Biomedical subjects

J Qiu

Publications and source records attributed to J Qiu.

At least 19 recordsLinked to original sources

PubMed2006

Soluble HLA-G expression and renal graft acceptance.

HLA-G is a potentially interesting molecule associated with immunosuppressive function. We survey here the presence of soluble HLA-G (sHLA-G) in serial serum samples of renal transplants. A total of 330 sera of from 65 patients were tested for sHLA-G with ELISA. IgG/IgM antibodies to HLA, and MICA antibodies were also previously tested. After serial analysis of the 65 patients' 330 sera, 50% of 26 patients in functioning group had consistent sHLA-G expression or became positive, in comparison to 20.5% among 39 patients who rejected their transplants (p=0.013). Thus sHLA-G was associated with functioning transplants. Eighty percent (77 of 96) of the HLA IgG positive sera had no sHLA-G expression, while 81.4% (83 of 102) of the HLA-G(+) sera had no HLA IgG (p=0.005), which showed a negative association between sHLA-G and the presence of HLA IgG antibodies (which was previously been shown to be associated with failure). In this preliminary survey, sHLA-G was found in the serum of about 30% of renal transplant patients. sHLA-G had a negative association with allograft failure from chronic rejection, and a negative relationship with the production of HLA IgG antibodies. The significance of sHLA-G in renal transplants remains to be determined.

Adult↗

PubMed2006

Decreased intranuclear mobility of acute myeloid leukemia 1-containing fusion proteins is accompanied by reduced mobility and compartmentalization of core binding factor beta.

Acute myeloid leukemia 1 (AML1) gene on chromosome 21 is involved in several chromosomal translocations, including t(8;21) and t(16;21), that produce chimeric fusion proteins AML1-eight twenty-one (ETO) and AML-myeloid transforming gene chromosome 16 (MTG16), which contribute to leukemogenesis. The molecular basis for the leukemogenic effects of these fusion proteins is incompletely understood. Using gel-shift assay, we showed that AML1-ETO and AML1-MTG16 bound to a series of AML1 consensus DNA-binding sites with different affinities. Using fluorescence recovery after photobleaching (FRAP), we demonstrated that a fusion of AML1 with ETO or MTG16 exhibits reduced intranuclear mobility compared with wild-type AML1 or either fusion partner. The dimerization domain (nervy homology region 2) of ETO is responsible for the reduced mobility of AML1-ETO. Dual FRAP studies revealed that CBFbeta colocalized with AML1-ETO within the nucleus, resulting in reduced mobility of CBFbeta. Therefore, AML1 fusion proteins may interfere with normal AML1 function due to aberrant nuclear dynamics, which leads to spatial and temporal sequestration of CBFbeta and perhaps other coregulators critical for myeloid differentiation.

Animals↗

PubMed2006

Interference of cyclosporine on glucose metabolism: potential role in chronic transplantation kidney fibrosis.

OBJECTIVES: To explore the fibrosis induction effects of cyclosporine (CsA) on renal tubular epithelial cells, as well as the potential mechanisms of CsA-mediated alterations of glucose metabolism in chronic allograft nephropathy (CAN). METHODS: The rat renal tubular epithelial cell line NRK-52E cells were cultured in medium with 50 mmol/L of D-glucose for 7, 14, or 28 days. The expression of TGF-beta1, CTGF, Smad3, and Smad7, which are involved in the fibrosis signal pathway, was detected by immunofluorescence and reverse-transcriptase polymerase chain reaction. Meanwhile, cells were cultured in various concentration of CsA in glucose-free medium for 24 hours followed by the addition of D-[3-(3)H]-glucose for 30 minutes; glucose uptake was detected by examining the radiation intensity. RESULTS: The expression of TGF-beta1, Smad3, Smad7, and CTGF of NRK-52E cells were up-regulated significantly (P < .05) after culture with 50 mmol/L glucose for 7 days compared with those in 25 mmol/L glucose. At 14 and 28 days, TGF-beta1 was slightly decreased compared with 7 days, but CTGF, Smad3, and Smad7 increased robustly (P < .05). CsA significantly stimulated glucose uptake at various concentrations, with an absorption peak at 60 minutes that decreased at 75 minutes. Furthermore, CsA may have dose-dependent effects on glucose uptake. CONCLUSION: High glucose concentrations may play an important role in the NRK-52E cell fibrosis during the CAN process due to an interference of CsA on glucose metabolism.

Animals↗

PubMed2006

Nuclear translocation of SMAD3 may enhance the TGF-beta/SMADS pathway in high glucose circumstances.

OBJECTIVES: Posttransplant diabetes mellitus is one of the most frequent complications after kidney transplantation. It is considered to be one cause of chronic allograft nephropathy. This study sought to investigate the effects of high glucose on the expression and nuclear translocation of Smad3, which is an important signal mediator involved in the fibrotic signal pathway. METHODS: The established rat renal mesangial cell line HBZY-1 was cultured in medium with various concentrations of glucose (4.5 mg/mL, 9.0 mg/mL, or 13.5 mg/mL), which was collected at 7, 14, or 21 days. The total expression of Smad3, including both inner and outer nucleus proteins was examined by Western blot analysis. The nuclear translocated Smad3, representing only the inner nucleus protein, was detected by immunofluorescence staining observed under a laser confocal scanning microscope. RESULTS: No significant difference in the total Smad3 expression was demonstrated by Western blot analysis among the three groups of HBZY-1 cells at various concentration of glucose after 7, 14, or 21 days. There was no fluorescence detected in the nucleus at day 7 by immunofluorescence staining; however, robust positive expression of Smad3 was detected at days 14 and 21. CONCLUSION: As a restricted Smads member, Smad3 protein might not be upregulated in the presence of high glucose. However, with prolonged culture time, Smad3 translocates from cytoplasm to nucleus, which may be a pivotal step in the fibrotic signal pathway.

Animals↗

PubMed2006

The C-terminal peptide of connective tissue growth factor blocks the full molecule binding to tubular epithelial cell.

OBJECTIVES: Interstitial fibrosis is a critical pathologic change in chronic allograft nephropathy. The cytokine connective tissue growth factor (CTGF, also CCN2) plays an important role in epithelial-mesenchymal transformation (EMT) of tubular epithelial cells to renal interstitial fibrosis. The hexadeca-peptide within the C-terminal of CTGF (named P2) contains the unique binding domain of CTGF to its potential receptor, integrin alphavbeta3. This study examined whether P2 bound preferentially to the receptor and served as an inhibitor of CTGF. METHODS: All studies used an established rat kidney tubular epithelial cell line NRK-52E. Chemically synthesized P2 was purified, and some of it labeled with FITC. The affinity of CTGF or P2 to NRK-52E cells was examined by a solid-phase cell adhesion assay. Competitive binding between P2 and CTGF to NRK-52E cells was examined with flow cytometric analysis. RESULTS: Both P2 and CTGF bound to the NRK-52E cells, mediating cell adhesion. When the cells were incubated in the mixture of P2 and CTGF, P2 bound to the cells preferentially. Furthermore, when cells were preincubated with excessive CTGF, it became difficult for subsequent P2 binding to occur. CONCLUSIONS: P2 and CTGF seemed to bind to cell membranes at the same binding domain. P2 competitively blocked CTGF binding, acting as a CTGF inhibitor.

Animals↗

PubMed2006

Characteristics of neoplasm occurrence and the therapeutic effect of sirolimus in South Chinese kidney transplant recipients.

Kidney transplantation (KTx) recipients are at a higher risk of oncogenesis when compared to the general population. Sirolimus (SRL), a potent immunosuppressant, has shown promising antineoplastic effects in vitro and in vivo. This study retrospectively analyzed the neoplasm occurrence and the efficiency of SRL on unresectable malignancies in South Chinese KTx recipients. Thirty-three (1.64%) of 2017 patients who received KTx from January 1984 to December 2004 developed neoplasms at 4 to 117 months posttransplant, mostly in digestive organs (33.3%), the hematologic system (15.2%), or the skin (12.1%). The most common type was liver cancer (24.2%), followed by skin cancer, lymphoma, and thyroid cancer (9.1%). The median survival times were 41.5 and 6.0 months for those who did (n = 10) receive radical surgery or did not (n = 23), respectively. The 20-month survival rates were 70.0% versus 13.0% (P < .01). For unresectable patients, the median survival time of those treated with SRL (n = 8) was 14.5 months compared to 3.0 months for those who did not (n = 15). The survival rates at 12(th) and 20(th) months were 75.0% and 37.5% in the SRL group and 6.7% and 0% in the non-SRL group (P < .05). In conclusion, when compared with Western studies, a lower incidence and unique location pattern (liver cancer-dominant) are characteristics of de novo posttransplant neoplasms in South Chinese KTx recipients. Early diagnosis and feasible radical surgery are favorable for prognosis, and SRL is a treatment of choice for KTx recipients with neoplasms.

China↗

PubMed2005

Simultaneous labeling of mast cell proteases and protease mRNAs at the bone-implant interface of aseptically loosened hip implants.

Human mast cells (MC) exhibit two distinct phenotypes based on the protease content of their secretory granules. MC(TC) cells express tryptase, chymase, cathepsin G, and mast cell carboxypeptidase, while MC(T) cells express only tryptase. Both mast cell phenotypes were assessed near regions of osteolysis in uninfected failed joint implants by immunohistochemistry with antibodies to tryptase and chymase, and by in situ hybridization with anti-sense RNA probes for the respective mRNA molecules. Specimens from the interface membrane of 32 aseptically loosened total hip implants were studied, 28 of which had mast cells of the MC(TC) type. Most of these mast cells were aligned along the bone-prosthesis interface adjacent to loosened implants, suggesting involvement in the chronic inflammatory response that leads to bone resorption. Further ultrastructural evidence of mast cells in tissues from failed joint interface membranes was shown by transmission electron microscopy, and detection by staining after magnetic activated cell sorting. The presence of MC at the periprosthetic interface of eroded bone suggests MC degranulation and activity are associated with osteolysis in failed joint prostheses.

Arthroplasty, Replacement, Hip↗

PubMed2005

Clinical pharmacokinetics of tacrolimus after the first oral administration in combination with mycophenolate mofetil and prednisone in Chinese renal transplant recipients.

INTRODUCTION: Data on tacrolimus pharmacokinetics in combination with mycophenolate mofetil and prednisone are scarce in Chinese renal transplantation recipients. The purpose of this study was to detect interpatient pharmacokinetic variability of tacrolimus and to assess the predictability of individual tacrolimus concentrations at various times for the area under the curve (AUC) seeking to find the best sampling time for an abbreviated AUC to predict the total body exposure of tacrolimus after the first oral dose in Chinese renal transplantation recipients. METHODS: Sixteen primary kidney transplant recipients were treated with methylprednisolone and antilymphocyte globulin for 3 days. The first tacrolimus oral dose (0.075 mg/kg) was given at day 3 posttransplant. Mycophenolate mofetil and prednisone were administered orally posttransplant. Blood samples were obtained at 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 8.0, and 12.0 hours after taking the first oral dose. Tacrolimus blood concentrations were measured by ELISA. Twelve-hour AUC (AUC12) for each patient was calculated using the linear trapezoid rule. Associations between the blood concentration at each sampling time point and the AUC12 were evaluated by Pearson correlation coefficients. Abbreviated sampling equations were derived by multiple, stepwise regression analyses performed using AUC12 as the dependent variables. The variance in the strength of association between predicted AUC (AUC(P)) and AUC12 was reflected by linear regression coefficients of multiple determinations. RESULTS: In 16 patients, AUC12 values were within the range of 44.40 ng x h/mL to 158.01 ng x h/mL (mean = 92.23 +/- 34.97 ng x h/mL). The area of the maximum AUC12 was almost fourfold higher than that of the minimum AUC12. C12 significantly correlated with AUC(12) after the first tarcrolimus oral dose (r = .846, P < .001). C5, C8, and C3 showed better correlations: r = .924, .924, and .911, respectively. From stepwise multiple regression, C5 seemed to be the best predictor of total body exposure to tacrolimus (r = .92, r2 = .85). Alternatively, the concentrations at 5 and 1.5 hours or 5, 1.5, and 3 hours as an abbreviated AUC were as good as a full pharmacokinetic study (r = .97, r2 = .94, and r = .99, r2 = .99, respectively). CONCLUSIONS: Tacrolimus AUC12 show remarkable interindividual variations after the first oral dose in combination with mycophenolate mofetil and prednisone in Chinese renal transplant recipients. Although C12 is a good predictor of efficacy, C5 might be the best predictor of the first AUC12. A two-point sampling method using C5 and C1.5 or three-point sampling method using C5, C1.5, and C3 might be the best abbreviated AUC for a cost-effective tacrolimus monitoring strategy.

Administration, Oral↗

PubMed2005

Municipal wastewater treatment with pond-constructed wetland system: a case study.

The performance of a pond-constructed wetland system in the treatment of municipal wastewater in Kiaochow city was studied; and comparison with oxidation ponds system was conducted. In the post-constructed wetland, the removal of COD, TN and TP is 24%, 58.5% and 24.8% respectively. The treated effluent from the constructed wetland can meet the Chinese National Agricultural and Irrigation Standard. The comparison between pond-constructed wetland system and oxidation pond system shows that total nitrogen removal in a constructed wetland is better than that in an oxidation pond and the TP removal is inferior. A possible reason is the low dissolved oxygen concentration in the wetland. Constructed wetlands can restrain the growth of algae effectively, and can produce obvious ecological and economical benefits.

China↗

PubMed2003

Novel potential ALL low-risk markers revealed by gene expression profiling with new high-throughput SSH-CCS-PCR.

The current systems of risk grouping in pediatric acute lymphoblastic leukemia (ALL) fail to predict therapeutic success in 10-35% of patients. To identify better predictive markers of clinical behavior in ALL, we have developed an integrated approach for gene expression profiling that couples suppression subtractive hybridization, concatenated cDNA sequencing, and reverse transcriptase real-time quantitative PCR. Using this approach, a total of 600 differentially expressed genes were identified between t(4;11) ALL and pre-B ALL with no determinant chromosomal translocation. The expression of 67 genes was analyzed in different cytogenetic ALL subgroups and B lymphocytes isolated from healthy donors. Three genes, BACH1, TP53BPL, and H2B/S, were consistently expressed as a significant cluster associated with the low-risk ALL subgroups. A total of 42 genes were differentially expressed in ALL vs normal B lymphocytes, with no specific association with any particular ALL subgroups. The remaining 22 genes were part of a specific expression profile associated with the hyperdiploid, t(12;21), or t(4;11) subgroups. Using an unsupervised hierarchical cluster analysis, the discriminating power of these specific expression profiles allowed the clustering of patients according to their subgroups. These genes could help to understand the difference in treatment response and become therapeutical targets to improve ALL clinical outcomes.

B-Lymphocytes↗

PubMed2003

Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.

Lactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 A cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its long-recognized antimicrobial properties.

Amino Acid Sequence↗

PubMed2003

Hyperleptinemia precipitates diet-induced obesity in transgenic mice overexpressing leptin.

Transgenic mice overexpressing leptin backcrossed to the C57BL/6J genetic background (LepTg) have a lean phenotype, characterized by a 95% reduction in adipose mass; reduced plasma levels of glucose, triglycerides, insulin, and IGF-1; and a 75% decrease in adipocyte size. High-fat diet treatment for 20 wk revealed that, compared with normal mice, the LepTg mice had an increased susceptibility to diet-induced obesity, as demonstrated by their rate of weight gain, higher accumulation of sc white adipose tissue mass, hypertrophy of adipocytes, and normalization of their reduced metabolic parameters. The stromal vascular fraction of white adipose tissue from the LepTg mice was highly cellular and contained cells capable of rapid lipid accumulation in primary cultures. The precipitous diet-induced obesity of the LepTg mice was accompanied with 10-fold and 1.6-fold elevations in insulin and IGF-1, respectively, suggesting that the trophic action of insulin and IGF-1 on the preadipocytes and small adipocytes may have caused them to rapidly differentiate and accumulate triacylglycerol stores. Other contributing factors may involve a shift in insulin sensitivity triggered by hyperleptinemia and a decrease in energy expenditure. These studies demonstrate that a chronic response to hyperleptinemia as in the LepTg mice is a predisposing factor to diet-induced obesity and suggest that individuals who are particularly lean because of increased leptin secretion may develop rapid obesity under conditions of a high-fat diet.

Adipocytes↗

PubMed2003

Echinococcosis on the Tibetan Plateau: prevalence and risk factors for cystic and alveolar echinococcosis in Tibetan populations in Qinghai Province, China.

Infections by larval stages of tapeworms of the genus Echinococcus (echinococcosis or hydatid disease) are zoonotic infections of major public health importance throughout much of the world. Humans become infected through accidental ingestion of eggs passed in faeces of canid definitive hosts. Tibetan populations of China have some of the highest documented levels of infections by both Echinococcus granulosus and E. multilocularis, the causes of cystic and alveolar echinococcosis, respectively. In this study we measured the prevalence of cystic (CE) and alveolar (AE) echinococcosis disease in Tibetan communities in Qinghai, Province, China, and identified putative risk factors for both infections in these communities. 3703 volunteers in three predominately Tibetan counties of Qinghai were surveyed between June 1997 and June 1998. Parasitic lesions were diagnosed by imaging of characteristic space-occupying lesions in abdominal organs (ultrasound) or the lungs (radiographs). Specific serodiagnostic assays (Dot-ELISA and Em2-ELISA) were performed on sera of positively imaged subjects to further distinguish the disease agent. All participants completed a questionnaire documenting age, sex, education level, occupation, lifestyle (nomadic or settled), slaughter practices, drinking water source, hygienic practice and association with dogs. Data were analyzed using SAS version 8. 6.6% of the volunteers had image-confirmed infection with E. granulosus (CE) and 0.8% had E. multilocularis (AE) infection. The significant univariate factors for echinococcal infection (both CE and AE) included livestock ownership, Tibetan ethnicity, female gender, low income, herding occupation, limited education, water source, age greater than 25 years old, poor hygienic practices, offal disposal practices and dog care. Multivariate analysis revealed that livestock ownership was a significant risk factor for both forms of the disease, as well as age greater than 25 years, female gender, herding occupation, and being nomadic (vs semi-nomadic or settled). No additional significant risk factors were identified among the 344 nomadic participants. Being female and being older than 25 years of age were significant factors among the 1906 semi-nomadic participants. Among the 1445 settled participants, allowing dogs to sleep indoors was statistically significant. Issues such as inadequate assessment of animal ownership, selection bias, disease misclassification, and loss of information may have led to reduction in strength of some risk factor associations and need to be addressed in future epidemiologic analysis of echinococcosis in this population.

Adolescent↗

PubMed2002

Building a mouse model hallmarking the congenital human cytomegalovirus infection in central nervous system.

To investigate the mechanisms that human cytomegalovirus (HCMV) can vertically transmit from the placenta of mice to infect their offspring in the central nervous system (CNS) and cause congenital anomalies, and in order to provide basic research for preparing HCMV vaccine, we have developed a new type of mouse model of HCMV congenital CNS infection. Pure strain mice were propagated after being infected with HCMV. Then the degree of infection by HCMV to offspring was determined. The experiment shows that in the infection groups the mortality of fetal mice and the fatality of neonatal mice in one week are higher than that of the control groups (P < or = 0.05). At the same time we investigated the CNS of fetus's mice whose mothers were infected by HCMV. Our results showed: 1. The virus was successfully isolated from their cerebral cortex. 2. The signal of HCMV hybridization print was found in their nervous cell through in situ hybridization. 3. Especially human herpes virus-like particles and inclusion bodies in the plasm of nerve cell were found in the tissue of their brain under the electron microscope. This new type of mouse model of HCMV inherent CNS infection will help prepare HCMV vaccine and research HCMV congenital infection in CNS.

Animals↗

PubMed2001

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia.

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid(-/-) neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid(-/-) mice (-67% and -41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.

Animals↗

PubMed2001

Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells.

Although the nongenomic effects of glucocorticoids have been well acknowledged, its precise intracellular signal transduction pathway remains to be elucidated. The present study using Western immunoblot and protein kinase activity assay, for the first time, showed that corticosterone (B) can induce a rapid activation of Erk1/2 mitogen-activated protein kinase (MAPK) in PC12 cells. The dose-response curve was bell shaped, with the maximal activation at 10(-9) M in 15 min. The results from immunofluorescence staining also revealed that the activated Erk1/2 MAPK was translocated from cytoplasm to nucleus of PC12 cells in 15 min. Activation of Erk1/2 MAPK by B was apparently not mediated by the classical cytosolic steroid receptors, for B-BSA can induce the phosphorylation of Erk1/2 MAPK, but the antagonist (RU38486) cannot block the phosphorylation of Erk1/2 MAPK induced by B. Phosphorylation of Erk1/2 MAPK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway did not involve the tyrosine kinase activity. On the other hand, protein kinase C activator (PMA) can activate and protein kinase C inhibitor (Gö6976) can block the activation of Erk1/2 MAPK induced by B. Taken together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate Erk1/2 MAPK through protein kinase C alpha in PC12 cells.

Active Transport, Cell Nucleus↗

PubMed2001

The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA.

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

Alternative Splicing↗

PubMed2001

Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors.

RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg(2+) and Mn(2+). Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.

Amino Acid Sequence↗