Biomedical subjects

Wei Yang

Publications and source records attributed to Wei Yang.

At least 91 records · Page 5Linked to original sources

PubMed2005

Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.

H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates K(ATP) channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of K(ATP) channels in INS-1E cells, an insulin-secreting cell line. In the present study, K(ATP) channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. K(ATP) channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mm) in the pipette solution inhibited K(ATP) channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1-5 mm) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal K(ATP) channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mm) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell K(ATP) channel currents at concentrations equal to or lower than 100 microm. H2S (100 microm) markedly increased open probability by more than 2-fold of single K(ATP) channels (inside-out recording) in native INS-1E cells (n = 4, P < 0.05). Single-channel conductance and ATP sensitivity of K(ATP) channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates K(ATP) channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the K(ATP) channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic beta-cells.

Adenosine Triphosphate↗

PubMed2005

Risks of human conotruncal heart defects associated with 32 single nucleotide polymorphisms of selected cardiovascular disease-related genes.

Investigating possible genetic polymorphisms and gene-environment interactions in the etiology of human conotruncal defects is a prudent research approach. In this study we explore gene-only and gene-environment effects of 32 single nucleotide polymorphisms (SNPs) on conotruncal defect risks. The genes bearing these SNPs participate in one of five pathogenetic processes, homocysteine metabolism, coagulation, cell-cell interaction, inflammatory response, and blood pressure regulation. We used DNA samples and data from a California population-based case-control interview study (1987-1988 birth cohort). We employed a multilocus allele-specific hybridization assay. Allelic variants were determined by genotyping 155 infants with conotruncal defects (cases) and 437 infants without malformations (controls). Among the 32 SNPs, four were associated with odds ratios of 2 or more, and two with odds ratios of 0.5 or less. The four SNPs were F2 G20210A (prothrombin) with an odds ratio of 2.5 (95% confidence interval; 0.9-7.0), F7 promoter (-323) 10-bp del/ins with an odds ratio of 2.3 (0.8-6.8), ITGB3 leu33pro (platelet glycoprotein IIIa) with an odds ratio of 2.2 (0.9-5.7), and NPPA T2238C (atrial natriuretic precursor peptide) with an odds ratio of 2.9 (0.8-10.1). Two SNPs were associated with decreased risks: TNF (tumor necrosis factor, G (-376A)) and ADD1 gly460trp (alpha adducin) with odds ratios of 0.5 (0.1-2.3) and 0.5 (0.2-1.9), respectively. Analyses that investigated a potential gene-nutrient interaction between maternal periconceptional vitamin use and MTHFR genotypes did not indicate that the CT or TT genotype contributed to conotruncal defect risk in infants even in the absence of maternal use of multivitamin supplements with folic acid. Analyses that investigated a potential interaction on risk between NOS3 genes and maternal cigarette smoking, revealed some evidence for higher risk of conotruncal defects in infants whose mothers smoked cigarettes periconceptionally and who had one of the variant alleles for NOS3 A(-922G) or NOS3 glu298asp compared to those infants whose mothers did not smoke and whose genotypes were wild-type. Our results provide some support for involvement of genetic variation of biologically relevant candidate genes for some birth defects whose pathogenesis may be related to altered vascular tone or integrity. In particular, NPPA appears to be a good candidate gene for conotruncal defects and warrants further investigation.

California↗

PubMed2005

[Prognostic factors on outcome of radiofrequency ablation of 172 primary hepatic tumors].

OBJECTIVE: To investigate the prognostic factors affecting outcome in Radiofrequency (RF) ablation of primary hepatic tumors by univariate and multivariate analyses, and to assess the therapeutic efficacy of Radio-frequency ablation. METHODS: A total of 172 patients with primary hepatic tumors underwent RF treatment in our department between 1999 and 2004. Among them, 150 patients were not the surgery candidates. The follow-up period ranged from 4 to 51 months and follow-up rate was 94.8% (163 patients). Kaplan-Meier model and log-rank test were used in univariate analyses and COX regression model was used in multivariate analyses, to identify prognostic factors for survival. RESULTS: The survival rates at half a year, 1 year, 2 year and 3 year were 91.5%, 77.4%, 55.7% and 51.2% respectively. Using univariate analyses, 24 potential factors were analyzed and 8 of them were found significant for survival. These 8 factors were Child-Pugh grade, UICC stage, AFP value before first RF, Portal hypertension, first treatment or treatment for recurrence, pathological grade, treatment purpose and method, ablative safe margin. For 116 patients who underwent RF when first diagnosis, the survival rates in patients with different tumor stage (UICC standard of hepatocellular carcinoma) at half a year, 1 year, 2 year and 3 year were estimated as follows: 96.7%, 92.3%, 81.6% and 65.3% in 33 patients of stage I-II; 91.2%, 76.3%, 56.6% and 51.4% in 83 patients of stage III-IV; There was significant difference in survival rates between patients of stage I-II and patients of stage III-IV. For 56 patients suffered from recurrent hepatic tumors, the survival rates at half a year, 1 year, 2 year and 3 year were 88.5%, 70.2%, 41.4% and 24.5%, being similar to those of stage III-IV. The 8 factors mentioned above were further analyzed by multivariate model and 3 of them were identified as prognostic factors for survival. These 3 factors included Child-Pugh grade, Portal hypertension, treatment purpose and method. CONCLUSION: Based on our statistical analysis, the prognostic factors on outcome in Radio-frequency ablation of hepatic tumors were grouped into two categories. Child-Pugh grade, and Portal hypertension were the characteristics of patients and uncontrollable. While treatment purpose and method also had significant impact on survival and was controllable during RF procedure. This information might be useful to improve treatment level of Radio-frequency ablation of hepatic tumors.

Adult↗

PubMed2005

Synthesis and properties of electrophosphorescent chelating polymers with iridium complexes in the conjugated backbone.

The synthesis of electrophosphorescent chelating polymers by Suzuki polycondensation of A-A- and B-B-type monomers is described, in which the fluorene-alt-carbazole (PFCz) segment is used as polymer backbone. By using alkyl-substituted ligands of iridium complex monomers, chelating copolymers with higher contents of iridium complex can be synthesized. Chemical and photophysical characterization confirm that the Ir complex is incorporated into the polymer backbone as one of the monomer repeat units by means of two 5-bromotolylpyridine ligands. Chelating polymers with Ir complexes in the conjugated polymer backbone show highly efficient energy transfer of excitons from the PFCz host segment to the Ir complex by an intramolecular trapping mechanism. The external quantum and luminous efficiencies of a device made with PFCzMppyIrhm4 copolymer reach 4.1 % ph/el (photons/electron) and 5.4 cd A(-1), respectively, at a current density of 32.2 mA cm(-2), an emission peak of 577 nm, and a luminance of 1730 cd cm(-2). Most important, the devices made from the chelating copolymers show no notable efficiency decay with increasing current density due to reduced concentration quenching and triplet-triplet (T-T) annihilation. This indicates that incorporation of the phosphorescent complex into the rigid conjugated polymer main chain is a new way to simultaneously realize high efficiency, long-term stability, and simple processing of phosphorescent polymer light-emitting diodes.

Journal Article↗

PubMed2005

Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis.

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.

Base Pair Mismatch↗

PubMed2005

Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.

To examine whether transplantation of human mesenchymal stem cells (MSCs) could reconstruct the corneal damage and also whether grafted MSCs could differentiate into corneal epithelial cells, we isolated MSCs from healthy donors. After growth and expansion on amniotic membrane, cells were transplanted into rat corneas 7 days after chemical burns. Reconstruction of the damaged cornea and the rat vision were measured once a week by slit lamp and by an optokinetic head-tracking instrument, respectively. Corneas were then cut out, fixed, and imbedded for immunofluorescent study of the expression of keratin 3 and keratin-pan as epithelial cell markers. Expression of CD45, interleukin 2, and metalloproteinase-2 was also investigated for inflammation and inflammation-related angiogenesis. The data showed that transplantation of MSCs, like limbal epithelial stem cells, successfully reconstructed damaged rat corneal surface. Interestingly, the therapeutic effect of the transplantation may be associated with the inhibition of inflammation and angiogenesis after transplantation of MSCs rather than the epithelial differentiation from MSCs. This study provides the first line of evidence that MSCs can be used for reconstruction of damaged corneas, presenting a new source for autotransplantation in the treatment of corneal disorders.

Animals↗

PubMed2005

Epidemiologic characteristics of anophthalmia and bilateral microphthalmia among 2.5 million births in California, 1989-1997.

There is a paucity of epidemiologic information about the eye malformations anophthalmia and microphthalmia. Using data from a large population-based registry, we explored prevalences and maternal/infant characteristics associated with anophthalmia and bilateral microphthalmia. Data were derived from the California Birth Defects Monitoring Program, a population-based active surveillance system for collecting information on infants and fetuses with congenital malformations using multiple source ascertainment. Approximately 2.5 million births (liveborn and stillborn) occurred during the ascertainment period, 1989-1997. Information on maternal and infant/fetal characteristics was obtained from California birth certificate and fetal death files. The prevalence per 10,000 livebirths and stillbirths for anophthalmia was 0.18 and for bilateral microphthalmia was 0.22. These estimates reflect prevalences among births without chromosomal anomalies. Relative risks of anophthalmia were modestly higher among women aged 40 or more (relative risk = 2.0, 95% confidence interval 0.5-8.6). Risks were substantially lower for those mothers with >12 years of education, even after adjusting for other study factors, relative risk = 0.6 (0.2-1.7). The risk of anophthalmia was approximately twofold among multiple births compared to singletons. Similar to results for anophthalmia, decreased risks for bilateral microphthalmia were observed for maternal education of 12 years or more and increased risks observed for multiple births. These data show descriptive epidemiologic features of anophthalmia and bilateral microphthalmia.

Adult↗

PubMed2005

[Radiofrequency ablation of recurrent hepatocellular carcinoma after hepatectomy].

OBJECTIVE: To assess the efficiency and safety of radiofrequency ablation (RFA) of recurrent hepatocellular carcinoma (RHCC) after hepatectomy and to investigate efficacy of RFA for patients with early and late phase recurrence, separately, setting 1 year as the cut-off between the early and late phases. METHODS: A total of 42 patients with 77 RHCC and a history of hepatic resection for hepatocellular carcinoma (HCC) underwent ultrasound-guided percutaneous radiofrequency ablation in our department and entered this study (RHCC group). The average diameter of RHCC was (3.8 +/- 1.4) cm (range, 1.5-6.6 cm). 21 of the 42 RHCC patients had Child-Pugh class A cirrhosis 19, class B and two, class C cirrhosis. The average interval between initial surgery and the diagnosis of recurrence was 22.8 months (range, 1-96 month). 42 RHCC patients were divided into two groups as early recurrence group including 20 patients with 40 RHCC, and late recurrence group including the other 22 patients with 37 RHCC according to the recurrence interval. During the same period 148 patients with 217 primary HCC were also treated by RF ablation and regarded as primary HCC group. The average diameter of primary HCC was (4.0 +/- 1.4) cm (range, 1.2-7.0 cm). Regular follow-up with enhanced CT was performed to evaluate the treatment results. Ablation was considered a success if no contrast enhancement was detected in the treated area on 1 month CT scans. RESULTS: The ablation success rate, local recurrence rate, new tumor incidence and mean survival in RHCC group were 90.5%, 14.3%, 38.1% and (28.0 +/- 3.5) months, respectively, which were similar to the corresponding results of 87.2%, 16.2%, 37.8% and (39.0 +/- 2.1) month in primary HCC group. However, when further comparison was performed between early recurrence group, late recurrence group and primary HCC group, there were some significant differences. The incidence of new tumors in early recurrence group was significantly higher than that in late recurrent group (60.0% vs. 18.2%, P = 0.005); early recurrence group survived shorter than primary HCC group [(15.4 +/- 2.3) vs. (39.0 +/- 2.1) months, P < 0.005]. The survival time was similar between late recurrence group and primary HCC group. One case was found haemorrhage after RFA and recovered with conservative treatment. No major complications occurred in the remaining 41 patients. CONCLUSIONS: RF ablation is generally effective and safe in treating RHCC. And it's more effective in late recurrence than in early recurrence.

Adult↗

PubMed2005

[Standard treatment of liver malignancies with radiofrequency ablation].

OBJECTIVE: To investigate the role of standard treatment with ultrasound-guided radiofrequency ablation (RFA) in improving the treatment level of liver malignancies. METHODS: 302 patients with 476 liver malignancies were treated with established protocol and adjuvant measures and subjected to efficiency analysis. In the 302 patients, 181 had 282 hepatocellular carcinomas (HCC) with a mean diameter of 4.2 cm, and 121 had 194 metastatic liver carcinomas (MLC) with a mean diameter of 3.9 cm. According to UICC-TNM system 50 patients (27.6%) were in stage I/II and 131 (72.4%) in stage III/IV (including 39 patients with recurrent HCC after surgical resection). A standard protocol and an individualized protocol were used to treat the tumors based on their size, shape and special location such as the distance from diaphragm, gallbladder and gastrointestinal tract. Needle placement method and operation skill for the tumor region adjacent to important structures were described. Some adjuvant measures such as supplementary fine needle localization, local saline injection and feeding vessel ablation were used to improve RFA efficacy in tumors with different features. Local ablation of bleeding site and haemostatic administration systemically were adopted to deal with bleeding. For the patients with tumor adjacent to gastrointestinal tract, prolonged fasting after the RFA procedure was required. the patients were followed up regularly to assess the treatment efficiency, and the tumor was considered complete necrosed if no viability was found on enhanced CT or enhanced US one month after RFA. RESULTS: The tumor necrosis rate was 95.7% (270/282 tumors) for HCC, 94.8% (184/194 tumors) for MLC, 91.1% (51/56 tumors) for tumor near gastrointestinal tract, 88.5% (69/78 tumors) for tumors near diaphragm, and 94.3% (49/52 tumors) for tumor near gallbladder. The local recurrence rate was 10.3% (29/282 tumors) for HCC and 14.4% (28/194 tumors) for MLC. The 1, 2 and 3 year overall survival rates were 87.6%, 67.4% and 58.6% in the HCC patients, and 87.4%, 48.2%, 25.3% in the MLC patients respectively. The 1, 2 and 3 year survival rates of 50 HCC patients in early (I-II) stages were 90.7%, 85.9% and 73.7%, respectively. The incidence of complications was 2.2% (13/583 sessions), including 5 cases of hemorrhage, 1 case colon perforation, 8 cases of injury of adjacent structures. CONCLUSION: Application of proper protocol and adjuvant measures plays an important role in improving tumor ablation rate. Knowledge about possible complications and their control may increase the treatment efficacy and help to promote the use of RFA technique.

Carcinoma, Hepatocellular↗

PubMed2005

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.

Arabidopsis Proteins↗

PubMed2005

[Efficacy of radiofrequency ablation of 343 patients with hepatic tumor and the relevant complications].

OBJECTIVE: To investigate the treatment efficacy of radiofrequency ablation (RFA) of hepatic tumors and the relevant complications. METHODS: A total of 343 patients with 778 hepatic tumors underwent ultrasound-guided RFA (582 procedures). There were 212 cases of hepatic cellular carcinoma (HCC) with 448 tumors, and the average largest diameter was 4.0 cm. Of all the patients, 63 (29.7%) were in the stage of I-II (UICC Systems) and 149 (70.3%) in stage of III-IV (including 43 patients with tumor recurrence after surgical resection). There were 131 cases of metastatic liver carcinoma (MLC), with 330 metastases in the liver, the average diameter was 3.9 cm, and the liver metastases of 91 patients (69.5%) came from gastrointestinal tract. The patients were treated using the relatively standard protocol. Crucial attention must be paid to monitoring the abnormal changes in ultrasound images as well as the vital signs of the patients to find the possible hemorrhage and peripheral structure injury in time. The tumors were considered ablated successfully if no viability was found on enhanced CT within 24 hours or 1 month after RFA. The patients were followed up for 2-62 months. RESULTS: The ablation success rate for HCC was 95.5% (428/448 tumors), and the rate for MLC was 96.4% (318/330 tumors). The local tumor recurrence rates for HCC and MLC were 8.5% (38/448 tumors) and 11.8% (39/330 tumors), respectively. A total of 138 patients (40.2%) underwent repeated ablations for 2-11 times because of tumor recurrence or metastasis. The first, second and third years survival rates were 87.7%, 67.4% and 56.8% for HCC patients, 81.6%, 50.8% and 27.2% for MLC patients, respectively. The survival rate from 63 early-stage HCC patients were 92.9%, 82.8% and 74.5%, respectively. The major complication rate in this study was 2.4% (14 of 582 procedures). The complications which consisted of mechanical and thermal injuries usually occurred during or shortly after the RFA treatment. There were 5 hemorrhages, 1 colon perforation, 5 injuries of adjacent structures, 2 bile leakages and 1 skin burn. CONCLUSION: RFA, as a minimally invasive local treatment, has become an effective and relatively safety alternative for the patients of hepatic tumors, even of advanced live tumor, tumor recurrence, liver metastases which are unresectable or difficult to treat with traditional therapies. Knowledge about possible complications and their control may increase the treatment efficacy and help to promote the use of RFA technique.

Adult↗

PubMed2005

Are the betaine-homocysteine methyltransferase (BHMT and BHMT2) genes risk factors for spina bifida and orofacial clefts?

Abnormalities in folate and/or homocysteine metabolism may adversely influence embryonic development, leading to the birth of infants with a variety of congenital malformations, including neural tube defects (NTDs) and craniofacial abnormalities. Based upon suggestive evidence that periconceptional folic acid supplementation is effective in preventing a significant proportion of the aforementioned birth defects, genetic variation in the folate biosynthetic pathways may influence the infant's susceptibility to these birth defects. The goal of our study was to investigate sequence variations in the betaine-homocysteine methyltransferase (BHMT) and betaine-homocysteine methyltransferase (BHMT2) genes as modifiers of risk of spina bifida, cleft palate, and cleft lip and palate. The results of this study indicated that individuals homozygous for the single nucleotide polymorphism R239Q in BHMT did not have elevated risks for spina bifida. Genotype frequencies for the BHMT2 rs626105 polymorphism also did not reveal any elevated risks for spina bifida, and only a modest, imprecise elevation of risk for orofacial clefts. The results of these experiments suggest that variants of the BHMT/BHMT2 genes in infants do not substantially contribute to the risk of spina bifida or orofacial clefts in our study population.

Betaine-Homocysteine S-Methyltransferase↗

PubMed2005

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein.

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.

Animals↗

PubMed2005

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn.

BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

Amnion↗

PubMed2005

Induction of apoptosis in mouse liver by microcystin-LR: a combined transcriptomic, proteomic, and simulation strategy.

Microcystins (MCs) are a family of cyclic heptapeptide hepatotoxins produced by freshwater species of cyanobacteria that have been implicated in the development of liver cancer, necrosis, and even deadly intrahepatic bleeding. MC-LR, the most toxic MC variant, is also the most commonly encountered in a contaminated aquatic system. This study presents the first data in the toxicological research of MCs that combines the use of standard apoptotic assays with transcriptomics, proteomic technologies, and computer simulations. By using histochemistry, DNA fragmentation assays, and flow cytometry analysis, we determined that MC-LR causes rapid, dose-dependent apoptosis in mouse liver when BALB/c mice are treated with MC-LR for 24 h at doses of either 50, 60, or 70 microg/kg of body weight. We then used gene expression profiling to demonstrate differential expressions (>2-fold) of 61 apoptosis-related genes in cells treated with MC-LR. Further proteomic analysis identified a total of 383 proteins of which 35 proteins were up-regulated and 30 proteins were down-regulated more than 2.5-fold when compared with controls. Combining computer simulations with the transcriptomic and proteomic data, we found that low doses (50 microg/kg) of MC-LR lead to apoptosis primarily through the BID-BAX-BCL-2 pathway, whereas high doses of MC-LR (70 microg/kg) caused apoptosis via a reactive oxygen species pathway. These results indicated that MC-LR exposure can cause apoptosis in mouse liver and revealed two independent pathways playing a major regulatory role in MC-LR-induced apoptosis, thereby contributing to a better understanding of the hepatotoxicity and the tumor-promoting mechanisms of MCs.

Animals↗

PubMed2005

Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.

Benzene is an established human leukemogen. The mechanism of benzene-induced leukemogenesis, however, remains unclear, but chromosomal damage is thought to play a critical role. We previously reported that the loss of chromosomes 5 and 7 (monosomy 5 and 7) and the gain of chromosomes 8 and 21 (trisomy 8 and 21) are significantly increased in benzene-exposed workers in comparison to matched controls. To determine if selective effects of benzene can occur, we employed three-color painting on an 8-square slide to screen numerical changes in all 24 human chromosomes (OctoChrome FISH) in a pilot study of 11 subjects (6 exposed to >5 ppm benzene and 5 age- and sex-matched controls). The effects of benzene on each chromosome were assessed as the incidence rate ratio (IRR) from a Poisson regression model with the strongest effects being reflected by the highest IRR values. Monosomy of chromosomes 5, 6, 7 and 10 had the highest IRRs and statistical significance in this preliminary study (IRR>2.5, p<0.01). On the other hand, the monosomy levels of six other chromosomes (1, 4, 9, 11, 22 and Y) were unchanged in the exposed workers with IRRs close to 1.0. Similarly, selective effects were also observed on trisomy induction with chromosomes 8, 9, 17, 21 and 22 (IRR>2.5, p<0.01). These results suggest that benzene has the capability of producing selective effects on certain chromosomes, which is supported by our in vitro findings showing that chromosomes 5 and 7 are more sensitive to loss than other chromosomes following exposure to benzene metabolites. We are currently investigating potential mechanisms for this induction of selective aneuploidy.

Adult↗

PubMed2005

Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA.

How DNA repair proteins distinguish between the rare sites of damage and the vast expanse of normal DNA is poorly understood. Recognizing the mutagenic lesion 8-oxoguanine (oxoG) represents an especially formidable challenge, because this oxidized nucleobase differs by only two atoms from its normal counterpart, guanine (G). Here we report the use of a covalent trapping strategy to capture a human oxoG repair protein, 8-oxoguanine DNA glycosylase I (hOGG1), in the act of interrogating normal DNA. The X-ray structure of the trapped complex features a target G nucleobase extruded from the DNA helix but denied insertion into the lesion recognition pocket of the enzyme. Free energy difference calculations show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site. The structure reveals a remarkably effective gate-keeping strategy for lesion discrimination and suggests a mechanism for oxoG insertion into the hOGG1 active site.

Base Sequence↗

PubMed2005

Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.

Bacterial Outer Membrane Proteins↗