parameters and performance requirements were defined, plan demonstration, drawing design, fabrication, debug and preliminary plant inedible residue-biodegradation tests of microorganisms were conducted. Result. The temperature, stirring speed and gas-supplying flow of bioreactor of the facility were controlled automatically, as well as the pH and dissolved oxygen concentration were measured automatically and controlled manually, testifying that its performance reached the requirements of predetermined technical indexes. The 15-d test showed that the facility ran smoothly, its above-mentioned parameters could be measured and controlled precisely, and the biodegradation rate of lettuce's inedible biomass approximately attained 90%. Conclusion. The facility holding reasonable technical indexes, smooth and dependable performances, is capable of being utilized to biodegrade plant inedible biomass. It is expected that if the above-mentioned parameter combinations are optimized further, the results should be better.

Objective. To study the tolerance of lettuce to elevated CO2 concentration in Controlled Ecological Life Support System (CELSS). Method. Lettuce was cultivated in the Ground-based Experimental Facility for Higher Plant Cultivation in Space (GEFHPCS), in which many parameters were kept unchanged, while concentration of CO2 was controlled at 5 different levels (2000-10000 micromoles mol-1). During the growing periods, the morphologies of lettuce were observed every day, the replenished amounts of CO2 to GEFHPCS and water to the nutrient fluid box as well as the amounts of condensed water collected from GEFHPCS were all recorded every day. After harvest, the output and photosynthetic rate were calculated and lots of constituents of lettuce were analyzed. Result. The growth of lettuce were relatively ideal when CO2 concentration was at 6000 micromoles mol-1, but an obviously withering appearance was found when CO2 concentration increased 10000 micromoles mol-1, this time the output and quality of lettuce were unsatisfactory. Conclusion. It would be optimal when CO2 concentration is controlled at about 6000 micromoles mol-1 in a lettuce-cultivating chamber.

Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cycle, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-induced HPV-6 E1 promoter and negatively regulates its activity in undifferentiated cells (W. Ai, E. Toussaint, and A. Roman, J. Virol. 73:4220-4229, 1999). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcriptional activator or repressor and that can also inhibit HPV replication in vitro, binds the HPV-6 E1 promoter. EMSAs, using subfragments of the promoter as competitors, showed that the YY1 binding site is located at the 5' end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, monitored with the reporter gene luciferase, was threefold greater than that of the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes showed decreased binding of YY1 to the wild-type promoter. Consistent with this, in differentiated keratinocytes, the activity of the transfected luciferase gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated keratinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated keratinocytes. Both the wild-type and mutated promoters could be negatively regulated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP appear to be negative regulators of the differentiation-induced HPV-6 E1 promoter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.

A ground-based experimental facility was developed for conducting initial ground-based simulation study of Controlled Ecological Life Support System (CELSS). The facility is composed of a main chamber, O2 and CO2 composition control subsystems, plant cultivation subsystem and whole data management subsystem. The growth room, being composed of a inner wall of mirror-face stainless steel, holds a volume of 1.8 m3 and a growing area of 1.2 m2; electronic fluorescent lamps were used as lighting sources and polyvinyl formal was used for root matrixes; the environmental parameters of the growing room such as temperature, relative humidity, O2 concentration, CO2 concentration, lighting period and irradiance intensity and the nutrient parameters such as pH, electrical conductivity, dissolved oxygen concentration, liquid level of nutrient storage tank and flow rate of nutrient were all controlled automatically; all of the above-mentioned parameters can be inspected, collected, stored and printed regularly and dynamically. The results of a combined debugging and preliminary plant cultivation verified that the technical target of the facility had reached its initial design requirements, it can be used to conduct ground-based simulation studies of space cultivation of higher plants.

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase.

Expression of human papillomavirus genes increases as the target cell, the keratinocyte, differentiates. CCAAT displacement protein (CDP) is a cellular protein which has been shown in other cell types to negatively regulate gene expression in undifferentiated cells but not in differentiated cells. We have previously shown that a 66-bp purine-thymidine-rich sequence (the 66-mer) binds CDP and negatively regulates the human papillomavirus type 6 (HPV-6) E6 promoter (S. Pattison, D. G. Skalnik, and A. Roman, J. Virol. 71:2013-2022, 1997). Cotransfection experiments with a plasmid expressing luciferase from the HPV-6 E6, E7, or E1 regulatory region and a plasmid carrying the CDP gene indicate that CDP represses transcription from all three HPV-6 promoters. Using electrophoretic mobility shift assays (EMSAs), we have shown that CDP binds HPV-6 both upstream and downstream of the E6, E7, and E1 transcription initiation start sites. Furthermore, when keratinocytes were induced to differentiate, all three promoter activities increased. Consistent with this, immunoblotting and EMSAs revealed that endogenous nucleus CDP and, correspondingly, DNA binding activity decreased when keratinocytes were induced to differentiate. The elevated promoter activities were abrogated by exogenously transfected CDP. Our data demonstrate that CDP fulfills the requirement of a differentiation-dependent negative regulator that could tie the HPV life cycle to keratinocyte differentiation.