Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of Show more
Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di- and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1alpha and HP1beta) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter. Show less
Imprinted genes are monoallelically expressed from either the maternal or paternal genome. Because cancer develops through genetic and epigenetic alterations, imprinted genes affect tumorigenesis depe Show more
Imprinted genes are monoallelically expressed from either the maternal or paternal genome. Because cancer develops through genetic and epigenetic alterations, imprinted genes affect tumorigenesis depending on which parental allele undergoes alteration. We have shown previously in a mouse model of neurofibromatosis type 1 (NF1) that inheriting mutant alleles of Nf1 and Trp53 on chromosome 11 from the mother or father dramatically changes the tumor spectrum of mutant progeny, likely due to alteration in an imprinted gene(s) linked to Nf1 and Trp53. In order to identify imprinted genes on chromosome 11 that are responsible for differences in susceptibility, we tested candidate imprinted genes predicted by a bioinformatics approach and an experimental approach. We have tested 30 candidate genes (Havcr2, Camk2b, Ccdc85a, Cntnap1, Ikzf1, 5730522E02Rik, Gria1, Zfp39, Sgcd, Jup, Nxph3, Spnb2, Asb3, Rasd1, Map2k3, Map2k4, Trp53, Serpinf1, Crk, Rasl10b, Itga3, Hoxb5, Cbx1, Pparbp, Igfbp4, Smarce1, Stat3, Atp6v0a1, Nbr1 and Meox1), two known imprinted genes (Grb10 and Impact) and Nf1, which has not been previously identified as an imprinted gene. Although we confirmed the imprinting of Grb10 and Impact, we found no other genes imprinted in the brain. We did, however, find strain-biased expression of Camk2b, 5730522E02Rik, Havcr2, Map2k3, Serpinf1, Rasl10b, Itga3, Asb3, Trp53, Nf1, Smarce1, Stat3, Cbx1, Pparbp and Cntnap1. These results suggest that the prediction of imprinted genes is complicated and must be individually validated. This manuscript includes supplementary data listing primer sequences for Taqman assays and Ct values for Taqman PCR. Show less
Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecul Show more
Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines. Show less
Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is Show more
Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC. Show less
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to Show more
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less
To investigate the effects of Ginkgo biloba extract (EGB) on the testosterone synthesis in the Leydig cells of type 2 diabetic rats. Thirty male SD rats were equally randomised into a normal control, Show more
To investigate the effects of Ginkgo biloba extract (EGB) on the testosterone synthesis in the Leydig cells of type 2 diabetic rats. Thirty male SD rats were equally randomised into a normal control, a type 2 diabetic and an EGB group. Morphological changes of Leydig cells were observed by light microscopy (LM) and transmission electron microscopy (TEM), concentrations of serum luteinizing hormone (LH) and testosterone (T) were determined by enzyme linked immunosorbent assay (ELISA), and the mRNA levels in the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17a-hydroxylase (P450c17), 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) from the Leydig cells were examined by RT-PCR. Compared with the normal control, there was a significant decrease in the number and volume of Leydig cells, the levels of serum LH and T and the expression of mRNA in StAR, P450scc, 17beta-HSD3 and 3beta-HSD1 in the type 2 diabetes group. And the expression of the P450c17 gene showed a tendency of descending, but with no significance. Compared with the type 2 diabetes group, 12 weeks of EGB treatment caused very slight pathological changes in the Leydig cells, significantly increased the concentrations of blood LH and T, markedly elevated the levels of mRNA in StAR and P450scc and induced an ascending tendency of the expressions of P450c17, 17beta-HSD3 and 3beta-HSD1. EGB enhances testosterone synthesis and secretion of Leydig cells by reducing the impairment of the testis in type 2 diabetic rats. Show less
LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes ce Show more
LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death. Show less
LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO Show more
LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO-1-Fc) inhibited low potassium-induced cerebellar granular neurons (CGNs) apoptosis. In the present study, we examined the neuroprotective mechanism of LINGO-1-Fc by Western blot and in situ GST pull-down assay. CGN cultures were preincubated in medium with LINGO-1-Fc or control protein at the concentration of 10 mug/ml for 2 h and then switched to low potassium medium in the presence of corresponding proteins. Cultures were harvested at indicated time intervals for successive analysis. Several apoptosis-associated signaling factors, GSK-3beta, ERK1/2, and Rho GTPases, were observed to be activated in response to potassium deprivation and the activation/dephosphorylation of GSK-3beta was suppressed by LINGO-1-Fc pretreatment compared with control group. Besides, the endogenous LINGO-1 expression level of CGN cultures was augmented by low potassium stimuli and restrained by LINGO-1 antagonist treatment. Although the protein level of p75(NTR) and Nogo-A were down-regulated in different patterns during apoptosis, neither of them was affected by LINGO-1-Fc application. Taken together, these results suggest a new mechanism of LINGO-1 antagonist regulated neuronal survival involving protein synthesis of LINGO-1 and inactivation of GSK-3 pathway. Show less
Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and a Show more
Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement. Show less
Guillain-Barré Syndrome (GBS) is a rare autoimmune inflammatory polyneuropathy with a high risk of respiratory failure and unclear pathogenesis. Currently, there are no valid biomarkers for diagnosis Show more
Guillain-Barré Syndrome (GBS) is a rare autoimmune inflammatory polyneuropathy with a high risk of respiratory failure and unclear pathogenesis. Currently, there are no valid biomarkers for diagnosis of GBS. We used 2-DE and MS to analyze the protein profiles of five pairs of cerebrospinal fluid (CSF) samples of the GBS patients and the patient controls. Three proteins (orosomucoid, haptoglobin and apolipoprotein A-IV) were up-regulated, and two proteins (prostaglandin D2 synthase and transthyretin) were down-regulated in the CSF of the GBS patients. The CSF haptoglobin level, quantified by enzyme-linked immunosorbent assay, was significantly higher in the GBS patients (12.44 ± 2.70 μg/mL) compared to the chronic inflammatory demyelinating polyradiculoneuropathy (2.82 ± 0.83 μg/mL), viral meningitis (3.57 ± 0.97 μg/mL) and control patients (1.44 ± 0.35 μg/mL, p<0.05). This study indicated that protein profile analysis using a combination of 2-DE and MS provides an effective strategy for elucidating the pathogenesis and identifying potential CSF biomarkers for GBS. The raised intrathecal synthesis of haptoglobin specifically only in GBS patients, but not in patients with other neurological diseases examined, provides evidence of central nervous system involvement in GBS, and may be used as a potential diagnostic marker for GBS. Show less
Increased protein level in the cerebrospinal fluid (CSF) is a characteristic of patients with Guillain-Barré syndrome (GBS), an acute inflammatory autoimmune disorder in the peripheral nervous system Show more
Increased protein level in the cerebrospinal fluid (CSF) is a characteristic of patients with Guillain-Barré syndrome (GBS), an acute inflammatory autoimmune disorder in the peripheral nervous system (PNS). However, the molecular mechanisms underlying the disease remain poorly understood and so far no reliable disease-related markers are available. By comparing the CSF proteome of GBS patients with control subjects suffering from other neurological disorders, it may be possible to identify proteins that involve in the disease process and thus to study the pathogenesis of GBS. We used two-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), to determine the abnormal CSF proteins in GBS patients. Our data showed that the levels of six proteins and their isoforms in CSF were significantly altered in GBS patients compared with controls. Haptoglobin, apolipoprotein A-IV and PRO2044 (unnamed protein) were considerably increased in the CSF of GBS patients, whereas transthyretin, apolipoprotein E and fibrinogen were considerably decreased. We concluded that these six proteins may be involved in the pathogenesis of GBS and call for further studying the role of these proteins in the pathogenesis of the disease. Show less
Apolipoprotein A5 (ApoA5) is present in human serum at a very low concentration. We developed a new method to determine ApoA5 concentration in human serum, and to investigate the correlation between s Show more
Apolipoprotein A5 (ApoA5) is present in human serum at a very low concentration. We developed a new method to determine ApoA5 concentration in human serum, and to investigate the correlation between serum ApoA5 and the lipid profiles in healthy subjects, and to analyze whether the correlation was affected by gender. All the subjects (total 92, male 50, female 42) were healthy subjects without any medication. Lipids were measured enzymatically. An ELISA performed by a couple of monoclonal antibodies was used to measure serum ApoA5. The average ApoA5 concentration was 182.7+/-104.7 ng/ml ranging from 5.4 to 455.6 ng/ml. Serum ApoA5 concentration was negatively correlated with TG in female (r=-0.496, P=0.001). In all subjects, ApoA5 concentration was positively correlated to HDL-C (r=0.453, P<0.001). This correlation was more predominant in female (r=0.617, P<0.001) than in male (r=0.289, P=0.042). ApoA5 concentration was negatively correlated to body mass index (BMI) with more significance in female than in male (r=-0.345, P=0.001 for all; r=-0.456, P=0.002 for female; r=-0.198, P=0.167 for male). The serum concentration of ApoA5 was very low. The concentration of ApoA5 was negatively correlated with TG and BMI, but positively correlated with HDL-C. The correlations were affected by gender. Show less
Bardet-Biedl syndrome (BBS) is an oligogenic syndrome whose manifestations include retinal degeneration, renal abnormalities, obesity and polydactylia. Evidence suggests that the main etiopathophysiol Show more
Bardet-Biedl syndrome (BBS) is an oligogenic syndrome whose manifestations include retinal degeneration, renal abnormalities, obesity and polydactylia. Evidence suggests that the main etiopathophysiology of this syndrome is impaired intraflagellar transport (IFT). In this study, we study the Bbs4-null mouse and investigate photoreceptor structure and function after loss of this gene. We find that Bbs4-null mice have defects in the transport of phototransduction proteins from the inner segments to the outer segments, before signs of cell death. Additionally, we show defects in synaptic transmission from the photoreceptors to secondary neurons of the visual system, demonstrating multiple functions for BBS4 in photoreceptors. Show less
Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is re Show more
Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Delta strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast. Show less
Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current tr Show more
Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS. Show less
MEKK3 is a mitogen-activated protein kinase kinase kinase that participates in various signaling pathways. One of its functions is to activate the ERK5 signal pathway by phosphorylating and activating Show more
MEKK3 is a mitogen-activated protein kinase kinase kinase that participates in various signaling pathways. One of its functions is to activate the ERK5 signal pathway by phosphorylating and activating MEK5. MEKK3 and MEK5 each harbors a PB1 domain in the N-terminus, and they form a heterodimer via PB1-PB1 domain interaction that was reported to be indispensable to the activation of MEK5. Using NMR spectroscopy, we show here that a prolyl isomerization of the Gln38-Pro39 bond is present in MEKK3 PB1, which is the first case of structural heterogeneity within PB1 domains. We have solved the solution structures of both isomers and found a major difference between them in the Pro39 region. Residues Gly37-Leu40 form a type VIb beta-turn in the cis conformation, whereas no obvious character of beta-turn was observed in the trans conformation. Backbone dynamics studies have unraveled internal motions in the beta3/beta4-turn on a microsecond-millisecond time scale. Further investigation of its binding properties with MEK5 PB1 has demonstrated that MEKK3 PB1 binds MEK5 PB1 tightly with a Kd of about 10(-8) M. Mutagenesis analysis revealed that residues in the basic cluster of MEKK3 PB1 contributes differently to the PB1-PB1 interaction. Residues Lys 7 and Arg 5 play important roles in the interaction with MEK5 PB1. Taken together, this study provides new insights into structural details of MEKK3 PB1 and its binding properties with MEK5 PB1. Show less
ApoAV, a newly discovered apolipoprotein, plays a key role in human triglyceride homeostasis; however, the structure-function correlation of apoAV is not clearly understood. To explore the relationshi Show more
ApoAV, a newly discovered apolipoprotein, plays a key role in human triglyceride homeostasis; however, the structure-function correlation of apoAV is not clearly understood. To explore the relationship, wild type and six deletion mutants, that is (AV (Delta(1-51)), AV (Delta(51-128)), AV (Delta(132-188)), AV (Delta(192-238)), AV (Delta(246-299)), AV (Delta(301-343))), of human apoAV expressed in Escherichia coli were studied. All the deleted regions together encompass almost the entire 343 amino acid sequence of wild type apoAV. Circular dichroism spectroscopy showed that the alpha helical content of lipid-free wild type apoAV was 46%. In comparison with wild type apoAV, AV (Delta(192-238)) and AV (Delta(301-343)) displayed significantly decreased lipid binding activities, confirming the importance of these two regions in lipid binding function of apoAV. While, the LPL activation function of apoAV remarkably impaired after deletion of residues 192-238. These findings suggested that the domain (192-238) is absolutely necessary for apoAV in lipid binding and lipoprotein lipase activation. Show less
Recently, a T/C polymorphism of the promoter region of the APOA5 gene at position -1131 and a G/T polymorphism at position 553 were found to be associated with increased levels of plasma triglyceride. Show more
Recently, a T/C polymorphism of the promoter region of the APOA5 gene at position -1131 and a G/T polymorphism at position 553 were found to be associated with increased levels of plasma triglyceride. Triglyceride plays a role in coronary artery disease (CAD), so this case-control study tested for a possible link between these two APOA5 polymorphisms, their common haplotypes and the risk of CAD. The subjects included 211 CAD patients and 677 unrelated controls. A significantly higher level of triglycerides and a lower level of high-density lipoprotein cholesterol (HDL-C) were noted for carriers with -1131C than for non-carriers (P<0.001 and 0.013, respectively) among controls. Plasma triglyceride levels were significantly higher (P=0.014) in controls with genotypes that contained the c.553T allele than in homozygotes for the G allele. Subjects homozygous for the wild-type haplotype had significantly lower triglyceride levels and higher HDL-C levels than subjects with all other haplotype pairs. The -1131C homozygous carriers and c.553T heterozygous carriers were found more frequently in 211 patients with CAD than in the 317 age/sex-matched controls (P=0.008 and 0.023, respectively) in univariate analysis. The significant association between c.553T allele carriers with CAD remained in multivariate regression analysis (OR, 1.79; CI, 1.07-3.00; P=0.028), after adjustments were made for other risk factors. Notably, haplotype analysis further verified that the APOA5 -1131C and c.553T bi-loci haplotype was significantly overpresented in CAD, as compared to the controls. These results indicate that the variants of APOA5 gene modulate plasma triglyceride and may use them to predict CAD susceptibility in Taiwanese Chinese. Show less
Chemotherapy (CT) resistance in ovarian cancer is related to multiple factors, and assessment of these factors is necessary for the development of new drugs and therapeutic regimens. In an effort to i Show more
Chemotherapy (CT) resistance in ovarian cancer is related to multiple factors, and assessment of these factors is necessary for the development of new drugs and therapeutic regimens. In an effort to identify such determinants, we evaluated the expression of approximately 21,000 genes using DNA microarray screening in paired tumor samples taken prior to and after CT treatment from 6 patients with predominantly advanced stage, high-grade epithelial ovarian cancer. A subset of differentially expressed genes was selected from all microarray data by initial filtering on confidence at p=0.05, followed by filtering on expression level (>or=2-fold). Using these selection criteria, we found 121 genes to be commonly up-regulated and 54 genes to be down-regulated in the post-CT tumors, compared to primary tumors. Up-regulated genes in post-CT tumors included substantial number of genes with previously known implication in mechanisms of chemoresistance (TOP2A, ETV4, ABCF2, PRDX2, COX2, COX7B, MUC1, MT3, MT2A), and tumorigenesis (SCGB2A2, S100A9, YWHAE, SFN, ATP6AP1, MGC5528, ASS, TACC3, ARHGAP4, SRA1; MGC35136, PSAP, SPTAN1, LGALS3BP, TUBA4, AMY2B, PPIA, COX1, GRB2, CTSL). Down-regulated genes in post-CT samples mostly included genes implicated in chemosensitivity (GRP, TRA1, ADPRTL1, TRF4-2), cell proliferation and cell cycle control (NGFRAP1, TPD52L1, TAX1BP1) and tumor suppression and apoptosis (SMOC2, TIMP3, AXIN1, CASP4, P53SCV). Additionally, gene clustering analysis revealed the existence of two distinct expression signatures of chemoresistant tumors, which was further confirmed by assessment of some genetic (p53 gene mutation status) and clinical parameters (CT regimens). Our data suggest that intrinsic and acquired chemoresistant phenotypes of post-CT tumors may be attributed to the combined action of different factors implicated in mechanisms of chemoresistance, tumor invasion/progression and control of cell proliferation. This type of molecular profiling could have important clinical implications in resolving chemoresistance and the development of novel treatment strategies designed to prevent its emergence. Show less
Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is c Show more
Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is characterized by the hallmarks of developmental and learning difficulties, post-axial polydactylia, obesity, hypogenitalism, renal abnormalities, retinal dystrophy, and several less frequently observed features. Eleven genes mutated in BBS patients have been identified, and more are expected to exist, since about 20-30% of all families cannot be explained by the known loci. To investigate the etiopathogenesis of BBS, we created a mouse null for one of the murine homologues, Bbs4, to assess the contribution of one gene to the pleiotropic murine Bbs phenotype. Bbs4 null mice, although initially runted compared to their littermates, ultimately become obese in a gender-dependent manner, females earlier and with more severity than males. Blood chemistry tests indicated abnormal lipid profiles, signs of liver dysfunction, and elevated insulin and leptin levels reminiscent of metabolic syndrome. As in patients with BBS, we found age-dependent retinal dystrophy. Behavioral assessment revealed that mutant mice displayed more anxiety-related responses and reduced social dominance. We noted the rare occurrence of birth defects, including neural tube defects and hydrometrocolpos, in the null mice. Evaluations of these null mice have uncovered phenotypic features with age-dependent penetrance and variable expressivity, partially recapitulating the human BBS phenotype. Show less
The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR l Show more
The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR ligands, such as transforming growth factor-alpha (TGF-alpha) or heparin-binding EGF (HB-EGF), is also often increased in gliomas resulting in an autocrine loop that contributes to the growth autonomy of glioma cells. Glioblastoma multiformes express a characteristic EGFR mutant (EGFRvIII, de 2-7) that does not bind ligand, signals constitutively, and is more tumorigenic than the wild-type receptor. However, the downstream signals that mediate this increased tumorigenicity are not well understood. We hypothesized that signals induced specifically by EGFRvIII and not the wild-type receptor are more likely to mediate its increased tumorigenic activity and examined the gene expression profiles resulting from inducible expression of comparable levels of either wild-type EGFR or EGFRvIII in a U251-MG glioma cell line. Expression of EGFRvIII resulted in specific up-regulation of a small group of genes. Remarkably, all these genes, which include TGFA, HB-EGF, EPHA2, IL8, MAP4K4, FOSL1, EMP1, and DUSP6, influence signaling pathways known to play a key role in oncogenesis and function in interconnected networks. Increased expression of EGFRvIII-induced genes was validated by real-time PCR. The mutant receptor does not bind ligand, and EGFRvIII-induced expression of TGF-alpha and HB-EGF suggests that EGFRvIII plays a role in generating an autocrine loop using the wild-type EGFR in glioma. It also raises the possibility that EGFRvIII may signal, at least in part, through the wild-type receptor. Indeed, we show that inhibiting the activity of HB-EGF, a potent mitogen, with neutralizing antibodies reduces cell proliferation induced by expression of EGFRvIII. This suggests that the EGFRvIII-HB-EGF-wild-type EGFR autocrine loop plays an important role in signal transduction by EGFRvIII in glioma cells. We also show by immunohistochemistry that HB-EGF expression correlates with the presence of EGFRvIII in glioblastoma multiforme. Thus, our study provides a new insight into oncogenic signaling by EGFRvIII and improves our understanding of how autocrine loops are generated in glioma. Show less
To explore the possible mechanism of apoptosis induced by photodynamic therapy (PDT) in human pancreatic cancer cells Capan-1 with 2-butylamino-2-demethoxy-hypocrellin B (BAHB) as photosensitizer. The Show more
To explore the possible mechanism of apoptosis induced by photodynamic therapy (PDT) in human pancreatic cancer cells Capan-1 with 2-butylamino-2-demethoxy-hypocrellin B (BAHB) as photosensitizer. The localization of BAHB in Capan-1 cells was studied, apoptosis was determined by DNA gel electrophoresis after PDT. The mitochondria membrane potential (DYm) and cytochrome C release were observed by laser scan confocal microscopy and Western blotting. The low concentration photosensitizer was mainly localized in mitochondria and also in lysosomes when the concentration is high. DNA ladder analysis showed characteristic of apoptosis. The mitochondria membrane potential (DYm) showed a loss of 30% around, after 6 hours by PDT under laser scan confocal microscopy, which is caused by a sudden increase in the permeability of mitochondria membrane accompanied with apoptosis. In Western blotting, cytochrome C release was observed from the mitochondria into the cytoplasm during BAHB-induced apoptosis. The research suggests that BAHB-induced apoptosis is related to photosensitization of mitochondria. Show less
Zhi-Guo Xie, Zheng-Mao Hu, Qian Pan+7 more · 2006 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics · added 2026-04-24
To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation. Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene muta Show more
To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation. Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation. By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control. The mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses. Show less
LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexe Show more
LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexes mediate the axonal growth inhibitory effects of Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) via RhoA activation. Soluble LINGO-1 (LINGO-1-Fc), which acts as an antagonist of these pathways by blocking LINGO-1 binding to NgR1, was administered to rats after dorsal or lateral hemisection of the spinal cord. LINGO-1-Fc treatment significantly improved functional recovery, promoted axonal sprouting and decreased RhoA activation and increased oligodendrocyte and neuronal survival after either rubrospinal or corticospinal tract transection. These experiments demonstrate an important role for LINGO-1 in modulating axonal outgrowth in vivo and that treatment with LINGO-1-Fc can significantly enhance recovery after spinal cord injury. Show less
To detect the disease-causing gene mutation of hypertrophic cardiomyopathy (HCM) in a Chinese family and to analyze the correlation of the genotype and the phenotype. One family affected with HCM was Show more
To detect the disease-causing gene mutation of hypertrophic cardiomyopathy (HCM) in a Chinese family and to analyze the correlation of the genotype and the phenotype. One family affected with HCM was studied. The clinical data including symptom, physical examination, echocardiography and electrocardiography were collected. The full encoding exons and flanking sequences of beta-myosin heavy chain gene (MYH7) and cardiac myosin-binding protein C gene (MYBPC3) were amplified with PCR and the products were sequenced. A G8887A mutation, which is an acceptor splicing site of intron 15 (IVS15-1G > A) in MYBPC3 (gi: Y10129) was identified in 6 out of 11 family members. Three mutation carriers developed HCM at 48 - 75 years old with mild chest pain, chest distress and asymmetric septal hypertrophy (13 - 14 mm) and remaining mutation carriers are free of HCM. No mutation was identified in MYH7 gene. HCM caused by the IVS15-1G > A mutation is a benign phenotype. It is helpful to screen MYBPC3 gene mutation in late-onset HCM patients with mild symptoms. Show less
Fission yeast capping protein SpCP is a heterodimer of two subunits (Acp1p and Acp2p) that binds actin filament barbed ends. Neither acp1 nor acp2 is required for viability, but cells lacking either o Show more
Fission yeast capping protein SpCP is a heterodimer of two subunits (Acp1p and Acp2p) that binds actin filament barbed ends. Neither acp1 nor acp2 is required for viability, but cells lacking either or both subunits have cytokinesis defects under stressful conditions, including elevated temperature, osmotic stress, or in combination with numerous mild mutations in genes important for cytokinesis. Defects arise as the contractile ring constricts and disassembles, resulting in delays in cell separation. Genetic and biochemical interactions show that the cytokinesis formin Cdc12p competes with capping protein for actin filament barbed ends in cells. Deletion of acp2 partly suppresses cytokinesis defects in temperature-sensitive cdc12-112 cells and mild overexpression of capping protein kills cdc12-112 cells. Biochemically, profilin has opposite effects on filaments capped with Cdc12p and capping protein. Profilin depolymerizes actin filaments capped by capping protein but allows filaments capped by Cdc12p to grow at their barbed ends. Once associated with a barbed end, either Cdc12p or capping protein prevents the other from influencing polymerization at that end. Given that capping protein arrives at the division site 20 min later than Cdc12p, capping protein may slowly replace Cdc12p on filament barbed ends in preparation for filament disassembly during ring constriction. Show less
Jun Gao, Yusheng Wei, Yue Huang+8 more · 2005 · The Journal of biological chemistry · American Society for Biochemistry and Molecular Biology · added 2026-04-24
The apoAI/CIII/AIV gene cluster is involved in lipid metabolism and has a complex pattern of gene expression modulated by a common regulatory element, the apoCIII enhancer. A new member of this cluste Show more
The apoAI/CIII/AIV gene cluster is involved in lipid metabolism and has a complex pattern of gene expression modulated by a common regulatory element, the apoCIII enhancer. A new member of this cluster, apolipoprotein (apo) AV, has recently been discovered as a novel modifier in triglyceride metabolism. To determine the expression of all four apo genes in combination and, most importantly, whether the transcription of apoAV is coregulated by the apoCIII enhancer in the cluster, we generated an intact transgenic line carrying the 116-kb human apoAI/CIII/AIV/AV gene cluster and a mutant transgenic line in which the apoCIII enhancer was deleted from the 116-kb structure. We demonstrated that the apoCIII enhancer regulated hepatic and intestinal apoAI, apoCIII, and apoAIV expression; however, it did not direct the newly identified apoAV in the cluster. Furthermore, human apo genes displayed integrated position-independent expression and a closer approximation of copy number-dependent expression in the intact transgenic mice. Because apoCIII and apoAV play opposite roles in triglyceride homeostasis, we analyzed the lipid profiles in our transgenic mice to assess the effects of human apoAI gene cluster expression on lipid metabolism. The triglyceride level was elevated in intact transgenic mice but decreased in mutant ones compared with nontransgenic mice. In addition, the expression of human apoAI and apoAIV elevated high density lipoprotein cholesterol in transgenic mice fed an atherogenic diet. In conclusion, our studies with human apoAI/CIII/AIV/AV gene cluster transgenic models showed that the apoCIII enhancer regulated expression of apoAI, apo-CIII, and apoAIV but not apoAV in vivo and showed the influences of expression of the entire cluster on lipid metabolism. Show less
To investigate the association between the -1131T/C and 56C/G polymorphism in the APOA5 gene as well as the -482C/T in the APOC3 gene and susceptibility to coronary artery disease (CAD) in a Chinese H Show more
To investigate the association between the -1131T/C and 56C/G polymorphism in the APOA5 gene as well as the -482C/T in the APOC3 gene and susceptibility to coronary artery disease (CAD) in a Chinese Han population. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polyacrylamide gel electrophoresis (PAGE) methods, we analyzed the genotypes in 312 CAD patients diagnosed by angiography and 317 healthy controls. The levels of serum lipid profiles were also studied by biochemical methods. The frequency of the APOA5 -1131 C allele in CAD patients was significantly higher than that of the control group (39.9% vs. 33.3%, P = 0.02). Compared with the wild type TT, CC homozygotes had a significantly increased CAD risk (OR = 1.93 and OR = 1.80 using unadjusted and adjusted logistic regression models, respectively). This association still existed after adjustment for the APOC3-482 variant. The APOA5-1131C allele also showed a correlation with increasing plasma TG levels (P < 0.01). The APOA5-1131T/C polymorphism but not APOC3-482C/T might contribute to an increased risk of CAD among Chinese accompanied by an elevation of serum TG levels; this effect was found to be independent of the APOC3-482C/T variant. Show less
Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 Show more
Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus. Show less
Haiyan Xu, Qing Yang, Minhui Shen+6 more · 2005 · The Journal of biological chemistry · American Society for Biochemistry and Molecular Biology · added 2026-04-24
Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosp Show more
Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. In insulin resistance and type II diabetes there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycemia. To search for novel genes that negatively regulate insulin signaling in controlling metabolic pathways, we screened a cDNA library derived from the white adipose tissue of ob/ob mice using a reporter system comprised of the PEPCK promoter placed upstream of the alkaline phosphatase gene. The mitogen-activated dual specificity protein kinase phosphatase 3 (MKP-3) was identified as a candidate gene that antagonized insulin suppression on PEPCK gene transcription from this screen. In this study, we showed that MKP-3 was expressed in insulin-responsive tissues and that its expression was markedly elevated in the livers of insulin-resistant obese mice. In addition, MKP-3 can activate PEPCK promoter in synergy with dexamethasone in hepatoma cells. Furthermore, ectopic expression of MKP-3 in hepatoma cells by adenoviral infection increased the expression of PEPCK and G6Pase genes and led to elevated glucose production. Taken together, our data strongly suggests that MKP-3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis. Therefore, dysregulation of MKP-3 expression and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes. Show less