The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness–induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix–induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling–mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.
lung fibrosis; transcription factor; α-smooth muscle actin
The Drosophila compound eye is a large sensory organ that places a high demand on oxygen supplied by the tracheal system. Although the development and function of the Drosophila visual system has been extensively studied, the development and contribution of its tracheal system has not been systematically examined. To address this issue, we studied the tracheal patterns and developmental process in the Drosophila visual system. We found that the retinal tracheae are derived from air sacs in the head, and the ingrowth of retinal trachea begin at mid-pupal stage. The tracheal development has three stages. First, the air sacs form near the optic lobe in 42-47% of pupal development (pd). Second, in 47-52% pd, air sacs extend branches along the base of the retina following a posterior-to-anterior direction and further form the tracheal network under the fenestrated membrane (TNUFM). Third, the TNUFM extend fine branches into the retina following a proximal-to-distal direction after 60% pd. Furthermore, we found that the trachea extension in both retina and TNUFM are dependent on the FGF(Bnl)/FGFR(Btl) signaling. Our results also provided strong evidence that the photoreceptors are the source of the Bnl ligand to guide the trachea ingrowth. Our work is the first systematic study of the tracheal development in the visual system, and also the first study demonstrating the interactions of two well-studied systems: the eye and trachea.
CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.
“Candidatus Neoehrlichia mikurensis” was detected by PCR in 4.0% (34/841) of the rodents tested in this study. The 34 rodents represented nine species from seven regions of China. Phylogenetic analyses based on the partial groEL and nearly entire 16S rRNA gene sequences of the agent revealed genetic diversity, which was correlated with its geographic origins.
Xanthium stramarium (XAS) and Psoralea corylifolia (PSC), phototoxic oriental medicinal plants, has been used in traditional medicines in Asian countries.
The effects of highly purified XAS or PSC extract combined with ultraviolet A1 (UVA1) irradiation on cell proliferation and transforming growth factor-beta1 (TGF-β1) expression of the keloid fibroblast were being investigated to define potential therapeutic uses for keloid treatments.
The keloid fibroblasts were treated with XAS or PSC alone or in the combination with UVA1 irradiation. The cell viability, apoptosis, and expression of TGF-β1 and collagen I were investigated.
XAS and PSC in combination with UVA1 irradiation suppressed cell proliferation and induced apoptosis of keloid fibroblasts. Furthermore, the XAS and PSC in combination with UVA1 irradiation inhibited TGF-β1 expression and collagen synthesis in keloid fibroblasts.
These findings may open up the possibility of clinically used XAS or PSC in combination with UVA1 irradiation for keloid treatments.
Apoptosis; Keloid; Psoralea corylifolia; Transforming growth factor-beta 1; Xanthium stramarium
The voltage-current (V-I) characteristics of tungsten (W) strips patterned by focused-ion-beam assisted deposition were studied at different temperatures and various magnetic fields applied perpendicular to the strips. At fields of 1 Tesla (T) and 2 T, a scaling behavior was observed in V-I isotherms, which can be fitted by quasi-three-dimensional (quasi-3D) vortex-glass (VG) to liquid transition theory.
To analyze proteomic and signal transduction alterations in irradiated melanoma cells.
We combined stable isotope labeling with amino acids in cell culture (SILAC) with highly sensitive shotgun tandem mass spectrometry (MS) to create an efficient approach for protein quantification. Protein-protein interaction was used to analyze relationships among proteins.
Energy metabolism protein levels were significantly different in glycolysis and not significantly different in oxidative phosphorylation after irradiation. Conversely, tumor suppressor proteins related to cell growth and development were downregulated, and those related to cell death and cell cycle were upregulated in irradiated cells.
Our results indicate that irradiation induces differential expression of the 29 identified proteins closely related to cell survival, cell cycle arrest, and growth inhibition. The data may provide new insights into the pathogenesis of uveal melanoma and guide appropriate radiotherapy.
melanoma cell; 2D-LC-MS/MS; stable isotope labeling with amino acids; proteomic analysis; X-ray irradiation; protein-protein interaction
Aspergillus biofilms were prepared from 22 strains of Aspergillus spp. via a 96-well plate-based method. Using a broth microdilution checkerboard technique with the XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] colorimetric assay, we demonstrated a synergistic antifungal activity against 18 of 22 Aspergillus biofilm strains with a combination of caspofungin and amphotericin B and against 13 of 22 strains with a combination of caspofungin and voriconazole. We did not observe antagonism.
Herbal medicine has long been viewed as a valuable asset for potential new drug discovery and herbal ingredients’ metabolites, especially the in vivo metabolites were often found to gain better pharmacological, pharmacokinetic and even better safety profiles compared to their parent compounds. However, these herbal metabolite information is still scattered and waiting to be collected.
HIM database manually collected so far the most comprehensive available in-vivo metabolism information for herbal active ingredients, as well as their corresponding bioactivity, organs and/or tissues distribution, toxicity, ADME and the clinical research profile. Currently HIM contains 361 ingredients and 1104 corresponding in-vivo metabolites from 673 reputable herbs. Tools of structural similarity, substructure search and Lipinski’s Rule of Five are also provided. Various links were made to PubChem, PubMed, TCM-ID (Traditional Chinese Medicine Information database) and HIT (Herbal ingredients’ targets databases).
A curated database HIM is set up for the in vivo metabolites information of the active ingredients for Chinese herbs, together with their corresponding bioactivity, toxicity and ADME profile. HIM is freely accessible to academic researchers at http://www.bioinformatics.org.cn/.
TCM; In vivo; Metabolism; Metabolite; Biotransformation; Structure search
De qi is a core concept of acupuncture and is necessary to produce therapeutic effect. In 2010, de qi has been received as a term in the official extension of the CONSORT Statement. However, there are few articles that discuss which factors have influences on obtaining de qi in clinical trials. This paper aims to explore these factors and give advice on trial design in order to optimize de qi in acupuncture RCTs.
The aim of this study was to evaluate the procedure for and efficacy of endoscopic subcutaneous mastectomy for gynecomastia. Endoscopic subcutaneous mastectomy was performed on 100 benign, palpable breast enlargements in 58 male patients who were followed-up for 15–63 months. The surgery was conducted with the insufflation of CO2 subdermally. No cases were converted to open surgery. The unilateral surgery time was 70–90 min. The mean volume of the resected tissue was 200 ml. All procedures were completed successfully, with satisfactory clinical effects and ideal esthetic results postoperatively. There were three cases (3%) of papillary epidermal partial necrosis; following removal of the dressing during the hospital stay, normal nipple sensation returned. Endoscopic subcutaneous mastectomy had good clinical effects and ideal cosmetic results and is an appropriate approach for gynecomastia.
gynecomastia; endoscopic; mastectomy
The tetraspanin CD151 acts as a promoter of metastasis and invasion in several tumors. However, the role of CD151 in human gastric cancer (HGC) remains unclear.
Twenty HGC specimens and matched nontumor samples, human gastric epithelial cells (HGEC), and four gastric cancer cell lines were used to analyze CD151 expression. Short hairpin RNA-mediated downregulation of CD151 expression in HGC cells was performed to examine the role of CD151 in the proliferation and metastasis/invasion of HGC cells in vivo and in vitro. The relationship of CD151 with integrin α3 in HGC cells was investigated by silencing integrin α3 followed by co-immunoprecipitation and immunofluorescence staining. Finally, the prognostic value of CD151 and integrin α3 was evaluated by immunohistochemistry in tissue microarrays of 76 HGC patients.
CD151 was expressed at higher levels in HGC tissues and HGC cells than in nontumor tissues and HGEC cells. Down-regulation of CD151 by vshRNA-CD151 impaired metastasis and invasion of HGC-27 cells, but did not affect cell proliferation. CD151 formed a complex with integrin α3 in HGC cells. CD151-cDNA transfection rescued the metastatic potential and invasiveness of HGC-27-vshCD151 cells, but not those of HGC-27-vshintegrin α3 cells in vitro. Clinically, CD151 overexpression was significantly correlated with high TNM stage, depth of invasion and positive lymph node involvement (p<0.05), and high levels of integrin α3 were associated with large tumor size, high TNM stage, depth of invasion and lymph node involvement (p<0.05). Importantly, the postoperative 5-year overall survival of patients with CD151low and/or integrin α3low was higher than that of patients with CD151high and/or integrin α3high.
CD151 is positively associated with the invasiveness of HGC, and CD151 or the combination of CD151 and integrin α3 is a novel marker for predicting the prognosis of HGC patients and may be potential therapeutic targets.
The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.
Hearing loss is the most prevalent sensory perception deficit in humans, affecting 1/500 newborns, can be syndromic or nonsyndromic and is genetically heterogeneous. Nearly 80% of inherited nonsyndromic bilateral sensorineural hearing loss (NBSNHI) is autosomal recessive. Although many causal genes have been identified, most are minor contributors, except for GJB2, which accounts for nearly 50% of all recessive cases of severe to profound congenital NBSNHI in some populations. More than 60% of children with a NBSNHI do not have an identifiable genetic cause. To identify genetic contributors, we genotyped 659 GJB2 mutation negative pediatric probands with NBSNHI and assayed for copy number variants (CNVs). After identifying 8 mild-moderate NBSNHI probands with a Chr15q15.3 deletion encompassing the Stereocilin (STRC) gene amongst this cohort, sequencing of STRC was undertaken in these probands as well as 50 probands and 14 siblings with mild-moderate NBSNHI and 40 probands with moderately severe-profound NBSNHI who were GJB2 mutation negative. The existence of a STRC pseudogene that is 99.6% homologous to the STRC coding region has made the sequencing interpretation complicated. We identified 7/50 probands in the mild-moderate cohort to have biallelic alterations in STRC, not including the 8 previously identified deletions. We also identified 2/40 probands to have biallelic alterations in the moderately severe-profound NBSNHI cohort, notably no large deletions in combination with another variant were found in this cohort. The data suggest that STRC may be a common contributor to NBSNHI among GJB2 mutation negative probands, especially in those with mild to moderate hearing impairment.
Bilateral sensorineural hearing loss; SNHI; Chr15q15.3; Stereocilin; STRC; DFNB16; SNP genotyping array; copy number variation; CNV
Cross-linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy, including allergic rhinitis, asthma, anaphylaxis, and immediate-type hypersensitivity reactions. Previous studies have demonstrated that the K+ channel, KCa3.1, plays a critical role in IgE-stimulated Ca2+ entry and degranulation in both human and mouse mast cells. We now have shown that the class II phosphatidylinositol-3-kinase C2β (PI3KC2β) is necessary for FcεRI-stimulated activation of KCa3.1, Ca2+ influx, cytokine production, and degranulation of bone marrow-derived mast cells (BMMC). In addition, we found that the E3 ubiquitin ligase, tripartite motif containing protein 27 (TRIM27), negatively regulates FcεRI activation of KCa3.1 and downstream signaling by ubiquitinating and inhibiting PI3KC2β. TRIM27−/− mice are also more susceptible in vivo to acute anaphylaxis. These findings identify TRIM27 as an important negative regulator of mast cells in vivo and suggest that PI3KC2β is a potential new pharmacologic target to treat IgE-mediated disease.
Extracorporeal shockwave therapy (ESWT) shows chondroprotective effect in osteoarthritis of the rat knees. However, the ideal number of ESWT is unknown. This study investigated the effects of different numbers of ESWT in osteoarthritis of the knee in rats.
Forty-five male Sprague-Dawley rats were divided into five groups. Group I underwent sham arthrotomy without anterior cruciate ligament transection (ACLT) or medial meniscectomy (MM) and received no ESWT. Group II underwent ACLT + MM and received no ESWT. Group III underwent ACLT + MM, and received ESWT once a week for one treatment. Group IV underwent ACLT + MM and received ESWT twice a week for 2 treatments. Group V underwent ACLT + MM and received ESWT three times a week for 3 treatments. Each treatment consisted of 800 impulses of shockwave at 14 Kv to the medial tibia condyle. The evaluations included radiographs of the knee, histomorphological examination and immunohistochemical analysis at 12 weeks.
At 12 weeks, group II and V showed more radiographic arthritis than groups I, III and IV. On histomorphological examination, the Safranin O matrix staining in groups III and IV are significantly better than in groups II and V, and the Mankin scores in groups III and IV are less than groups II and V. Groups III and IV showed significant decreases of Mankin score and increase of Safranin O stain as compared to group I. Group V showed significant increases of Mankin score and a decrease of Safranin O stain as compared to group II. In articular cartilage, group II showed significant increase of MMP13 and decrease of collagen II as compared to group I. Groups III and IV showed significant decrease of MMP13 and increase of collagen II as compared to group I. Group V showed significant increase of MMP13 and decrease of collagen II as compared to group II. In subchondral bone, vWF, VEGF, BMP-2 and osteocalcin significantly decreased in groups II and V, but increased in groups III and IV relative to group I.
ESWT shows a number of treatment related chondroproctective effect in osteoarthritis of the knee in rats.
Shockwave; Number of treatment; Chondroprotective; Osteoarthritis; Knee; Rats
Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP-bound and inactive GDP-bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two-hybrid system. We found that Rab13 binds to the C-terminus of Endospanin-2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin-2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin-1, a close homolog of Endospanin-2. Rab13 and Endospanin-2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin-2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13–Endospanin-2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin-2 and Endospanin-1. The role of this interaction in cell physiology, however, remains to be elucidated.
▸ Rab13 and Rab8 both interact with Endospanin-2 and Endospanin-1. ▸ Rab13 and Rab8 binding to endospanins is specific; Rabs 7, 10, 11 and 32 do not bind. ▸ Rab13 binding to Endospanin-2 is nucleotide-dependent. ▸ Rab13 and Endospanin-2 colocalise in perinuclear vesicles and at the cell periphery.
Vesicle trafficking; Rab13; Rab effector; Protein interaction; Endospanin; Osteoclast; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GHR, growth hormone receptor; GST, glutathione-S-transferase; HA, human influenza hemagglutinin; MBP, maltose binding protein; OB-R, leptin receptor; VPS55, vacuolar protein sorting 55.
Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intra-genic, extra-genic and inter-genic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated non-coding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into the transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.
DNA methylation at proximal promoters facilitates lineage restriction by silencing cell type–specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such nonproximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.
Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity1. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state2,3. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance2. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.
Radiotherapy is used in locally advanced pancreatic cancers where it can improve survival in combination with gemcitabine. However, prognosis is still poor in this setting where more effective therapies remain needed. MLN4924 is an investigational small molecule currently in Phase I clinical trials. MLN4924 inhibits NAE (NEDD8 Activating Enzyme), a pivotal regulator of the E3 ubiquitin ligase SCF (SKP1, Cullins, and F-box protein), that has been implicated recently in DNA repair. In this study, we provide evidence that MLN4924 can be used as an effective radiosensitizer in pancreatic cancer. Specifically, MLN4924 (20–100 nM) effectively inhibited cullin neddylation and sensitized pancreatic cancer cells to ionizing radiation in vitro with a sensitivity enhancement ratio (SER) of ~1.5. Mechanistically, MLN4924 treatment stimulated an accumulation of several SCF substrates, including CDT1, WEE1 and NOXA, in parallel with an enhancement of radiation-induced DNA damage, aneuploidy, G2/M phase cell cycle arrest and apoptosis. RNAi-mediated knockdown of CDT1 and WEE1 partially abrogated MLN4924-induced aneuploidy, G2/M arrest, and radiosensization, indicating a causal effect. Further, MLN4924 was an effective radiosensitizer in mouse xenograft models of human pancreatic cancer. Our findings offer proof of concept for use of MLN4924 as a novel class of radiosensitizer for the treatment of pancreatic cancer.
NAE inhibitor; MLN4924; CRL/SCF E3 ubiquitin ligase; radiosensitization; DNA damage; pancreatic cancer cells
To examine the relationship between angiogenesis and lymphangigenesis in recurrent pterygia.
Tissues from 34 excised recurrent pterygia (including 12 Grade 1, 10 Grade 2, and 12 Grade 3) were involved in the study and tissues from 7 nasal epibulbar conjunctivae segments were used as controls. Sections from each pterygium were immunostained with CD31 and LYVE-1 monoclonal antibodies to evaluate lymphatic microvessel density (LMVD) and blood microvessel density (BMVD), and the relationship between LMVD and BMVD in the pterygium was examined.
There was a large number of CD31(+)LYVE-1(−) blood vessels but only a few CD31(+)LYVE-1(+) lymphatic vessels in grades 1 and 2 pterygium. However, lymphatic vessels were dramatically increased in grade 3 pterygium. LMVD correlated closely with BMVD in all pterygia, including grades 1, 2 and 3 peterygium patients (all P values <0.01). Although both the density of blood and lymphatic vessels increased in recurrent pterygia, lymphatic vessels developed much faster than blood vessels, especially in grade 3 pterygia.
There is a significant but not parallel relationship between angiogenesis and lymphangiogenesis in recurrent pterygium. The outgrowth of blood and lymphatic vessels provide evidence that immunological mechanism may play a role in the development and recurrence of pterygium.
angiogenesis; lymphangiogenesis; recurrent pterygium
SAG/RBX/ROC protein is an essential RING component of SCF E3 ubiquitin ligase. The role of SAG during embryogenesis remains unknown. We report here a critical role for SAG in controlling vascular and neural development by modulating RAS activity via promoting degradation of neurofibromatosis type 1 (NF1). Mice mutant for Sag died at embryonic day 11.5-12.5 with severe abnormalities in vascular and nervous system. Sag inactivation caused Nf1 accumulation and Ras inhibition, which blocks embryonic stem (ES) cells from undergoing endothelial differentiation and inhibits angiogenesis and proliferation in teratomas. Simultaneous Nf1 deletion fully rescues the differentiation defects in Sag−/− ES cells, and partially rescues vascular and neural defects in Sag−/− embryos, suggesting that the effects of Sag deletion may not be solely explained by Nf1 misregulation. Collectively, our study identifies NF1 as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase and establishes a ubiquitin-dependent regulatory mechanism for the NF1-RAS pathway during embryogenesis.
Apoptosis; Endothelial differentiation; mouse knockout; NF1-RAS; SAG-CUL1-FBXW7 E3 ubiquitin ligase; vascular and neural development
Binding of IP3 (inositol 1,4,5-trisphosphate) to the IP3-binding core (residues 224–604) of IP3Rs (IP3 receptors) initiates opening of these ubiquitous intracellular Ca2+ channels. The mechanisms are unresolved, but require conformational changes to pass through the suppressor domain (residues 1–223). A calmodulin-binding peptide derived from myosin light chain kinase uncouples these events. We identified a similar conserved 1-8-14 calmodulin-binding motif within the suppressor domain of IP3R1 and, using peptides and mutagenesis, we demonstrate that it is essential for IP3R activation, whether assessed by IP3-evoked Ca2+ release or patch-clamp recoding of nuclear IP3R. Mimetic peptides specifically inhibit activation of IP3R by uncoupling the IP3-binding core from the suppressor domain. Mutations of key hydrophobic residues within the endogenous 1-8-14 motif mimic the peptides. Our results show that an endogenous 1-8-14 motif mediates conformational changes that are essential for IP3R activation. The inhibitory effects of calmodulin and related proteins may result from disruption of this essential interaction.
1-8-14 motif; calcium signalling; calmodulin; inositol 1,4,5-trisphosphate receptor; myosin light chain kinase (MLCK); BCR, B-cell receptor; CaBP1, Ca2+-binding protein 1; CaM, calmodulin; CLM, cytosol-like medium; IP3, inositol 1,4,5-trisphosphate; IBC, IP3-binding core; IP3R, IP3 receptor; MLCK, myosin light chain kinase; NT, N-terminus; RyR, ryanodine receptor; SD, suppressor domain
HIV infection related to commercial sexual contact is a serious public health issue in China. The objectives of the present study are to explore the predictors of condom use among female sex workers (FSWs) in China and examine the relationship between Health Belief Model (HBM) constructs.
A cross-sectional study was conducted in two cities (Wuhan and Suizhou) in Hubei Province, China, between July 2009 and June 2010. A total of 427 FSWs were recruited through mediators from the ‘low-tier’ entertainment establishments. Data were obtained by self-administered questionnaires. Structural equation models were constructed to examine the association. We collected 363 valid questionnaires. Within the context of HBM, perceived severity of HIV mediated through perceived benefits of condom use had a weak effect on condom use (r = 0.07). Perceived benefits and perceived barriers were proximate determinants of condom use (r = 0.23 and r = −0.62, respectively). Self-efficacy had a direct effect on perceived severity, perceived benefits, and perceived barriers, which was indirectly associated with condom use behaviors (r = 0.36).
The HBM provides a useful framework for investigating predictors of condom use behaviors among FSWs. Future HIV prevention interventions should focus on increasing perceived benefits of condom use, reducing barriers to condoms use, and improving self-efficacy among FSWs.