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1.  Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase 
G. zeae extracellular lipase has been overexpressed, purified and crystallized. Diffraction data were collected to 2.8 Å resolution.
Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 Å resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P212121, with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 Å, α = β = γ = 90°. The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 Å3 Da−1.
doi:10.1107/S1744309108019283
PMCID: PMC2531274  PMID: 18765911
extracellular lipases; Fusarium graminearum; Gibberella zeae; fusarium head blight
2.  Genome-wide association scans identified CTNNBL1 as a novel gene for obesity 
Human Molecular Genetics  2008;17(12):1803-1813.
Obesity is a major public health problem with strong genetic determination; however, the genetic factors underlying obesity are largely unknown. In this study, we performed a genome-wide association scan for obesity by examining approximately 500 000 single-nucleotide polymorphisms (SNPs) in a sample of 1000 unrelated US Caucasians. We identified a novel gene, CTNNBL1, which has multiple SNPs associated with body mass index (BMI) and fat mass. The most significant SNP, rs6013029, achieved experiment-wise P-values of 2.69 × 10−7 for BMI and of 4.99 × 10−8 for fat mass, respectively. The SNP rs6013029 minor allele T confers an average increase in BMI and fat mass of 2.67 kg/m2 and 5.96 kg, respectively, compared with the alternative allele G. We further genotyped the five most significant CTNNBL1 SNPs in a French case–control sample comprising 896 class III obese adults (BMI ≥ 40 kg/m2) and 2916 lean adults (BMI < 25 kg/m2). All five SNPs showed consistent associations with obesity (8.83 × 10−3 < P < 6.96 × 10−4). Those subjects who were homozygous for the rs6013029 T allele had 1.42-fold increased odds of obesity compared with those without the T allele. The protein structure of CTNNBL1 is homologous to β-catenin, a family of proteins containing armadillo repeats, suggesting similar biological functions. β-Catenin is involved in the Wnt/β-catenin-signaling pathway which appears to contribute to maintaining the undifferentiated state of pre-adipocytes by inhibiting adipogenic gene expression. Our study hence suggests a novel mechanism for the development of obesity, where CTNNBL1 may play an important role. Our study also provided supportive evidence for previously identified associations between obesity and INSIG2 and PFKP, but not FTO.
doi:10.1093/hmg/ddn072
PMCID: PMC2900891  PMID: 18325910
3.  Laforin Negatively Regulates Cell Cycle Progression through Glycogen Synthase Kinase 3β-Dependent Mechanisms▿ §  
Molecular and Cellular Biology  2008;28(23):7236-7244.
Glycogen synthase kinase 3β (GSK-3β) represses cell cycle progression by directly phosphorylating cyclin D1 and indirectly regulating cyclin D1 transcription by inhibiting Wnt signaling. Recently, we reported that the Epm2a-encoded laforin is a GSK-3β phosphatase and a tumor suppressor. The cellular mechanism for its tumor suppression remains unknown. Using ex vivo thymocytes and primary embryonic fibroblasts from Epm2a−/− mice, we show here a general function of laforin in the cell cycle regulation and repression of cyclin D1 expression. Moreover, targeted mutation of Epm2a increased the phosphorylation of Ser9 on GSK-3β while having no effect on the phosphorylation of Ser21 on GSK-3α. In the GSK-3β+/+ but not the GSK-3β−/− cells, Epm2a small interfering RNA significantly enhanced cell growth. Consistent with an increased level of cyclin D1, the phosphorylation of retinoblastoma protein (Rb) and the levels of Rb-E2F-regulated genes cyclin A, cyclin E, MCM3, and PCNA are also elevated. Inhibitors of GSK-3β selectively increased the cell growth of Epm2a+/+ but not of Epm2a−/− cells. Taken together, our data demonstrate that laforin is a selective phosphatase for GSK-3β and regulates cell cycle progression by GSK-3β-dependent mechanisms. These data provide a cellular basis for the tumor suppression activity of laforin.
doi:10.1128/MCB.01334-08
PMCID: PMC2593373  PMID: 18824542
4.  TSC–mTOR maintains quiescence and function of hematopoietic stem cells by repressing mitochondrial biogenesis and reactive oxygen species 
The Journal of Experimental Medicine  2008;205(10):2397-2408.
The tuberous sclerosis complex (TSC)–mammalian target of rapamycin (mTOR) pathway is a key regulator of cellular metabolism. We used conditional deletion of Tsc1 to address how quiescence is associated with the function of hematopoietic stem cells (HSCs). We demonstrate that Tsc1 deletion in the HSCs drives them from quiescence into rapid cycling, with increased mitochondrial biogenesis and elevated levels of reactive oxygen species (ROS). Importantly, this deletion dramatically reduced both hematopoiesis and self-renewal of HSCs, as revealed by serial and competitive bone marrow transplantation. In vivo treatment with an ROS antagonist restored HSC numbers and functions. These data demonstrated that the TSC–mTOR pathway maintains the quiescence and function of HSCs by repressing ROS production. The detrimental effect of up-regulated ROS in metabolically active HSCs may explain the well-documented association between quiescence and the “stemness” of HSCs.
doi:10.1084/jem.20081297
PMCID: PMC2556783  PMID: 18809716
5.  Upregulation of the constitutively expressed HSC70 by KLF4 
Cell Stress & Chaperones  2008;13(3):337-345.
Krüppel-like factor 4 (KLF4) is a transcription factor that is abundantly expressed in various organisms from bacteria to mammals. It has been demonstrated that KLF4 regulates the expression of a wide range of genes. Analysis of KLF4 target genes reveals its diverse regulatory functions in cell growth, proliferation, differentiation, embryogenesis, and inflammation. However, the regulation of the expression of inducible heat shock protein 70 (HSP72) and heat shock cognate 70 (HSP73) by KLF4 is not defined. In our previous study, a complementary deoxyribonucleic acid microarray assay showed that KLF4 overexpression led to dramatic upregulation of HSP73 messenger ribonucleic acid (mRNA) in murine C2C12 myoblast cells, suggesting that HSP73 is a potential target gene regulated by KLF4. The effect of KLF4 on the expression of HSP72 and HSP73 was further examined by reverse transcriptase polymerase chain reaction and Western blot in KLF4-overexpressing or KLF4-deficient cells. The results showed the upregulation of the HSP73 constitutive expression by KLF4 overexpression in both C2C12 cells and murine RAW264.7 macrophages; in response to heat stress, however, few changes were observed in the levels of HSP73 by KLF4 overexpression. In addition, knockdown of endogenous KLF4 expression by morpholino antisense oligonucleotides significantly decreased both HSP73 mRNA and protein levels under normal conditions. Conversely, KLF4 had no effect on the expression of HSP72. Taken together, these findings suggest an important role for KLF4 as a novel regulator of the constitutive expression of HSP73.
doi:10.1007/s12192-008-0033-5
PMCID: PMC2673948  PMID: 18379898
KLF4; HSP73; HSP72; Gene expression
6.  Identification of an antifungal metabolite produced by a potential biocontrol Actinomyces strain A01 
Brazilian Journal of Microbiology  2008;39(4):701-707.
Actinomyces strain A01 was isolated from soil of a vegetable field in the suburb of Beijing, China. According to the morphological, cultural, physiological and biochemical characteristics, and 16S rDNA sequence analysis, strain A01 was identified as Streptomyces lydicus. In the antimicrobial spectrum test strain A01 presented a stable and strong inhibitory activity against several plant pathogenic fungi such as Fusarium oxysporum, Botrytis cinerea, Monilinia laxa, etc. However, no antibacterial activity was found. In pot experiments in greenhouse, the development of tomato gray mold was markedly suppressed by treatment with the fermentation broth of the strain A01, and the control efficacy was higher than those of Pyrimethanil and Polyoxin. A main antifungal compound (purity 99.503%) was obtained from the fermentation broth of strain A01 using column chromatography and HPLC. The chemical structural analysis with U V, IR, MS, and NMR confirmed that the compound produced by the strain A01 is natamycin, a polyene antibiotic produced by S. chattanovgensis, S. natalensis, and S. gilvosporeus, widely used as a natural biological preservative for food according to previous reports. The present study revealed a new producing strain of natamycin and its potential application as a biological control agent for fungal plant diseases.
doi:10.1590/S1517-838220080004000020
PMCID: PMC3768479  PMID: 24031293
Streptomyces lydicus; natamycin; fungal plant disease; biological control
7.  (E)-1-Ferrocenyl-3-(3-nitro­phen­yl)prop-2-en-1-one 
In the title compound, [Fe(C5H5)(C14H10NO3)], one cyclo­penta­diene ring is substituted and one is unsubstituted. The two rings are almost parallel and are eclipsed and ordered. The conjugated substituent is slightly twisted with respect to the cyclo­penta­diene ring. The crystal structure contains four inter­molecular C—H⋯O hydrogen-bonds in the range 3.324 (3)–3.539 (3) Å and one π(aryl ring)–π (Cp ring) stacking inter­action with a ring–centroid distance of 3.894 (2) Å.
doi:10.1107/S1600536808027815
PMCID: PMC2959240  PMID: 21200999
8.  Methyl 2-[(E)-(4-nitro­phen­yl)hydrazono]-3-oxobutyrate 
The mol­ecule of the title compound, C11H11N3O5, exists as the E isomer as it is stabilized by an intra­molecular hydrogen bond. Except for the methyl H atoms, all atoms lie in special positions on a mirror plane and form a large conjugated system; the methyl H atoms are disordered about the mirror plane. In the crystalline state, bifurcated intra- and inter­molecular N—H⋯O hydrogen bonds and four inter­molecular C—H⋯O hydrogen bonds link the mol­ecules into large perfectly planar sheets. Along the c axis, the N—N bond center approaches the phenyl-ring centroids of its neighbouring mol­ecules above and below to give π–π overlap (at a distance of ca 3.57 Å), thus fusing the mol­ecules into a three-dimensional framework.
doi:10.1107/S160053680802312X
PMCID: PMC2962099  PMID: 21203301
9.  (E)-1-Ferrocenyl-3-phenyl­prop-2-en-1-one 
The title compound, [Fe(C5H5)(C14H11O)], exists as the E isomer, and the substituent is fully conjugated with the attached five-membered ring. In the ferrocene unit, the substituted cyclo­penta­dienyl ring (Cps) plane and unsubstituted cyclo­penta­dienyl ring (Cp) plane are almost parallel, and the C atoms in Cp and Cps are in an eclipsed conformation. In the crystal structure, mol­ecules are linked into C(5) chains via inter­molecular C—H⋯O hydrogen bonds, and neighbouring chains are assembled into sheets by inter­molecular C—H⋯π(arene) hydrogen bonds along the c axis.
doi:10.1107/S1600536808020059
PMCID: PMC2961926  PMID: 21202997
10.  N-[(E,Z)-1,3-Diphenyl­prop-2-enyl­idene]-N′-(1,3-dithio­lan-2-yl­idene)hydrazine 
Mol­ecules of the title compound, C18H16N2S2, exist as the (2E, 1′Z)-isomer. The 1,3-dithiol­ane ring has an envelope conformation; the atoms of the C—C bond are disordered over two positions with occupancies of 0.47 (7) and 0.53 (7). The structure exhibits inter­molecular C—H⋯S and C—H⋯π(arene) hydrogen bonds.
doi:10.1107/S1600536808018606
PMCID: PMC2961686  PMID: 21202962
11.  Torsional ultrasound modality for hard nucleus phacoemulsification cataract extraction 
Zeng, M | Liu, X | Liu, Y | Xia, Y | Luo, L | Yuan, Z | Zeng, Y | Liu, Y
The British Journal of Ophthalmology  2008;92(8):1092-1096.
Aim:
To evaluate the efficacy and safety of phacoemulsification using torsional modality with different parameter settings for hard nucleus cataract extraction.
Design:
A prospective, randomised clinical study.
Methods:
A clinical practice study conducted at the Cataract Service, Zhongshan Ophthalmic Center, Sun-Yat-Sen University, and Guangzhou. One eye each from 198 consecutive patients with cataract density grade IV according to the Emery–Little system classification system, requiring phacoemulsification and intraocular lens implantation, was included. Eyes were randomly assigned to the Linear Torsional combined with Ultrasound power group (Linear Tor+US group, n = 66), 100% Fixed Torsional group (Fixed Tor group, n = 65) and conventional Ultrasound burst group (US group, n = 67). All surgeries were performed by a single experienced surgeon and outcomes evaluated by another surgeon masked to treatment. Intraoperative parameters were Ultrasound Time (UST), Cumulative Dissipated Energy (CDE) and surgical complications. Patients were examined on post-op days 1, 7 and 30. Postoperative outcomes were final best corrected visual acuity (BCVA), average central and incisional corneal thickness and central endothelial cell counts.
Results:
The mean UST was lower in the Fixed Tor group than in the US group and in the Lin US+Tor group (p⩽0.0001). The mean CDE was lower in the Lin Tor+US group and in the Fixed Tor group than in the US group (p⩽0.0001). Comparing with the two Tor group, the US group had a lower average BCVA on post-op 1, 7 (p⩽0.0001) and 30 (p>0.01), greater average central corneal and incisional thickness on days 1, 7 (p⩽0.0001) and 30 (p>0.01), and higher average corneal endothelial cell losses on day 7 and 30 days (p⩽0.0001).
Conclusions:
Torsional combined with ultrasound power or high fixed torsional amplitude can yield more effective hard nucleus phacoemulsification than conventional ultrasound modality.
doi:10.1136/bjo.2007.128504
PMCID: PMC2569137  PMID: 18567650
12.  Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China 
Background
The pathogenesis of nasopharyngeal carcinoma (NPC) is a complicated process involving genetic predisposition, Epstein-Bar Virus infection, and genetic alterations. Although some oncogenes and tumor suppressor genes have been previously reported in NPC, a complete understanding of the pathogenesis of NPC in the context of global gene expression, transcriptional pathways and biomarker assessment remains to be elucidated.
Methods
Total RNA from 32 pathologically-confirmed cases of poorly-differentiated NPC was divided into pools inclusive of four consecutive specimens and each pool (T1 to T8) was co-hybridized with pooled RNA from 24 normal non-cancerous nasopharyngeal tissues (NP) to a human 8K cDNA array platform. The reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and immunohistochemistry.
Results
Stringent statistical filtering parameters identified 435 genes to be up-regulated and 257 genes to be down-regulated in NPC compared to NP. Seven up-regulated genes including CYC1, MIF, LAMB3, TUBB2, UBE2C and TRAP1 had been previously proposed as candidate common cancer biomarkers based on a previous extensive comparison among various cancers and normal tissues which did not, however, include NPC or NP. In addition, nine known oncogenes and tumor suppressor genes, MIF, BIRC5, PTTG1, ATM, FOXO1A, TGFBR2, PRKAR1A, KLF5 and PDCD4 were identified through the microarray literature-based annotation search engine MILANO, suggesting these genes may be specifically involved in the promotion of the malignant conversion of nasopharyngeal epithelium. Finally, we found that these differentially expressed genes were involved in apoptosis, MAPK, VEGF and B cell receptor signaling pathways and other functions associated with cell growth, signal transduction and immune system activation.
Conclusion
This study identified potential candidate biomarkers, oncogenes/tumor suppressor genes involved in several pathways relevant to the oncogenesis of NPC. This information may facilitate the determination of diagnostic and therapeutic targets for NPC as well as provide insights about the molecular pathogenesis of NPC.
doi:10.1186/1479-5876-6-32
PMCID: PMC2443113  PMID: 18570662
13.  Transcriptome Analysis of a cDNA Library from Adult Human Epididymis 
Mammalian Gene Collection (MGC) verified over 9000 human full-ORF genes and FLJ Program reported 21 243 cDNAs of which 14 409 were unique ones and 5416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20 000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website www.sdscli.com.
doi:10.1093/dnares/dsn005
PMCID: PMC2650638  PMID: 18390568
human epididymis cDNA library; transcriptomes for human epididymis; sperm maturation
14.  Plasmacytoid dendritic cells induce NK cell–dependent, tumor antigen–specific T cell cross-priming and tumor regression in mice 
The Journal of Clinical Investigation  2008;118(3):1165-1175.
A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-γ production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8+ T cells.
doi:10.1172/JCI33583
PMCID: PMC2230660  PMID: 18259609
15.  Curcumin prevents and reverses murine cardiac hypertrophy 
Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase–dependent (p300-HAT–dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT–dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-κB, and TGF-β–Smad signaling pathways.
doi:10.1172/JCI32865
PMCID: PMC2248327  PMID: 18292803
16.  H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation 
The Journal of General Virology  2008;89(Pt 3):697-702.
Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.
doi:10.1099/vir.0.83419-0
PMCID: PMC2885753  PMID: 18272760
17.  4-(5-tert-Butyl-1,3-dithian-2-yl)-5-chloro-2-phenyl-1,3-oxazole 
In the title mol­ecule, C17H20ClNOS2, the phenyl and oxazole rings are nearly coplanar with an average deviation of 0.022 Å from the mean plane (M). The 1,3-dithiane ring adopts a chair conformation and is twisted in such a way that the C—CBu fragment lies in M (deviations are 0.031 and 0.010 Å, respectively, for the two C atoms).
doi:10.1107/S1600536808002821
PMCID: PMC2960807  PMID: 21201886
18.  Effects of Yinchenhao Tang and related decoctions on DMN-induced cirrhosis/fibrosis in rats 
Chinese Medicine  2008;3:1.
Background
Chinese medicine decoctions such as Yinchenhao Tang (YCHT), Xiayuxue Tang (XYXT), Huangqi Tang (HQT), Yiguan Jian (YGJ) and Xiaochaihu Tang (XCHT)) were used to treat liver cirrhosis. The present study evaluates the effects of these decoctions on fibrosis in rats induced by dimethylnitrosamine (DMN).
Methods
DMN solution (0.5%) was injected to rats for three consecutive days per week for four weeks. At the beginning of week 3, rats were randomly divided into 4-week DMN control group, YCHT, XYXT, HQT, YGJ, XCHT and vehicle groups. Each group was orally administered with specific decoctions daily for two weeks. Rats in the vehicle group were orally administered with only water.
Results
Liver fibrosis and cirrhosis were observed in weeks 2 and 4 in DMN-intoxicated rats. Compared with normal rats, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities and level of total bilirubin acid (TBA) in serum and content of Hydroxyproline (Hyp) in liver tissue of model group rats rose significantly. However, the albumin (Alb) level in serum decreased significantly. Compared with the 4-week DMN group, the pathological conditions and functions of the liver in the YCHT group improved significantly, and the content of Hyp decreased remarkably: only one rat in this group developed liver cirrhosis and the ratio of cirrhosis was only 8.3%. On the other hand, the other decoctions did not show remarkable effects. YCHT inhibited α-SMA activation, including its gene expression into mRNA and protein.
Conclusion
Among the five Chinese medicine decoctions, YCHT exerted the most significant therapeutic effects on DMN-induced cirrhosis/fibrosis in rats.
doi:10.1186/1749-8546-3-1
PMCID: PMC2267793  PMID: 18237412
19.  Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits 
Molecular Vision  2008;14:2404-2412.
Purpose
To explore lens crystallin characteristics and morphology of rabbit regenerated lenses in comparison with wild type natural lenses by means of proteomic analysis and histological assay.
Methods
The lens regeneration model of the New Zealand rabbit was established, and lens regeneration was observed by slit lamp examination and photography. A histological assay was evaluated under light microscopy and transmission electron microscopy (TEM). Protein samples of regenerated lenses were collected from experimental rabbit eyes 2, 4, and 16 weeks after surgery. Two-dimensional gel electrophoresis (2-DE) was performed. Image analyses was done using the ImageMaster 2D Elite 3.01 software package. The protein spots were trypsinized and identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry.
Results
Lens regeneration began in the periphery of the capsule bag about one to two weeks after the surgery and proceeded to regenerate toward the center. The regenerated lens appeared spherical in shape with a fairly translucent cortical structure and a nuclear opacity. Histological findings showed that the remnant lens epithelial cells differentiate at the lens capsule equator and new lens fibers form in a concentric pattern in a manner similar to that observed in natural lenses. However, TEM showed morphological changes in the epithelial cells of the regenerated lenses as compared with natural lenses. 2-D electrophoresis revealed that the patterns of protein spots from regenerated lenses (two weeks, four weeks, and 16 weeks) were analogous to those of 16-week-old natural lenses but were substantially different from those of two-week-old natural lenses, particularly when the two-week-old regenerated lenses were compared with the two-week-old natural lenses.
Conclusions
Proteomic analysis revealed that crystallin expression in regenerated rabbit lenses was analogous to that of natural lenses of adult rabbits but was different from that of very young rabbits (two weeks old), and TEM revealed the presence of morphological changes in the epithelial cells of regenerated lenses. These results suggest that the regrowth of lens materials in the lens capsule after endocapsular phacoemulsification might actually represent the regeneration of “mature” lens substances, which have led us to the conclusion that the regenerative process does not exactly mimic embryonic development.
PMCID: PMC2605429  PMID: 19098996
20.  Differential expression of the catalytic subunits for PP-1 and PP-2A and the regulatory subunits for PP–2A in mouse eye 
Molecular Vision  2008;14:762-773.
Purpose
Reversible protein phosphorylation is a fundamental regulatory mechanism in all biologic processes. Protein serine/threonine phosphatases-1 (PP-1) and 2A (PP-2A) account for 90% of serine/threonine phosphatase activity in eukaryote cells and play distinct roles in regulating multiple cellular processes and activities. Our previous studies have established the expression patterns of the catalytic subunits for PP-1 (PP-1cs) and PP-2A (PP-2Acs) in bovine and rat lenses. In the present study, we have determined the expression patterns of PP-1cs (PP-1α and PP-1β) and PP-2Acs (PP-2Aα and PP-2Aβ) in the retina and cornea along with the ocular lens of the mouse eye. Moreover, since the function of PP-2A is largely relied on its regulatory subunits, we have also analyzed the expression patterns of the genes encoding the scaffold A subunits of PP-2A, PP2A-Aα and PP2A-Aβ, and the regulatory B family subunits of PP-2A, PP2A-Bα, PP2A-Bβ, and PP2A-Bγ. In addition, we have also demonstrated the differential protections of PP-1 and PP-2A in mouse lens epithelial cell line, αTN4–1, against oxidative stress-induced apoptosis.
Methods
Total RNAs and proteins were extracted from the retina, lens epithelium, lens fiber cells, and cornea of the mouse eye. Reverse transcription polymerase chain reaction (RT–PCR) and real time PCR were used to detect the mRNA expression. Western blot and immunohistochemistry analysis were applied to examine the protein expression and distribution. Stable clones of αTN4–1 cells expressing either PP-1α or PP-2Aα were used to analyze the differential protections against oxidative stress-induced apoptosis.
Results
PP-1 is more abundant than PP-2A in the mouse eye. The catalytic subunits for PP-1 and PP-2A display similar expression patterns in the retina and cornea but much reduced in the lens. The mRNAs for all five isoforms of PP2A-A and PP2A-B subunits are highly expressed in the retina, but only three out of the five mRNAs are expressed in the cornea. In the ocular lens, only PP2A-Aβ and PP2A-Bγ mRNAs are clearly detectable. The A and B subunit proteins of PP-2A are highly expressed in the retina and cornea but are much reduced in the ocular lens. PP2A-Aα/β are differentially distributed in the mouse retina.When transfected into mouse lens epithelial cells, αTN4–1, PP-1α and PP-2Aα display differential protection against oxidative stress-induced apoptosis.
Conclusions
Our results lead to the following conclusions regarding PP-1 and PP-2A in mouse eye: 1) PP1 is a more abundant phosphatase than PP-2A; 2) both PP-1 and PP-2A may play important roles, and the functions of PP-2A appear to be highly regulated by various regulatory subunits; and 3) the genes encoding PP-1α/β, PP-2Aα/β, PP-2A-Aα/β, and PP-2A-B α/β/γ are all differentially expressed.
PMCID: PMC2324119  PMID: 18432318
21.  Benzo(a)pyrene-Caused Increaesd G1-S Transition Requires the Activation of c-Jun through p53-Dependent PI-3K/Akt/ERK Pathway in Human Embryo Lung Fibroblasts 
Toxicology letters  2008;178(3):167-175.
doi:10.1016/j.toxlet.2008.03.012
PMCID: PMC3759236  PMID: 18448277
B(a)P; Signaling Pathway; Cell cycle; c-Jun; Akt; p53
22.  Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells 
Gastroenterology  2008;136(1):320-330.e2.
Background & Aims
Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC).
Methods
MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats.
Results
PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL.
Conclusions
Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.
doi:10.1053/j.gastro.2008.09.066
PMCID: PMC3724240  PMID: 19013163
microparticles; exosomes; liver cirrhosis; hedgehog; sinusoidal endothelial cells; nitric oxide
23.  Dual actions involved in arsenite-induced oxidative DNA damage 
Chemical research in toxicology  2008;21(9):1806-1813.
Arsenic is a recognized human carcinogen, but the mechanism of carcinogenesis is not well understood. Oxidative stress and inhibition of DNA damage repair have been postulated as potential carcinogenic actions of arsenic. The present study tests the hypothesis that arsenite not only induces oxidative stress, but also inhibits the activity of the DNA base excision repair protein, poly(ADP-ribose) polymerase-1 (PARP-1), leading to exacerbation of the oxidative DNA damage induced by arsenic. HaCat cells were treated with arsenite for 24 hrs before measuring 8-hydroxyl-2′-deoxyguanosine (8-OHdG), PARP-1 activity, and reactive oxygen species (ROS). Zinc supplementation and PARP-1 siRNA were used to increase or decrease, respectively, the PARP-1 protein’s physiological function. At high concentrations (10μM or higher), arsenite greatly induced oxidative DNA damage, as indicated by 8-OHdG formation. At lower concentrations (1μM), arsenite did not produce detectable 8-OHdG, but was still able to effectively inhibit PARP-1 activity. Zinc supplementation reduced the formation of 8-OHdG, restored the PARP-1 activity inhibited by arsenite, but did not decrease ROS production. SiRNA knockdown of PARP-1 did not affect the 8-OHdG level induced by arsenic, while it greatly increased the 8-OHdG level produced by hydrogen peroxide indicating that PARP-1 is a molecular target of arsenite. Our findings demonstrate that in addition to inducing oxidative stress at higher concentrations, arsenite can also inhibit the function of a key DNA repair protein, PARP-1, even at very low concentrations, thus exacerbating the overall oxidative DNA damage produced by arsenite, and potentially, by other oxidants as well.
doi:10.1021/tx8001548
PMCID: PMC3606021  PMID: 18707137
arsenic; PARP-1; DNA damage repair; 8-OHdG; ROS; carcinogenesis
24.  Growth Inhibitory and Tumor- Suppressive Functions of p53 Depend on its Repression of CD44 Expression 
Cell  2008;134(1):62-73.
SUMMARY
The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a non-canonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting de-repressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and non-tumorigenic cells, CD44’s expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the de-repression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant, cells.
doi:10.1016/j.cell.2008.06.006
PMCID: PMC3222460  PMID: 18614011
25.  GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth 
Cell research  2008;18(9):900-910.
We report here the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal Ring finger domain, which is mostly conserved with those of the IAP family members, and which is required for its E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases since the caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockade of JNK activation inhibits GIDE-induced release of cytochrome c and Smac and apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE-induced apoptosis. These proapoptotic properties of GIDE require its E3 ligase activity. When somewhat over or underexpressed, GIDE slows or hastens cell growth respectively. These pro-apoptotic or growth rate effects of GIDE may account for its absence from tumor cells.
doi:10.1038/cr.2008.75
PMCID: PMC3156110  PMID: 18591963
GIDE; apoptosis; E3 ligase; mitochondria; caspase; JNK

Results 1-25 (2583)