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.     

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.     

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.     

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.     

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.     

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.     

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.     

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His₆-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.     

A newly identified 50 kDa protein, which is associated with phosphodiesterase 3B, is phosphorylated by insulin in rat adipocytes

Phosphodiesterase 3B (PDE3B), a major PDE isoform in adipocytes, plays a pivotal role in the anti-lipolytic action of insulin. Insulin phosphorylates and activates PDE3B in a phosphatidylinositol 3-kinase-dependent manner. We identified a new 50 kDa protein that is phosphorylated by insulin and is co-immunoprecipitated with PDE3B by anti-PDE3B antibodies in rat adipocytes. The insulin-induced phosphorylation of the 50 kDa protein was also detected in a cell free system against the N-terminal and the catalytic regions, which are more than 700 amino acids apart recognize the 50 kDa protein, suggesting that it is not a proteolytic product, but an associated protein with PDE3B. Phosphoamino acid analysis indicated that both serine and threonine residues in the 50 kDa protein were phosphorylated, but only serine residues in PDE3B were phosphorylated. Therefore, it appears likely that this is a new protein which is associated with PDE3B.     

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20 q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3′ end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3′ end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitiative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5′ flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.     

Identification of mouse MARVELD1 as a microtubule associated protein that inhibits cell cycle progression and migration

MARVEL domain-containing 1 (MARVELD1) is a newly identified nuclear protein; however its function has not been clear until now. Here, we report that mouse MARVELD1 (mMARVELD1), which is highly conserved between mice and humans, exhibits cell cycle-dependent cellular localization. In NIH3T3 cells, MARVELD1 was observed in the nucleus and at the perinuclear region during interphase, but was localized at the mitotic spindle and midbody at metaphase, and a significant fraction of mMARVELD1 translocated to the plasma membrane during anaphase. In addition, treatment of cells with colchicine, a microtubuledepolymerizing agent, resulted in translocation of mMARVELD1 to the plasma membrane, and association of mMARVELD1 and α-tubulin was confirmed by co-immunoprecipitation. Finally, overexpression of mMARVELD1 resulted in a remarkable inhibition of cell proliferation, G1-phase arrest, and reduced cell migration. These findings indicate that mMARVELD1 is a microtubule-associated protein that plays an important role in cell cycle progression and migration.     

EB病毒潜伏膜蛋白1的B细胞表位预测

目的 预测EB病毒潜伏膜蛋白1(Latent Membrane Protein 1,LMPl)的B细胞表位.方法 基于EB病毒基因组序列,采用DNAStar Lasergene软件包中的Protean软件,对LMP1的亲水性,表面可能性,抗原指数及其二级结构中的柔性区域进行分析,并结合吴玉章的抗原指数预测法预测其B细胞表位.结果 B细胞表位最有可能位于潜伏膜蛋白N端第356-358,2-19,249-314区段或其附近,而潜伏膜蛋白N端第185-223区段内或附近也可能存在B细胞表位.结论 用多参数预测EB病毒LMP1的B细胞表位,为鼻咽癌的筛查及抗肿瘤转移靶向治疗的分子免疫学研究奠定基础.     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as eta subunit of chaperonin containing T-complex protein 1.

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Phosphatidylserine translocation into brain mitochondria: Involvement of a fusogenic protein associated with mitochondrial membranes

Data reported in the literature indicate that lipid movement between intracellular organelles can occur through contacts and close physical association of membranes (Vance, J.E. 1990. J Biol Chem 265: 7248-7256). The advantage of this mechanism is that the direct interaction of membranes provides the translocation event without the involvement of lipid-transport systems. However, pre-requisite for the functioning of this machinery is the presence of protein factors controlling membrane association and fusion. In the present work we have found that liposomes fuse to mitochondria at acidic pH and that the pre-treatment of mitochondria with pronase inhibits the fusogenic activity. Mixing of ¹⁴C-phosphatilyserine (PS) labeled liposomes with mitochondria at pH 6.0 results in the translocation of ¹⁴C-PS into mitochondria and in its decarboxylation to¹⁴ C-phosphatidylethanolamine through the PS decarboxylase activity localized on the outer surface of the inner mitochondrial membrane. Incorporation of ¹⁴C-PS is inhibited by the pre-treatment of mitochondria with pronase or with EEDQ, a reagent for the derivatization of the protonated form of carboxylic groups. These results indicate the presence of a protein associated with mitochondria which is able to trigger the fusion of liposomes to the mitochondrial membrane. A partial purification of a mitochondrial fusogenic glycoprotein is described in this work. The activity of the fusogenic protein appears to be dependent on the extent of protonation of the residual carboxylic groups and is influenced by the glucidic moiety, as demonstrated by its interaction with Concanavalin A. The purifed protein is able to promote the recover of the¹⁴ C-PS import from liposomes to pronase-treated mitochondria. Therefore, the protein is candidate to be an essential component in the machinery for the mitochondrial import of PS. (Mol Cell Biochem 175: 71–80, 1997)