Differential expression of genes in fetal brain as a consequence of maternal protein deficiency and nematode infection

Maternal dietary protein deficiency and gastrointestinal nematode infection during early pregnancy have negative impacts on both maternal placental gene expression and fetal growth in the mouse. Here we used next-generation RNA sequencing to test our hypothesis that maternal protein deficiency and/or nematode infection also alter the expression of genes in the developing fetal brain. Outbred pregnant CD1 mice were used in a 2 × 2 design with two levels of dietary protein (24% versus 6%) and two levels of infection (repeated sham versus Heligmosomoides bakeri beginning at gestation day 5). Pregnant dams were euthanized on gestation day 18 to harvest the whole fetal brain. Four fetal brains from each treatment group were analyzed using RNA Hi-Seq sequencing and the differential expression of genes was determined by the edgeR package using NetworkAnalyst. In response to maternal H. bakeri infection, 96 genes (88 up-regulated and eight down-regulated) were differentially expressed in the fetal brain. Differentially expressed genes were involved in metabolic processes, developmental processes and the immune system according to the PANTHER classification system. Among the important biological functions identified, several up-regulated genes have known neurological functions including neuro-development (Gdf15, Ing4), neural differentiation (miRNA let-7), synaptic plasticity (via suppression of NF-κβ), neuro-inflammation (S100A8, S100A9) and glucose metabolism (Tnnt1, Atf3). However, in response to maternal protein deficiency, brain-specific serine protease (Prss22) was the only up-regulated gene and only one gene (Dynlt1 a) responded to the interaction of maternal nematode infection and protein deficiency. In conclusion, maternal exposure to GI nematode infection from day 5 to 18 of pregnancy may influence developmental programming of the fetal brain.     

1H, 13C and 15N resonance assignments of domain 1 of receptor associated protein

The 39 kDa receptor associated protein (RAP) is a modular protein consisting of multiple domains. There has been no x-ray crystal structure of RAP available and the full-length protein does not behave well in a NMR tube. To elucidate the 3D structure of the RAP, we undertook structure determination of individual domains of the RAP. As the first step, here we report the nearly complete assignments of the ¹H, ¹³C and ¹⁵N chemical shift signals of domain 1 of the RAP.     

The LGI1/epitempin gene encodes two protein isoforms differentially expressed in human brain

The leucine-rich, glioma inactivated 1 (LGI1)/Epitempin gene has been linked to two phenotypes as different as gliomagenesis and autosomal dominant lateral temporal epilepsy. Its function and the biochemical features of the encoded protein are unknown. We characterized the LGI1/Epitempin protein product by western blot analysis of mouse and human brain tissues. Two proteins of about 60 and 65 kDa were detected by an anti-LGI1 antibody within the expected molecular mass range. The two proteins appeared to reside in different subcellular compartments, as they were fractionated by differential centrifugation. The specificity of both polypeptides was validated by cell transfection assay and mass spectrometry analysis. Immunoblot analysis of protein distribution in various zones of the human brain revealed variable amounts of both proteins. Notably, these proteins were more abundant in the temporal neocortex than in the hippocampus, the difference in abundance of the 65-kDa product being particularly pronounced. These results suggest that the two protein isoforms encoded by LGI1/Epitempin are differentially expressed in the human brain, and that higher expression levels of these proteins in the lateral temporal cortex may underlie the susceptibility of this brain region to the epileptogenic effects of LGI1/Epitempin mutations.     

Hamster contraception associated protein 1 (CAP1)

Based on cDNA and amino acid sequence, we demonstrate that hamster contraception associated protein 1 (CAP1) protein (an homolog of DJ-1 in mouse, CAP1/SP22/RS in rat and DJ-1/RS in human) is conserved during evolution. Through solubilization studies, it was demonstrated that hamster CAP1 has a peripheral membrane localization. SDS-PAGE analysis revealed that the migration pattern for hamster CAP1 compared to the other rodent counterparts, rat and mouse was different; indicating species-specific differences in the protein (possibly due to post-translational modifications). This protein also shows a ubiquitous presence in both somatic and germ tissues, and has been localized to the sperm tail. It was noticed that hamster CAP1 was lost from the mid piece of spermatozoa during capacitation. Interestingly, following in vitro treatment with ornidazole, CAP1 was lost from the spermatozoa and immunofluorescence studies showed that the major loss was from the mid piece of the spermatozoa. Another interesting feature highlighted about hamster CAP1 is its tendency to exist in two pI isoforms. Summarily, hamster CAP1 appears to exhibit species-specific differences compared to its rodent counterparts with respect to its unique peripheral localization, its size, two pI isoforms, and fate during capacitation, which may have implications in its functions.     

血清乙肝病毒大蛋白与乙肝前S1抗原联合检测在HBeAg阴性乙肝患者诊断中的意义

目的 探讨乙型肝炎e抗原阴性[HBeAg（－）]乙肝患者血清乙肝病毒大蛋白（HBV-LP）和乙肝前S1抗原（PreS1-Ag）联合检测的临床意义。方法 采用酶联免疫吸附（ELISA）法测定300例慢性乙肝患者的血清HBV-LP和PreS1-Ag浓度;实时荧光定量PCR（qRT-PCR）法检测血清HBV-DNA表达量;比较不同HBV-M模式下HBV-LP、PreS1-Ag与HBV-DNA的阳性检出率;分析以HBV-DNA表达量作为HBV感染及复制的金标准时,血清HBV-LP和PreS1-Ag单独检测及联合检测对HBeAg阴性乙肝患者的阳性预测值和阴性预测值。结果 （1）115例HBeAg（+）血清中,HBV-LP和PreS1-Ag的阳性率均与HBV-DNA阳性率差异无统计学意义（Ps＞0.05）;120例HBeAg（－）HBeAb（+）血清中,HBV-LP阳性率（64.2%）明显高于HBV-DNA阳性率（P＜0.05）,而PreS1-Ag阳性率与HBV-DNA阳性率差异无统计学意义（P＞0.05）;65例HBeAg（－）HBeAb（－）血清中,HBV-LP阳性率（72.3%）和PreS1-Ag阳性率（67.7%）均明显高于HBV-DNA阳性率（Ps＜0.05）;（2）以185例HBeAg（－）乙肝患者的HBV-DNA表达量为参考标准,HBV-LP、PreS1-Ag的阳性预测值分别为72.6%、71.6%,阴性预测值分别为93.4%、84.1%;HBV-LP和PreS1-Ag联合检测,HBV-LP/PreS1-Ag双阳性中的HBV-DNA阳性率（66.0%）显著高于HBV-LP/PreS1-Ag双阴性（P＜0.05）。结论 血清HBV-LP和PreS1-Ag水平与HBV-DNA表达量有关,二者联合检测可灵敏地反映HBeAg（－）乙肝患者HBV的复制状态,预测HBV-DNA水平。     

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.     

The S100B protein in biological fluids: more than a lifelong biomarker of brain distress

S100B is a calcium-binding protein concentrated in glial cells, although it has also been detected in definite extra-neural cell types. Its biological role is still debated. When secreted, S100B is believed to have paracrine/autocrine trophic effects at physiological concentrations, but toxic effects at higher concentrations. Elevated S100B levels in biological fluids (CSF, blood, urine, saliva, amniotic fluid) are thus regarded as a biomarker of pathological conditions, including perinatal brain distress, acute brain injury, brain tumors, neuroinflammatory/neurodegenerative disorders, psychiatric disorders. In the majority of these conditions, high S100B levels offer an indicator of cell damage when standard diagnostic procedures are still silent. The key question remains as to whether S100B is merely leaked from injured cells or is released in concomitance with both physiological and pathological conditions, participating at high concentrations in the events leading to cell injury. In this respect, S100B levels in biological fluids have been shown to increase in physiological conditions characterized by stressful physical and mental activity, suggesting that it may be physiologically regulated and raised during conditions of stress, with a putatively active role. This possibility makes this protein a candidate not only for a biomarker but also for a potential therapeutic target.     

Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐ras^G12C mutation) and PC9‐BrM3 (EGFR^Δexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐ras^G12C mutation) and PC9 (EGFR^Δexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.     

Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus

The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness.     

Benzoquinones from Ardisia japonica with inhibitory activity towards human protein tyrosine phosphatase 1B (PTP1B)

From the whole plant of Ardisia japonica, four [1,4]benzoquinones were isolated by means of bioassay-directed fractionation of the EtOH extract. Apart from the two known compounds maesanin (1) and its congener 2, two new benzoquinones, i.e., 5-ethoxy-2-hydroxy-3-[(10Z)-pentadec-10-en-1-yl][1,4]benzoquinone (3) and 5-ethoxy-2-hydroxy-3-[(8Z)-tridec-8-en-1-yl][1,4]benzoquinone (4), were identified. All compounds showed significant in vitro bioactivities against the PTP1B enzyme, with IC50 values in the range of ca. 3-19 microM.     

Evaluation of licorice flavonoids as protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator in insulin- and leptin-signaling cascades as well as a positive regulator in tumorigenesis, and much attention has been paid to PTP1B inhibitors as potential therapies for diabetes, obesity, and cancer. In the present study, the screening of a compound library of licorice flavonoids allowed for the discovery of several compounds, including licoagrone (3), licoagrodin (4), licoagroaurone (5), and isobavachalcone (6), as new PTP1B inhibitors. It was revealed that these compounds inhibit the activity of PTP1B in different modes and with different selectivities and that they exhibit different cellular activity in the insulin-signaling pathway. Glycybenzofuran (1), a competitive PTP1B inhibitor, showed both excellent inhibitory selectivity against PTP1B and cellular activity on the insulin-stimulated Akt phosphorylation level. The similarity of its action profiling in the insulin-signaling pathway suggested its potential as a new anti-insulin-resistant drug candidate.     

Microtubule-associated protein 1B: a neuronal binding partner for gigaxonin

Giant axonal neuropathy (GAN), an autosomal recessive disorder caused by mutations in GAN, is characterized cytopathologically by cytoskeletal abnormality. Based on its sequence, gigaxonin contains an NH2-terminal BTB domain followed by six kelch repeats, which are believed to be important for protein-protein interactions (Adams, J., R. Kelso, and L. Cooley. 2000. Trends Cell Biol. 10:17-24.). Here, we report the identification of a neuronal binding partner of gigaxonin. Results obtained from yeast two-hybrid screening, cotransfections, and coimmunoprecipitations demonstrate that gigaxonin binds directly to microtubule-associated protein (MAP)1B light chain (LC; MAP1B-LC), a protein involved in maintaining the integrity of cytoskeletal structures and promoting neuronal stability. Studies using double immunofluorescent microscopy and ultrastructural analysis revealed physiological colocalization of gigaxonin with MAP1B in neurons. Furthermore, in transfected cells the specific interaction of gigaxonin with MAP1B is shown to enhance the microtubule stability required for axonal transport over long distance. At least two different mutations identified in GAN patients (Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. Nat. Genet. 26:370-374.) lead to loss of gigaxonin-MAP1B-LC interaction. The devastating axonal degeneration and neuronal death found in GAN patients point to the importance of gigaxonin for neuronal survival. Our findings may provide important insights into the pathogenesis of neurodegenerative disorders related to cytoskeletal abnormalities.     

Anti-human placental antigen complex X-P2 (hPAX-P2) anti-serum recognizes C-terminus of huntingtin-associated protein 1A common to 1B as a determinant marker for the stigmoid body

The anti-serum against an unknown human placental antigen complex X-P2 (hPAX-P2) immunohistochemically recognizes three putative molecules (hPAX-P2S, hPAX-P2N, and hPAX-P2R), each of which is associated with the stigmoid bodies (STBs), necklace olfactory glomeruli (NOGs), or reticulo-filamentous structures (RFs) in the rat brain. The STBs also contain huntingtin-associated protein 1 (HAP1), and the HAP1-cDNA transfection induces STB-like inclusions in cultured cells. In order to clarify the relationship between hPAX-P2S and HAP1 isoforms (A/B), we performed Western blotting, immuno-histo/cytochemistry for light- and electron-microscopy and pre-adsorption tests with HAP1 deletion fragments. The results showed that the anti-hPAX-P2 anti-serum recognizes HAP1^474–577 of HAP1A/B in Western blotting and strongly immunostains HAP1A-induced STB-like inclusions but far weakly detects HAP1B-induced diffuse structures in HAP1-transfected HEK 293 cells. In the rat brain, immunoreactivity of the anti-hPAX-P2 anti-serum for the STBs was eliminated by pre-adsorption with HAP1^474–577, whereas no pre-adsorption with any different HAP1 fragments can suppress immunoreactivity for the NOGs and RFs, which were not immunoreactive to anti-HAP1 anti-serum. These findings indicate that hPAX-P2S, which is distinct from hPAX-P2N and hPAX-P2R, is identical with STB-constituted HAP1 and that the HAP1-induced/immunoreactive inclusions correspond to the hPAX-P2-immunoreactive STBs previously identified in the brain.     

Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.     

Epitope mapping of a single repetitive unit of the B13 Trypanosoma cruzi antigen as fusions to Escherichia coli LamB protein

B13, one of the immunodominant antigens of Trypanosoma cruzi, is composed of repeats of a 12-amino-acid motif. Using synthetic peptides, the sequence FGQAAAGDK was previously shown to contain the B13 immunodominant epitope recognized by chagasic patients sera. To investigate the effects of neighboring sequences in the immunodominance, we tested serum recognition of two B13 sequences fused to LamB. GDKPSPFGQAAA-LamB and FGQAAAGDKPSP-LamB were recognized, respectively, by 15% and 80% of 80 sera reactive to B13 antigen. Recognition of FGQAAAGDKPSP-LamB was inhibited by AAAGDK-containing synthetic peptides. FGQAAAGDKPSP-LamB competed with a B13 recombinant protein containing 16.6 repeats for binding to chagasic antibodies. These results strengthen previous conclusions on the immunodominant epitope of B13 and provide a comparison of two methods for epitope mapping.     

Dibenzocyclooctadiene lignans: a class of novel inhibitors of multidrug resistance-associated protein 1

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.