ARHGAP20 Expression Inhibited HCC Progression by Regulating the PI3K-AKT Signaling Pathway.

INTRODUCTION: One of the most common cancers is hepatocellular carcinoma (HCC), which is an aggressive cancer that is associated with high mortality. The expression and role of ARHGAP20 in HCC remain unclear. MATERIALS AND METHODS: The expression and clinical role of ARHGAP20 were investigated using online databases and HCC samples from Meizhou People's Hospital. Wound healing assays, transwell migration/invasion assays, and lung metastasis models were performed using nude mice. Gene set enrichment analyses were used to further explore the potential mechanisms. RESULTS: Inspired by expression analyses of three different public databases (ie, TIMER, Oncomine, and HCCDB database), we confirmed that ARHGAP20 was downregulated in clinical HCC tumors compared with normal controls. ARHGAP20 expression inhibited HCC migration and invasion in vitro and in vivo. Based on GSEA results, we tested markers of the PI3K-AKT signaling pathway. Interestingly, while ARHGAP20 upregulation suppressed HCC migration/invasion and phosphorylation of AKT/PI3K molecules, exposure to the PI3K-AKT pathway agonist rhIGF-1 partially rescued these phenomena. ARHGAP20 also showed a close correlation with certain components in the HCC immune microenvironment. Furthermore, we revealed that downregulated ARHGAP20 was significantly correlated with larger tumor size and vascular invasion, and could be used as an adverse independent prognostic factor for HCC OS but not RFS. CONCLUSION: ARHGAP20 was identified for the first time as a tumor suppressor gene that could inhibit HCC progression by regulating the PI3K-AKT signaling pathway and the immune microenvironment in HCC.

Combined toxicity of outdoor air pollution on kidney function among adult women in Mianyang City, southwest China.

Environmental pollution is a risk factor for kidney dysfunction. However, the combined toxicity of air pollutants on kidney function is scarce. We estimated the relationship between combined toxicity of air pollutants and kidney function among adult women (n = 7071, 18-65 years old) in Mianyang City, Southwest China. We measured serum concentrations of uric acid, urea, creatinine, and cystatin C, and we calculated the individual estimated glomerular filtration rate (eGFR) using a cystatin C-based equation developed specifically for Chinese patients with CKD equation. Air pollution data were collected to calculate the individual average daily dose (ADD) of pollutants based on the air quality complex index (AQCI). Mean AQCI was higher in winter and lower in summer, and followed the monthly and seasonal trends of air pollutants. Concomitantly, individual ADD was also higher in winter and lower in summer, and the seasonal differences were reflected in the levels of kidney biomarkers (including uric acid, urea, creatinine, cystatin C, and eGFR). With an interquartile range (IQR: 1.04-1.50 m3/day/kg) increases of ADD, the serum concentrations of uric acid, urea, creatinine, and cystatin C increase [B (95%CI): 1.774 (0.318, 3.231) umol/L, 0.218 (0.1888, 0.247) mmol/L, 1.501 (1.016, 1.986) umol/L, and 0.006 (0.003, 0.009) mg/L, respectively], whereas eGFR decreases [B (95%CI): -0.776 (-1.106, -0.446) mL/min/1.73 m2]. Totally, the relationship between combined toxicity of air pollutants and kidney function in Chinese adult women suggests that the toxicity of combined air pollutants inversely affects kidney function, which might accelerate the risk of CKD.

Upregulation of miR-200b Inhibits Hepatocellular Carcinoma Cell Proliferation and Migration by Targeting HMGB3 Protein.

HMGB3 belongs to the high-mobility group box subfamily and has been found to be overexpressed in gastric cancer. However, the expression and the role of HMGB3 in human hepatocellular carcinoma remain unknown. Here, we report that HMGB3, which is suppressed by miR-200b, contributes to cell proliferation and migration in human hepatocellular carcinoma. After analyzing The Cancer Genome Atlas data of 371 patients with hepatocellular carcinoma, we identified HMGB3 to be upregulated in human hepatocellular carcinoma tissue. Knockdown of HMGB3 in the hepatocellular carcinoma cell line suppressed cell proliferation and migration. TargetScan analysis showed miR-200b to be a possible regulator for HMGB3. Subsequent luciferase assays indicated that HMGB3 was a direct target of miR-200b. In addition, upregulation of miR-200b inhibited hepatocellular carcinoma cell growth and migration. HMGB3 overexpression or miR-200b downregulation was associated with poor prognosis. Our findings suggest HMGB3 may serve as an important oncoprotein whose expression is negatively regulated by miR-200b in hepatocellular carcinoma.

Risk factors for the evaluation of potential central nervous system metastasis in Burkitt's lymphoma: a case study and literature review.

Burkitt's lymphoma (BL) is a malignancy of B lymphocytes. The rapid growth rate and frequent systemic spread result in most patients presenting with advanced disease at diagnosis. Cerebrospinal fluid cytology is the gold standard (with very high accuracy) for diagnosing BL central nervous system (CNS) metastasis; however, the low sensitivity of this method limits its clinical applications. Here, we report a case of BL with CNS metastasis. The levels of vascular endothelial growth factor (VEGF)-A and VEGF-C in the serum and cerebrospinal fluid were used to evaluate the status of BL remission and recurrence. Comparisons were made between VEGF and the other risk factors used in evaluating CNS metastasis. Although not in strict accordance, VEGF levels mirrored the disease course. Therefore, VEGF may reflect the status of BL CNS metastasis. Understanding the role of VEGF in CNS metastasis may help to improve the staging and risk classification of BL as well as the investigation of targeted therapy.

Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure.

Distribution of EV71 receptors SCARB2 and PSGL-1 in human tissues.

The aim of this study was to investigate the distribution of Enterovirus 71 (EV71) receptors SCARB2 and PSGL-1 in human tissues. The samples were chosen from archived specimens, and the profiles of two receptors were detected in the gastrointestinal tract, lung, and brain in situ by immunohistochemistry. SCARB2 was detected in all the tissues studied, and strong staining was observed in the gastric fundus gland, mucosal and glandular epithelia of the intestine. Similar expression was found in bronchial epithelia and pneumocytes. In addition, SCARB2 was observed in the esophagus/gastric mucosal epithelia, neuron, glial cells, and blood vessels and the perivascular tissues of the brain. By comparison, PSGL-1 was expressed weakly in the mucosal and glandular epithelia of the small intestine and colon. PSGL-1 was expressed in a few bronchial epithelia, and weak staining was observed in the pneumocytes. However, PSGL-1 was found easily in the lamina propria of all the tissues studied, and strong staining of PSGL-1 was also observed in the neurons and glial cells. The distribution of the SCARB2 and PSGL-1 in human gastrointestinal tract, lung, and brain tissues correlated with the distribution of pathological changes seen in EV71 infection. The widespread prevalence of these receptors may help explain the multiple organ involvement in infection with EV71.

Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB.

Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well-known carcinogens. Here, we report that in CdCl2-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 µM) of Cd can significantly upregulate the protein levels of eukaryotic translation initiation factor 5A1 (eIF5A1) and redox-sensitive transcription factor NF-κB p65. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF5A1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF5A1 in Cd cytotoxicity. As eIF5A1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF5A1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF5A1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF5A1 promoter region of Cd-treated BEAS-2B cells. Taken together, our findings suggest for the first time a regulatory mechanism for the pro-apoptotic protein eIF5A1 in which its level is possibly modulated by NF-κB in human lung cells.

Quercetin triggers apoptosis of lipopolysaccharide (LPS)-induced osteoclasts and inhibits bone resorption in RAW264.7 cells.

AIMS: Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption. METHODS: RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors. RESULTS: LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), and Bax, while inhibited Bcl-2 expression. CONCLUSIONS: Quercetin could supress LPS-induced osteoclast bone resorption through blocking RANK signaling and inhibiting the expression of osteoclast-related genes. Quercetin also promoted LPS-induced osteoclast apoptosis via activation of the MAPK apoptotic signaling pathway. These findings suggest that quercetin could be of potential use as a therapeutic agent to treat bacteria-induced bone resorption.

Post-translational modification of human heat shock factors and their functions: a recent update by proteomic approach.

Heat shock factors (HSFs) are vital for modulating stress and heat shock-related gene expression in cells. The activity of HSFs is controlled largely by post-translational modifications (PTMs). For example, basal phosphorylation of HSF1 on three serine sites suppresses the heat shock response, and hyperphosphorylation of HSF1 on several other serine and threonine sites by stress-activated kinases results in its activation, while acetylation on K80 inhibits its DNA-binding ability. Sumoylation of HSF2 on K82 regulates its DNA-binding ability, whereas sumoylation of HSF4B on K293 represses its transcriptional activity. With the advancement of proteomic technology, novel PTM sites on various HSFs have been identified with the use of tandem mass spectrometry (MS/MS), but the functions of many of these PTMs are still unclear. Yet, it should be noted that the discovery of these novel PTM sites provided the necessary evidence for the existence of these PTM marks in vivo. Followed by subsequent functional analysis, this would ultimately lead to a better understanding of these PTM marks. MS/MS-based proteomic approach is becoming a gold standard in PTM validation in the field of life science. Here, the recent literature of all known PTMs reported on human HSFs and the resulting functions will be discussed.

Agonist-dependent potentiation of vanilloid receptor transient receptor potential vanilloid type 1 function by stilbene derivatives.

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.

Panax notoginseng saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK and P38 MAPK signaling pathways.

The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. Here, we investigated whether PNS could promote osteogenesis of bone marrow stromal cells (BMSCs) through modulating the MAPK signaling pathways, which are implicated in BMSC osteogenesis. We found that PNS markedly increased the mineralization of BMSCs by alizarin red S assays and stimulate alkaline phosphatase activity of these cells. Additionally, PNS significantly increased the mRNA levels of alkaline phosphatase, core-binding factor a1, and bone sialoprotein while decreasing PPARγ2 mRNA levels. Furthermore, inhibitors of ERK, PD98059, and p38, SB203580 inhibited the osteogenesis-potentiating effects by PNS. PNS stimulated the activation of ERK and p38 as evidenced by increased phosphorylation of these proteins, which was inhibited by PD98059 and SB203580. Our findings indicate that PNS could promote BMSC osteogenesis by activating the ERK and p38 signaling pathways.

Panax notoginseng saponins promotes proliferation and osteogenic differentiation of rat bone marrow stromal cells.

AIM OF THE STUDY: Panax notoginseng saponins (PNS) is the main effective component of Panax notoginseng, have various pharmacologic activities such as antioxidant, anti-inflammatory, and estrogen-like bioactivities, have been shown to be an effective agent on anti-osteoporosis. Bone marrow stromal cells (BMSCs) play a crucial homeostatic role in skeletal modeling and remodeling due to their capability to differentiate into osteooblasts. Whether PNS has effect on osteogenic differentiation of BMSCs are unknown. This study was designed to investigate the effects of PNS on the proliferation and osteogenic differentiation of BMSCs in vitro. MATERIALS AND METHODS: When BMSCs cultivated in the basal medium or the osteogenic induction medium (OS with or without PNS), cell proliferation was analyzed using an MTT assay, the mineralization was assessed using Alizarin red S staining, the alkaline phosphatase activity was measured using a commercial kit, the mRNA level of osteogenic gene and PPARγ2 gene were determined using RT-PCR, the protein level of PPARγ2 was analyzed by Western blotting. RESULTS: BMSCs cultured in the basal medium with PNS caused a significant increase in proliferation. PNS treatment increased ALP activity, Alizarin red S staining and mRNA level of ALP, Cbfa 1, OC, and BSP, whereas decreased the mRNA level and protein expression of PPARγ2 during osteogenic induction. In addition, the effects of PNS treatment were dose-dependent relationship. CONCLUSION: PNS could stimulate BMSCs proliferation and promote their osteogenic differentiation by up-regulation expression of osteogenic marker gene and down-regulation expression of adipogenic marker gene in a dose-dependent manner. Thus, PNS may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Telmisartan attenuates aortic hypertrophy in hypertensive rats by the modulation of ACE2 and profilin-1 expression.

Profilin-1 has recently been linked to vascular hypertrophy and remodeling. Here, we assessed the hypothesis that angiotensin (Ang) II type I receptor antagonist telmisartan improves vascular hypertrophy by modulation of expression of profilin-1 and angiotensin-converting enzyme 2 (ACE2). Ten-week-old male spontaneously hypertensive rats (SHR) were received oral administration of telmisartan (5 or 10mg/kg; daily) or saline for 10 weeks. Compared with Wistar-Kyoto (WKY) rats, there were marked increases in systolic blood pressure and profilin-1 expression and reduced ACE2 and peroxisome proliferator activated receptor-γ (PPARγ) levels in aorta of SHR, associated with elevated extracellular-signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) phosphorylation signaling and aortic hypertrophy characterized with increased media thickness, which were strikingly reversed by telmisartan. In cultured human umbilical artery smooth muscle cells (HUASMCs), Ang II induced a dose-dependent increase in profilin-1 expression, along with decreased ACE2 protein expression and elevated ERK1/2 and JNK phosphorylation. In addition, blockade of ERK1/2 or JNK by either specific inhibitor was able to abolish Ang II-induced ACE2 downregulation and profilin-1 upregulation in HUASMCs. Importantly, treatment with telmisartan (1 or 10 µM) or recombinant human ACE2 (2mg/ml) largely ameliorated Ang II-induced profilin-1 expression and ERK1/2 and JNK phosphorylation and augmented PPARγ expression in the cultured HUASMCs. In conclusion, telmisartan treatment attenuates vascular hypertrophy in SHR by the modulation of ACE2 and profilin-1 expression with a marked reversal of ERK1/2 and JNK phosphorylation signaling pathways.

Cisplatin induces apoptosis via upregulating Wrap53 in U-2OS osteosarcoma cells.

Wrap531α, a newly identified natural antisense transcript of p53, can regulate p53 expression upon DNA damage. We sought to investigate changes in Wrap53 and p53 levels in an osteosarcoma cell line (U-2OS) exposed to cisplatin and to study apoptosis before and after knockdown of Wrap53. Our RT-PCR analysis showed a dose- dependent 3 to 40-fold increase in Wrap53 mRNA transcript levels in U-2OS exposed to 5 to 20 µM cisplatin. An approximate 2-fold increase was also observed in transcript levels of p53 mRNA. Furthermore, transient knockdown of Wrap53 by siRNAs in U-2OS cells treated with 10 µM cisplatin reduced p53 mRNA transcript levels by up to 50% of those of controls. Immunoblotting analysis showed that in U-2OS cells treated with siRNA against exon 4 of the Wrap53 gene, the protein level of p53 was also markedly reduced. Our findings suggest that cisplatin upregulates the expression of p53 in osteosarcoma cells by upregulating the transcript levels of Wrap53. Finally, measurement of apoptotic cell death by flow cytometry showed that knockdown of Wrap53 reduced apotosis in U-2OS cells induced by cisplatin.

Bioinformatic analysis of the human DHRS4 gene cluster and a proposed mechanism for its transcriptional regulation.

BACKGROUND: The human DHRS4 gene cluster consists of three genes, DHRS4, DHRS4L2 and DHRS4L1. Among them, DHRS4 encodes NADP(H)-dependent retinol dehydrogenase/reductase. In a previous study, we investigated the alternative splicing of DHRS4 and DHRS4L2. DHRS4L1 was added to the gene cluster recently, but little is known about its structure and expression. To reveal the regulatory mechanism of the DHRS4 gene cluster expression, we studied the structure and transcription of DHRS4L1 in the context of the transcriptional behaviors of the human DHRS4 gene cluster. Based on the results of bioinformatics analysis, we propose a possible mechanism for the transcriptional regulation of the human DHRS4 gene cluster. RESULTS: The homologous comparison analysis suggests that DHRS4, DHRS4L2 and DHRS4L1 are three homologous genes in human. DHRS4L1 and DHRS4L2 are paralogues of DHRS4, and DHRS4L2 is the most recent member of the DHRS4 gene cluster. In the minus strand of the human DHRS4 gene cluster, a gene transcribed in an antisense direction was found containing a 5' sequence overlapping the region of exon 1 and promoter of DHRS4. By cloning the full length of RNA variants through 5'RACE and 3'RACE, we identified two transcription start sites, within exon a2 and exon 1, of this newly named gene DHRS4L1 using neuroblastoma cell line BE(2)-M17. Analysis of exon composition in the transcripts of DHRS4 gene cluster revealed that exon 1 was absent in all the transcripts initiated from exon a1 of DHRS4L2 and exon a2 of DHRS4L1. CONCLUSIONS: Alternatively spliced RNA variants are prevalent in the human DHRS4 gene cluster. Based on the analysis of gene transcripts and bioinformatic prediction, we propose here that antisense transcription may be involved in the transcriptional initiation regulation of DHRS4 and in the posttranscriptional splicing of DHRS4L2 and DRHS4L1 for the homologous identity of DHRS4 gene cluster. Beside the alternative transcriptional start sites, the antisense RNA is novel possible factor serving to remove exon 1 from the transcripts initiated from exon a1 and exon a2.

Panax notoginseng saponins potentiate osteogenesis of bone marrow stromal cells by modulating gap junction intercellular communication activities.

AIMS: The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. We investigated the effects of PNS on gap junction intercellular communication (GJIC) and osteogenesis-associated genes in rat bone marrow stromal cells (BMSCs). METHODS AND RESULTS: Our MTT assays demonstrated that PNS enhanced BMSC proliferation under basal medium culture in vitro. Alkaline phosphatase (ALP) assays and alizarin Red staining showed that PNS stimulated ALP activity and calcium deposition by BMSCs. Measurement of the traversing of Lucifer yellow through intercellular junctions revealed that PNS significantly stimulated GJIC activities. RT-PCR assays further showed that PNS augmented the increase in the mRNA levels of ALP, core-binding factor a1, and bone sialoprotein while decreasing the mRNA level of PPARγ2. PNS also reduced RANKL levels and increased osteoprotegerin levels. Gap junction inhibitor, 18a-glycyrrhetinic acid, could partially reverse the actions of PNS on BMSCs. CONCLUSIONS: Our findings indicate that PNS could promote osteogenesis of BMSCs by targeting osteogenesis-associated genes, which could be mediated by their actions on GJIC.

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.

Expression of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/reductase with deletion of exon 3 in cervical squamous carcinoma.

NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) plays an important role in maintaining the homeostasis of retinoid. Aberrations in retinoid metabolism are considered as early events in carcinogenesis. We identified a novel alternatively spliced variant, NRDRB1, in HeLa cell and human cervical squamous carcinoma tissues, which is characterized by a complete deletion of exon 3. The latter resulted in changes in subcellular localization of NRDRB1 when compared with the peroxisomal localization of NRDR. To clarify the clinical significance of NRDRB1, we investigated its mRNA and protein expressions in normal cervical and cervical squamous carcinoma tissues, using RT-PCR, quantitative real-time PCR, Gateway expressing system, immunoprecipitation, immunoblotting, MALDI-TOF mass spectrometry and immunohistochemistry. We detected NRDRB1 mRNA in 14 of 26 (53.9%) cervical cancer tissues, but in none of the 12 normal cervical tissues. NRDRB1 protein was expressed in NRDRB1 mRNA-positive cases. While the full-length NRDR mRNA was observed in both normal and neoplastic cervical tissues, its protein was only expressed in normal cervical epithelium. The results presented here provide evidence that metabolic disturbances of retinal and retinoic acid, due to abnormal splicing and functional disorder of NRDR, may be involved in cervical tumorigenesis.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A metabolism in mice.

This study aimed to clarify the effects of single and repeated administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities or expression of some metabolic enzymes of retinoids and the influence of supplemental vitamin A on changed vitamin A homeostasis by TCDD. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight, with or without continuous administration of 2,500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, and 28. In Experiment II, the mice were daily given 0.1 microg TCDD/kg body weight, with or without supplemental 2,000 IU vitamin A/kg body weight, and were killed on day 14, 28, and 42. In both experiments, TCDD significantly decreased the hepatic all-trans-retinol level and increased the hepatic all-trans-retinoic acid (RA) content, increased the mRNA and enzymatic activities of retinal oxidase. In TCDD + vitamin A mice, the all-trans retinol content was significantly higher, and the retinal oxidase mRNA was significantly lower on day 3 or 7 in Experiment I and on day 14 in Experiment II, compared to TCDD-treated mice. The induction of the retinal oxidase may contribute to the decrease in hepatic all-trans-retinol level and the increase in hepatic all-trans-RA caused by TCDD. Supplemental vitamin A might decelerate the effect of TCDD on retinal oxidase mRNA.

[Expression and biological activity of human angiogenesis inhibitor angiostatin in insect cells].

BACKGROUND & OBJECTIVE: Angiogenesis is the essential process for tumor growth and metastasis. Angiostatin, a potent endogenous inhibitor of angiogenesis, could selectively inhibit proliferation of vascular endothelial cells. This study was to construct recombinant angiostatin-baculovirus, and explore the expression and biological activity of recombinant angiostatin in insect cells. METHODS: The angiostatin baculovirus transfer vector pBlueBacHis2B was co-transfected with virus DNA into insect Sf9 cells to construct recombinant baculovirus. The recombinant virus was screened by plaque assay, and confirmed and amplified by polymerase chain reaction (PCR) to produce large scale, high-titer virus stock. The expression of the recombinant protein at different time points was detected by SDS-PAGE and Western blot. The recombinant protein was purified by ProBond purification system. Inhibitory effect of recombinant angiostatin on human umbilical vein endothelial cells (HUVECs) was examined by MTT assay. Its anti-angiogenesis effect was confirmed by in vivo chorioallantoic membrane (CAM) test. RESULTS: Recombinant angiostatin baculovirus with a high virus titer (2 x 10(8) pfu/ml) was constructed successfully. Recombinant angiostatin (53 ku) was effectively expressed in Sf9 cells, and its pure degree was about 90% of insect cellular total soluble proteins. The recombinant angiostatin obviously inhibited proliferation of endothelial cells with the 50% inhibitory concentration (IC(50)) of 2.3 microg/ml, and remarkably inhibited the growth of vessels in CAM. CONCLUSIONS: The baculovirus expression system could be used to construct high-titer recombinant angiostatin-virus stock, and highly express the recombinant angiostatin in insect cells. In vitro and in vivo experiments confirmed that angiostatin could inhibit proliferation of vascular endothelial cells.