5‐Aminoimidazole‐4‐carboxamide ribonucleoside‐mediated adenosine monophosphate‐activated protein kinase activation induces protective innate responses in bacterial endophthalmitis

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate‐activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl‐CoA carboxylase is down‐regulated in the SA‐infected retina. Intravitreal administration of an AMPK activator, 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl‐CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA‐infected eyes by inhibiting NF‐kB and MAP kinases (p38 and JNK) signalling. The anti‐inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA‐infected microglia and bone marrow‐derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA‐induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow‐derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of low Ca2+ stress‐induced embryo apoptosis response genes in Arachis hypogaea by SSH‐associated library lift (SSHaLL)

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca²⁺ deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up‐regulated cell wall hydrolases and down‐regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down‐regulated under Ca²⁺‐deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8′‐hydroxylases, key enzymes for ABA catabolism, were up‐regulated by 21‐fold under Ca²⁺‐deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over‐expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca²⁺ deficiency‐induced embryo abortion via ABA‐mediated apoptosis. The results elucidated the mechanism of low Ca²⁺‐induced embryo abortion and described the method for other fields of study.     

Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal‐regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC‐3) cells by blocking cell‐cycle progression. The present aim was to identify cell‐cycle‐related genes regulated by OvUS in PC‐3 cells using the quantitative human cell‐cycle RT² Profiler? PCR array. Cells were cultured ±200 µg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell‐cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down‐regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G₁ (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G₁ (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down‐regulated the cell proliferation marker gene MKI67, which is absent in cells at G₀. Results showed that OvUS blocks cell‐cycle progression through upregulation of cell‐cycle checkpoint and arrest genes and down‐regulation of genes involved in cell‐cycle progression. J. Cell. Biochem. 107: 1182–1188, 2009. © 2009 Wiley‐Liss, Inc.     

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid‐gastrulation

Transgenic mice with a defined cell‐ or tissues‐specific expression of Cre‐recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre‐expression from Cdx1 regulatory elements. The expression of Cre‐recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre‐mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development. genesis 47:204–209, 2009. © 2009 Wiley‐Liss, Inc.     

Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

Salvianolic acid B‐induced microRNA‐152 inhibits liver fibrosis by attenuating DNMT1‐mediated Patched1 methylation

Epithelial‐mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial‐mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down‐regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA‐mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl₄‐treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha‐smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up‐regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B‐treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR‐152 in Sal B‐treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR‐152. Further studies showed that the miR‐152 inhibitor reversed Sal B‐mediated PTCH1 up‐regulation and DNMT1 down‐regulation. Collectively, miR‐152 induced by Sal B, contributed to DNMT1 down‐regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.     

Knockdown of IGF‐binding protein 7 inhibits transformation of the endometrial gland in an in vitro model

Uterine endometrial glands and their secretory products are critical for the implantation and survival of the peri‐implantation embryo, and for the establishment of uterine receptivity. We previously reported that insulin‐like growth factor binding protein 7 (IGFBP7) is abundantly expressed in uterine glandular epithelial cells during the secretory phase of the menstrual cycle. In the present study, we used a cultured glandular epithelial cell line of human (EM1) to investigate the significance of IGFBP7 in the function of endometrial glands. EM1 cells formed a mesh‐like structure on Matrigel, which was accompanied by elevated levels of intracellular cyclic AMP. However, these morphological changes were blocked by treatment with protein kinase A (PKA) inhibitor (H89). IGFBP7 knockdown using specific short interference RNA (siRNA) inhibited the formation of the mesh‐like structure on Matrigel. Cyclic AMP analogs, dibutyryl‐cAMP, and N⁶‐phenyl‐cAMP induced the expression of leukemia inhibitory factor (LIF) which is essential for the onset of implantation. Enhanced LIF expression was suppressed by IGFBP7 siRNA treatment. Western blot analysis revealed that IGFBP7 knockdown results in the aberrant, constitutive expression of the MAPK signaling pathway. These results suggest that IGFBP7 regulates morphological changes of glandular cells by interfering with the normal PKA and MAPK signaling pathways that are associated with the transformation and/or differentiation of endometrial glands. Mol. Reprod. Dev. 77: 265–272, 2010. © 2009 Wiley‐Liss, Inc.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Enantiopure cyclic O‐substituted phenylphosphonothioic acid: Synthesis and chirality‐recognition ability

As a new acidic selector (resolving agent), we synthesized an enantiopure O‐alkyl phenylphosphonothioic acid with a seven‐membered ring ((R)‐ 5 ), which was designed on the basis of the results for the enantioseparation of 1‐arylethylamine derivatives with acyclic O‐ethyl phenylphosphonothioic acid ( I ). The phosphonothioic acid (R)‐ 5 showed unique chirality‐recognition ability in the enantioseparation of 1‐naphthylethylamine derivatives, aliphatic secondary amines, and amino alcohols; the ability was complementary to that of I . The X‐ray crystallographic analyses of the less‐ and more‐soluble diastereomeric salts showed that hydrogen‐bonding networks in the salt crystals are 2₁‐column‐type with a single exception which is cluster‐type. In the cases of the 2₁‐column‐type crystals, stability of the crystals is firstly governed by hydrogen bonds to form a 2₁‐column and secondly determined by intra‐columnar T‐shaped CH/π interaction(s), intra‐columnar hydrogen bond(s), inter‐columnar van der Waals interaction and/or inter‐columnar T‐shaped CH/π interaction(s). In contrast, the cluster‐type salt crystal is stabilized by the assistance of inter‐cluster T‐shaped CH/π and van der Waals interactions. To realize still more numbers of intra‐ and inter‐columnar and ‐cluster T‐shaped CH/π interactions, the seven‐membered ring of (R)‐ 5 plays a considerable role. Chirality 23:438–448, 2011. © 2009 Wiley‐Liss, Inc.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3⁺ and CD49b⁺ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009. © 2009 Wiley‐Liss, Inc.