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.

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.

[The expression of mammalian target of rapamycin and its substrates in autogenous vein graft in rats].

OBJECTIVE: To investigate the expression of mammalian target of rapamycin (mTOR) and its substrates including p70s6k and 4E-BP1 in autogenous vein graft. METHODS: Autogenous vein graft model was established in 64 Wistar rats by transplanting the right common jugular vein to infrarenal abdominal aorta. Vein graft samples were harvested 6 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery. The mRNA expression of mTOR, p70s6k and 4E-BP1 were measured by RT-PCR and in situ hybridization. Western blot and immunohistochemistry methods also were used to detect the protein expression of mTOR, p70s6k and 4E-BP1. Proliferating cell nuclear antigen (PCNA) was also detected at the same time. RESULTS: The mRNA expression of mTOR and p70s6k increased soon after vein graft transplanting, rose quickly and reached the peak 3 days to 2 weeks after surgery, which recovered 6 to 8 weeks after surgery. The expression of 4E-BP1 mRNA decreased soon after surgery and reached the lowest at 1 week, then rose to the peak 4 to 6 weeks after transplantation. Protein expression of mTOR and p70s6k reached the peak 2 to 4 weeks and recovered to normal level 8 weeks after surgery, but the expression of 4E-BP1 decreased to the lowest during 1 to 2 weeks and reached the peak 4 to 6 weeks after transplanting. The positive cells mostly located in vascular smooth muscle cell (VSMC) just like PCNA. CONCLUSIONS: The expression of mTOR and its substrates were activated in vein graft soon after transplantation, which means that mTOR and its substrates might become new targets for the prevention and therapy of stenosis or obliteration after vein graft transplanting.

[Mechanism of reversal of multidrug resistance in human renal carcinoma cells by protein kinase C inhibitor].

OBJECTIVE: To explore the mechanism of reversal of multidrug resistance in renal carcinoma cells by protein kinase C inhibitor. METHODS: RT-PCR, Western blot and inverted fluorescent microscopy were used to determine the expression of PKCalpha and MDR related gene MDR1, MRP1, LRP in RCC cells transferred by PKCalpha cDNA. Also effects of activator and inhibitor of PKC in combination with adriamycin on multidrug resistance in RCC cells were evaluated by MTT. RESULTS: The results of semi-quantitative RT-PCR analysis showed that the expression level of MDR1 was higher in RCC cells transferred by PKCalpha cDNA than in RCC cells, the reversal effectiveness of PKC inhibitors in combination with adriamycin (ADM) was apparently favorable. IC(50) of ADM in 786 - 0 cells was 7.8015e(-7) (5.7046e(-7) to 1.0669e(-6)); IC(50) of ADM in PKCalpha/786 - 0 cells was 1.6588e(-6) (1.1621e(-6) to 2.3677e(-6)); IC(50) of ADM in combination with PMA in PKCalpha/786 - 0 cells was 2.6794e(-6) (2.0521e(-6) to 3.4983e(-6)); IC(50) of ADM in combination with calphostin C in PKCalpha/786 - 0 cells was 9.2506e(-8) (5.9337e(-8) to 1.4422e(-7)). CONCLUSION: PKC inhibitors can reverse multidrug resistance in renal carcinoma cells in vitro via changes of expression of MDR1.

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.

[The role of protein kinase C alpha in recurrence of superficial bladder carcinoma].

OBJECTIVE: To investigate the relationship of protein kinase C-alpha (PKCalpha) expression/activation with tumor differentiation and resistance to chemotherapy drugs in superficial bladder carcinoma. METHODS: Expression of PKCalpha was measured by Western-blot analysis in 76 samples including tumor and normal tissues, respectively. A human RT4 bladder cancer cell line stably expressing green fluorescent protein (GFP)-PKCalpha (RT4/PKCalpha) was established. The sensitivity of the RT4/PKCalpha and parental cells to adriamycin (ADM) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The change of sensitivity of the RT4/PKCalpha to ADM were observed under the conditions of PKC activation and inhibition, respectively. RESULTS: Total level of PKCalpha expression and the ratio of the amount of PKCalpha expression or PKC activity in membrane to that in cytosol (M/C) were all more higher in cancerous tissues than in normal tissues (P < 0.01); With the increase of tumor grade, the relative level of PKCalpha expression significantly increased in membrane (P < 0.01) and decreased in cytosol (P < 0.01), M/C of PKCalpha was significantly elevated (P < 0.01), and total relative level of PKCalpha expression significantly increased (P < 0.01). Thirty-eight cases recurred during the follow-up period in total seventy cases. Multivariate analysis showed that high M/C of PKCalpha was independent prognostic factor for tumor recurrence after standard ADM treatment in the 2-year follow-up (RR = 3.98, 95% CI 1.22-5.68, P = 0.03). Transfection of PKCalpha increased resistance of RT4 cells to ADM [resistance index (RI): 6.97, t = 3.24, P < 0.01]. PKCalpha activation further greatly promoted the resistance (RI: 148.11, t = 5.18, P < 0.001) while inhibition of PKCalpha did conversely (RI: 1.6, t = 1.29, P > 0.05). CONCLUSION: The abnormal activation and expression level of PKCalpha closely correlate with both tumor grade and intrinsic resistance to ADM in patients with superficial bladder carcinoma.

[The protective effect of recombinant adenovirus of catalase on oxidative damaged rat lens].

OBJECTIVES: To investigate the validity of catalase recombinant adenovirus on the treatment of oxidative cataract. METHODS: The coding sequence of catalase was cloned and the catalase recombinant adenovirus was constructed. The expression time course of catalase gene in rat lens infected by recombinant adenovirus was determined by Western blotting. Cultured rat lens were randomly divided into 3 groups: the control group, the group treated by hydrogen peroxide and the group treated by hydrogen peroxide combined with catalase recombinant adenovirus. The transparence and apoptosis ratio of lens on the time points of 6, 12, 18, 24 hours were determined by image analysis and double colour flowcytometry. RESULTS: The coding sequence of catalase was cloned and recombinant adenovirus was successfully constructed. The expression of catalase in cultured rat lens infected by recombinant adenovirus reached peak point on 9 hours post infection and maintained the level in the whole experiment period. The transparence of the lens in the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was higher than that of group treated by hydrogen peroxide and lower than that of the control group on the time points of 6, 12, 18, 24 hours post infection. The differences among groups were statistically significant (P < 0.05). On the same time points, the apoptosis ratio of the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was lower than that of the group treated by hydrogen peroxide and higher than the control group. The differences among groups were statistically significant (P < 0.05). CONCLUSION: The catalase recombinant adenovirus, which can inhibit the turbidity and cell apoptosis of lens caused by oxidant, may be used as the gene therapy of oxidative cataract.

Inhibitory effects of vitamin A on TCDD-induced cytochrome P-450 1A1 enzyme activity and expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potent environmental contaminant that produces a wide range of adverse biological effects, including the induction of cytochrome P450 1A1(CYP1A1) that may enhance the toxic effects of TCDD. Several studies indicated that concurrent supplementation of vitamin A could reduce the toxicity, and potentially inhibit CYP1A1 activity (measured as ethoxyresorufin-O-deethylase [EROD] activity). In the present study, we investigated the in vivo effects of vitamin A on EROD activities and the expression of CYP1A1 in the liver of TCDD-treated mice. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight with or without the continuous administration of 2500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, or 28. In Experiment II, the mice were given daily an oral dose of 0.1 mug TCDD/kg body weight with or without supplement of 2000 IU vitamin A/kg body weight, and were killed on day 14, 28, or 42. In both experiments, TCDD caused liver damage and increase in relative liver weights, augmented the EROD activities and CYP1A1 expression, and increased the aryl hydrocarbon receptor (AhR) mRNA expression, but did not alter the AhR nuclear translocator (ARNT) mRNA expression. CYP1A1 mRNA expression and AhR mRNA expression showed a similar time course. The liver damage in TCDD + vitamin A-treated mice was less severe than that in TCDD-treated mice. EROD activities, CYP1A1 expression, and AhR mRNA expression in vitamin A + TCDD-treated mice were lower than those in TCDD-treated mice, indicating that supplementation of vitamin A might attenuate the liver damage caused by TCDD.

Effects of all-trans retinoic acid on orphan receptor APJ signaling in spontaneously hypertensive rats.

OBJECTIVE: Studies show general agreement that all-trans retinoic acid (atRA) has been linked to the regulation of G protein-coupled receptor (GPCRs) signaling. To further validate effects of atRA on the cardiovascular GPCRs, the present study was designed to assess whether atRA will modulate orphan receptor APJ, a homologue of angiotensin II type 1 (AT(1)) receptor. METHODS: Real-time polymerase chain reaction and Western blot methods were performed to examine the expression of APJ and its endogenous ligand apelin in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats after chronic atRA treatment. RESULTS: APJ and apelin expression were markedly depressed in placebo-treated SHR, compared with WKY rats (p<0.01). However, in atRA-treated SHR, a significant upregulation of APJ and apelin expression was observed in both heart and aorta (p<0.05), accompanied by a reduction of AT(1) expression, an elevation of serum nitric oxide levels and a subsequent decrease of blood pressure. CONCLUSIONS: Chronic atRA treatment activates gene and protein expression of APJ and apelin and reduces blood pressure in SHR, suggesting that atRA may regulate the balance between apelin-APJ and angiotensin II-AT(1) signaling and have potential clinical value in the prevention and treatment of human hypertension.

CDNA cloning of a short isoform of human liver NADP (H) -dependent retinol dehydrogenase/reductase and analysis of its characteristics.

In this report we found a new short PCR product when we amplified a 635 bp of NRDR fragment by RT-PCR. With 3'-Race and 5'-Race,we obtained two full-length cDNA sequences from human liver tissue,one 1 261 bp NRDR cDNA,another 1 003 bp NRDR isoform (NRDRiso,GenBank accession number:AY071856). The NRDR gene comprises eight exons and seven introns. The NRDRiso cDNA is produced by alternative splicing of NRDR cDNA, with the removal of 4, 5, 6 exons composed of consecutive 258 bp. The open reading frames of the NRDRiso cDNA predict a single polypeptide of 174 amino acids with the calculated minimum molecular mass of 18.6 kDa.

Upregulation of angiotensin-converting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats.

There is increasing evidence that all-trans retinoic acid (atRA) influences gene expression of components of renin-angiotensin system (RAS), which plays a pivotal role in the pathophysiology of essential hypertension. To further validate effects of atRA on the RAS and to assess the possibility that atRA affects the activity of angiotensin-converting enzyme 2 (ACE2), gene, and protein expression of ACE2 have been examined by real-time polymerase chain reaction and Western blot methods in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Rats were treated with atRA (10 or 20 mg x kg(-1) x day(-1)) or placebo given as daily intraperitoneal injection for 1 month. ACE2 expression was markedly decreased in placebo-treated SHR when compared with WKY rats. However, in atRA-treated SHR, a significant upregulation of ACE2 expression was observed in heart and kidney. In conclusion, chronic atRA treatment increases gene and protein expressions of ACE2, resulting in the reduction of blood pressure and the attenuation of myocardial damage in SHR, which suggests that atRA may be an attractive candidate for the potential prevention and treatment of human essential hypertension.

[Sequencing and bioinformatic analysis of NRDRiso cDNA].

This study describes the cDNA sequencing and the bioinformatic analysis of a novel NADP(H)-dependent retinol dehydrogenase/reductases isoform (NRDRiso). Based upon the concensus sequences of human and mouse NRDR coding region, we have identified a short 377 bp RT-PCR product from human liver tissue. The cDNA sequence of a NRDR isoform was then isolated using RACE approach and its sequence was analysed. The full-length cDNA is 1,003bp in length and was submitted to GenBank as NADP-dependent retinol dehydrogenase/reductase short isoform (NRDRiso). The open reading frames of NRDRiso cDNA is 525 bp.