Multiple Human Tissues Research Articles

Identifying Candidate Genes for Type 2 Diabetes Mellitus and Obesity through Gene Expression Profiling in Multiple Tissues or Cells

Type 2 Diabetes Mellitus (T2DM) and obesity have become increasingly prevalent in recent years. Recent studies have focused on identifying causal variations or candidate genes for obesity and T2DM via analysis of expression quantitative trait loci (eQTL) within a single tissue. T2DM and obesity are affected by comprehensive sets of genes in multiple tissues. In the current study, gene expression levels in multiple human tissues from GEO datasets were analyzed, and 21 candidate genes displaying high percentages of differential expression were filtered out. Specifically, DENND1B, LYN, MRPL30, POC1B, PRKCB, RP4-655J12.3, HIBADH, and TMBIM4 were identified from the T2DM-control study, and BCAT1, BMP2K, CSRNP2, MYNN, NCKAP5L, SAP30BP, SLC35B4, SP1, BAP1, GRB14, HSP90AB1, ITGA5, and TOMM5 were identified from the obesity-control study. The majority of these genes are known to be involved in T2DM and obesity. Therefore, analysis of gene expression in various tissues using GEO datasets may be an effective and feasible method to determine novel or causal genes associated with T2DM and obesity.

Detection of a common chimeric transcript between human chromosomes 7 and 16

Interchromosomal chimeric RNA molecules are often transcription products from genomic rearrangement in cancerous cells. Here we report the computational detection of an interchromosomal RNA fusion between ZC3HAV1L and CHMP1A from RNA-seq data of normal human mammary epithelial cells, and experimental confirmation of the chimeric transcript in multiple human cells and tissues. Our experimental characterization also detected three variants of the ZC3HAV1L-CHMP1A chimeric RNA, suggesting that these genes are involved in complex splicing. The fusion sequence at the novel exon-exon boundary, and the absence of corresponding DNA rearrangement suggest that this chimeric RNA is likely produced by trans-splicing in human cells.ReviewersThis article was reviewed by Rory Johnson (nominated by Fyodor Kondrashov); Gal Avital and Itai Yanai

TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy.

Exon 2 of the TFPI Gene Is a Translational Repressor of TFPIβ Protein Expression

AbstractAbstract 3358 Background:Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein located on endothelium and within platelets. Two TFPI isoforms, termed TFPIα and TFPIβ, are generated through alternative splicing toward the 3’ end of the TFPI gene. Alternative splicing also occurs within the 5’ untranslated region (UTR) of TFPI mRNA, resulting in the removal of exon 2 (X2). Previous studies have demonstrated that TFPIα and TFPIβ mRNA are produced, on average, in a ratio of 10:1 (TFPIα:TFPIβ) in human tissues. However, this does not appear to correlate with protein expression, suggesting that TFPI protein isoform production is regulated during translation. Biochemical studies were undertaken to determine how alternative splicing within the 5’ or 3’ UTRs of TFPI mRNA may function in the translational regulation of TFPI protein isoform production. Methods:Northern analysis of human lung RNA using probes directed toward Exon 1 (X1) or Exon 2 (both 5’ UTR), Exon 6 (common to both TFPIα and TFPIβ), Exon 8 (TFPIβ specific) or Exons 9 and 10 (TFPIα specific), and RT-PCR analysis of multiple human tissue cDNAs using an exon 1 specific forward primer and TFPIα or TFPIβ specific reverse primers, were used to investigate isoform specific alternative splicing of exon 2. Polysome profiling of HUVEC and EA.hy926 cellular RNA was performed to investigate translational control of TFPI isoform expression. Luciferase (Luc) reporter assays were established to quantify the effect of exon 2 on TFPI isoform protein expression. Constructs were produced where both splice variants of the 5’ UTR, and the TFPIα or TFPIβ specific 3’ UTRs, were inserted at their respective ends of a Gaussia Luc expression cassette. Luc activity was assessed in stably transfected CHO or EA.hy926 cells and normalized to GFP production or Luc mRNA. Results:Northern analysis revealed the presence of two TFPIα specific bands at 4.1 and 1.4 kb, produced via alternative polyadenylation, and one TFPIβ specific band at 1.1 kb. Exon 1 and 2 probes bind both TFPIα species, demonstrating that 5’ UTR alternative splicing occurs in TFPIα mRNA. RT-PCR analysis of human placenta, lung, and heart cDNA confirmed this, and demonstrated that exon 2 is also alternatively spliced in TFPIβ message. Polysome analysis identified TFPIβ-specific translational repression by exon 2. Luciferase reporter assays quantified this repression at 93% and 86% in CHO and EA.hy926 cells, respectively (see Table 1 for normalized data). In contrast, exon 2 had minimal effect on luciferase expression when the TFPIα 3’ UTR was present. RT-PCR analysis of 15 human tissues revealed that testis, thymus, brain, and spleen had the highest relative amount of exon 2 containing TFPIβ message, although all tissues had more exon 2-absent TFPIβ message compared to that containing exon 2. Conclusion:Exon 2 is a strong translational repressor of TFPIβ production, and a much weaker repressor of TFPIα. To our knowledge, this is the first time that alternative splicing within a 5’ UTR has been demonstrated to affect a specific protein isoform produced via alternative splicing at the 3’ end of the same gene. While the mechanism of repression has yet to be elucidated, the repression of TFPIβ, and not TFPIα, by exon 2 suggests that regions within both exon 2 and the 3’ UTR are required for this effect and is consistent with the hypothesis of mRNA circularization via interactions between the 5’ and 3’ UTRs. We hypothesize that the physiological relevance of the demonstrated repression will include tissue-specific regulation of TFPIβ expression and cellular regulation of TFPIβ expression in inflammatory disease.Table 1Normalised Luciferase activities determined via luciferase reporter assayCell LineMean Normalised Luc Activity [CPS/(RFU/μg protein)]Mean Normalised Luc Activity (CPS/Relative Luc mRNA)X1-Luc-TFPIβX1+X2-Luc-TFPIβX1-Luc-TFPIαX1+X2-Luc-TFPIαCHO94.96.3**426.0331.5#EA.hy926194.826.3*39316.536450.5#**- p < 0.0001 vs X1-Luc-TFPIβ;*- p < 0.01 vs X1-Luc-TFPIβ;#- p < 0.01 vs X1-Luc-TFPIα. Disclosures:Mast:Novo Nordisk A/S: Research Funding.

Feature Selection and Clustering of Gene Expression Profiles Using Biological Knowledge

In this paper, a novel feature selection algorithm, which is governed by biological knowledge, is developed. Gene expression data being high dimensional and redundant, dimensionality reduction is of prime concern. We employ the algorithm clustering large applications based on RAN-domized search (CLARANS) for attribute clustering and dimensionality reduction based on gene ontology (GO) study. Feature selection with unsupervised learning is a difficult problem, with neither class labels present nor any guidance available to the search. Determination of the optimal number of clusters is another major issue, and has an impact on the resulting output. The use of GO analysis helps in the automated selection of biologically meaningful partitions. Tools such as Eisen plot and cluster profiles of these clusters help establish their coherence. Important representative features (or genes) are extracted from each correlated set of genes in such partitions. The algorithm is implemented on high-dimensional Yeast cell-cycle, Human Multiple Tissues, and Leukemia microarray data. In the second pass, clustering on the reduced gene space validates preservation of the inherent behavior of the original high-dimensional expression profiles. While the reduced gene set forms a biologically meaningful gene space, it simultaneously leads to a decrease in computational burden. External validation of the reduced subspace, using various well-known classifiers, establishes the effectiveness of the proposed methodology.

HORMAD2/CT46.2, a novel cancer/testis gene, is ectopically expressed in lung cancer tissues

Recently, it has been shown that ectopic expression of several germ line genes, such as piwi, vasa, nos and aub, drives the growth of malignant brain tumors in Drosophila. A genome-wide association study (GWAS) of ~18 000 subjects indicated that genetic variation at 22q12.2, the location of the conserved meiotic gene HORMAD2, may contribute to the risk of lung cancer in Han Chinese individuals. The expression pattern of human HORMAD2 in multiple human tissues was determined by RT-PCR. HORMAD2 protein expression and localization were analyzed in the human testis using immunohistochemistry. The expression of HORMAD2 in lung cancer and normal tissues was studied using quantitative RT-PCR and immunohistochemistry. HORMAD2 is predominantly expressed in human testis and weakly expressed in liver. HORMAD2 is ectopically expressed in nearly 10% of lung cancer samples from Chinese Han individuals. In conclusion, HORMAD2 is a novel cancer/testis gene discovered by GWAS, which may provide a new target for lung cancer research.

Developmental expression and function of DKKL1/Dkkl1 in humans and mice

BackgroundExperiments were designed to identify the developmental expression and function of the Dickkopf-Like1 (DKKL1/Dkkl1) gene in humans and mice.MethodsMouse testes cDNA samples were collected at multiple postnatal times (days 4, 9, 18, 35, and 54, as well as at 6 months) and hybridized to Affymetrix mouse whole genome Genechips. To further characterize the homologous gene DKKL1 in human beings, the expression profiles between human adult testis and foetal testis were compared using Affymetrix human Genechips. The characteristics of DKKL1/Dkkl1 were analysed using various cellular and molecular biotechnologies.ResultsThe expression of Dkkl1 was not detected in mouse testes on days 4 or 9, but was present on days 18, 35, and 54, as well as at 6 months, which was confirmed by RT-PCR and Western blot results. Examination of the tissue distribution of Dkkl1 demonstrated that while Dkkl1 mRNA was abundantly expressed in testes, little to no expression of Dkkl1 was observed in the epididymis or other tissues. In an in vitro fertilization assay, a Dkkl1 antibody was found to significantly reduce fertilization. Human Genechips results showed that the hybridization signal intensity of DKKL1 was 405.56-fold higher in adult testis than in foetal testis. RT-PCR analysis of multiple human tissues indicated that DKKL1 mRNA was exclusively expressed in the testis. Western blot analysis also demonstrated that DKKL1 was mainly expressed in human testis with a molecular weight of approximately 34 kDa. Additionally, immunohistochemical staining showed that the DKKL1 protein was predominantly located in spermatocytes and round spermatids in human testes. An examination of the expression levels of DKKL1 in infertile male patients revealed that while no DKKL1 appeared in the testes of patients with Sertoli cell only syndrome (SCOS) or cryptorchidism, DKKL1 was observed with variable expression in patients with spermatogenic arrest.ConclusionsThese results, together with previous studies, suggest that DKKL1/Dkkl1 may play an important role in testicular development and spermatogenesis and may be an important factor in male infertility.

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3Î"TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Optical Spectroscopy: Current Advances and Future Applications in Cancer Diagnostics and Therapy

Optical spectroscopy (OS) is a tissue-sensing technique that could enhance cancer diagnosis and treatment in the near future. With OS, tissue is illuminated with a selected light spectrum. Different tissue types can be distinguished from each other based on specific changes in the reflected light spectrum that are a result of differences on a molecular level between compared tissues. Therefore, OS has the potential to become an important optical tool for cancer diagnosis and treatment monitoring. In recent years, significant progress has been made in the discriminating abilities of OS techniques between normal and cancer tissues of multiple human tissue types. This article provides an overview of the advances made with diffuse reflectance, fluorescence and Raman spectroscopy techniques in the field of clinical oncology, and focuses on the different clinical applications that OS could enhance.

Array comparative genomic hybridization in male infertility

Male infertility caused by a maturation arrest of spermatogenesis is a condition with an abrupt stop in spermatogenesis, mostly at the level of primary spermatocytes. The etiology remains largely unknown. We focused on patients with a complete arrest at the spermatocyte level (n = 9) and used array comparative genomic hybridization to screen for deletions or duplications that might be associated with maturation arrest. Interesting copy number variations (CNVs) were further examined by using quantitative PCR. Where appropriate, the expression pattern was analyzed in multiple human tissues including the testis. A total of 227 CNVs were detected in the patient group. After the elimination of CNVs that were also present in the control group or that were not likely to be involved in male infertility, the remaining 11 regions were investigated more in detail. We first determined the expression pattern of seven genes, for which expression had not been reported to be investigated in testicular tissue, after which one region could be eliminated. Next, all 10 promising candidate regions were analyzed by quantitative PCR in a control population. Eight deletions/duplications were absent in our control group, and therefore might be linked with the male infertility in our patients. One of these alterations, however, has been detected in a proven fertile father group. Further research is necessary to determine the relationship between the observed genomic alterations and maturation arrest of spermatogenesis. Furthermore, several of the above genes have not been studied at the functional level and consequently, more research is required to determine their role in spermatogenesis.

Aging effects on DNA methylation modules in human brain and blood tissue

BackgroundSeveral recent studies reported aging effects on DNA methylation levels of individual CpG dinucleotides. But it is not yet known whether aging-related consensus modules, in the form of clusters of correlated CpG markers, can be found that are present in multiple human tissues. Such a module could facilitate the understanding of aging effects on multiple tissues.ResultsWe therefore employed weighted correlation network analysis of 2,442 Illumina DNA methylation arrays from brain and blood tissues, which enabled the identification of an age-related co-methylation module. Module preservation analysis confirmed that this module can also be found in diverse independent data sets. Biological evaluation showed that module membership is associated with Polycomb group target occupancy counts, CpG island status and autosomal chromosome location. Functional enrichment analysis revealed that the aging-related consensus module comprises genes that are involved in nervous system development, neuron differentiation and neurogenesis, and that it contains promoter CpGs of genes known to be down-regulated in early Alzheimer's disease. A comparison with a standard, non-module based meta-analysis revealed that selecting CpGs based on module membership leads to significantly increased gene ontology enrichment, thus demonstrating that studying aging effects via consensus network analysis enhances the biological insights gained.ConclusionsOverall, our analysis revealed a robustly defined age-related co-methylation module that is present in multiple human tissues, including blood and brain. We conclude that blood is a promising surrogate for brain tissue when studying the effects of age on DNA methylation profiles.

P2-04-02: Identification of a Novel Glycosyltransferase-Like Gene as an Autophagic Inducer in Human Mammary Carcinoma Cells Via Down Regulation of BCL-2.

Abstract Here we report the identification of a previously undescribed glycosyltransferase-like gene, GLTx, and its cellular effect on mammary carcinoma cells. GLTx codes for a novel human protein which is highly conserved in primates. Data obtained from the mRNA expression database Oncomine, demonstrate that GLTx is differentially expressed in a variety of cancers including breast cancer. Western blot analysis of a multiple human tissue blot using a rabbit polyclonal antibody against GLTx demonstrated that GLTx was highly expressed in liver, moderately in kidney, intestine and stomach and undetectable in any other tissues. To functionally characterize GLTx, we attempted to establish stable cell lines with forced expression. However, forced expression of GLTx was completely lethal and resulted in severe cell death in the mammary carcinoma cell lines, MCF-7 and BT549, as well as in any other cell lines tested. Staining of MCF-7 cells transiently transfected with a GLTx expression plasmid by either Hoechst 33258 or fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that the observed cell death was not caused by apoptosis. Further investigation confirmed that GLTx expressing cells exhibited cytoplasmic accumulation of autophagosomes, consistent with the induction of autophagy, as measured by the fluorescently labelled autophagosome marker LC3. In addition we have shown that GLTx downregulated BCL-2, an inhibitor of autophagy, as measured by BCL-2 gene promoter luciferase activity assay and western blot assay following transient transfection of a GLTx expressing plasmid. Thus, we have demonstrated that GLTx is a novel autophagic inducer that causes cell death by down regulating BCL-2. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-04-02.

CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT)

Recently, we identified a microduplication in chromosomal band 1q21.1 encompassing the CHD1L/ALC1 gene encoding a chromatin-remodelling enzyme in congenital anomalies of the kidneys and urinary tract (CAKUT) patient. To explore the role of CHD1L in CAKUT, we screened 85 CAKUT patients for mutations in the CHD1L gene and performed functional analyses of the three heterozygous missense variants detected. In addition, we quantitatively determined CHD1L expression in multiple human fetal and adult tissues and analysed expression of CHD1L protein in human embryonal, adult and hydronephrotic kidney sections. Two of three novel heterozygous missense variants identified in three patients were not found in >400 control chromosomes. All variants lead to amino acid substitutions in or near the CHD1L macro domain, a poly-ADP-ribose (PAR)-binding module interacting with PAR polymerase 1 (PARP1), and showed decreased interaction with PARP1 by pull-down assay of transfected cell lysates. Quantitative messenger RNA analysis demonstrated high CHD1L expression in human fetal kidneys, and levels were four times higher than in adult kidneys. In the human embryo at 7-11 weeks gestation, CHD1L immunolocalized in the early ureteric bud and the S- and comma-shaped bodies, critical stages of kidney development. In normal postnatal sections, CHD1L was expressed in the cytoplasm of tubular cells in all tubule segments. CHD1L expression appeared higher in the hydronephrotic kidney of one patient with a hypofunctional CHD1L variant than in normal kidneys, recapitulating high fetal levels. Our data suggest that CHD1L plays a role in kidney development and may be a new candidate gene for CAKUT.

Immunophilins and Cardiovascular Complications

Immunophilins belong to a highly conserved family of proteins with cis-trans peptidyl-prolyl isomerase activity, generally classified by their ability to selectively bind specific immunosuppressive drugs, thereby regulating their activity. Immunophilins include Cyclophilins (CyPs), which are specific targets of the immunosuppressant drug cyclosporin A (CsA); FKBPs (FK506-binding proteins), that are sensitive to both FK506 (tacrolimus) and rapamycin (sirolimus); and FCBPs which are sensitive to CsA and FK506. Immunophilins are expressed in multiple human tissues, including brain, heart, kidney, liver and lung and regulate functions as diverse as intracellular calcium signaling, protein transport, protein folding and gene transcription. In particular, immunophilins play key functional roles in the cardiovascular system, where they can associate with proteins such as ryanodine and IP3 receptors (RyR and IP3R), calcineurin, and mitochondrial permeability transition pore (MPTP) and Heat-shock proteins-caveolin-cholesterol complex and regulate their function. The biological importance of immunophilins is further revealed by the pathophysiology, as they have been implicated in several cardiovascular diseases, including vascular stenosis, atherosclerosis, heart failure and arrhythmias. This review summarizes some of the most recent studies on immunophilins and focuses on their roles in the mechanisms underlying the cardiovascular disease.

Tissue-specific gene expression templates for accurate molecular characterization of the normal physiological states of multiple human tissues with implication in development and cancer studies

BackgroundTo elucidate the molecular complications in many complex diseases, we argue for the priority to construct a model representing the normal physiological state of a cell/tissue.ResultsBy analyzing three independent microarray datasets on normal human tissues, we established a quantitative molecular model GET, which consists of 24 tissue-specific Gene Expression Templates constructed from a set of 56 genes, for predicting 24 distinct tissue types under disease-free condition. 99.2% correctness was reached when a large-scale validation was performed on 61 new datasets to test the tissue-prediction power of GET. Network analysis based on molecular interactions suggests a potential role of these 56 genes in tissue differentiation and carcinogenesis.Applying GET to transcriptomic datasets produced from tissue development studies the results correlated well with developmental stages. Cancerous tissues and cell lines yielded significantly lower correlation with GET than the normal tissues. GET distinguished melanoma from normal skin tissue or benign skin tumor with 96% sensitivity and 89% specificity.ConclusionsThese results strongly suggest that a normal tissue or cell may uphold its normal functioning and morphology by maintaining specific chemical stoichiometry among genes. The state of stoichiometry can be depicted by a compact set of representative genes such as the 56 genes obtained here. A significant deviation from normal stoichiometry may result in malfunction or abnormal growth of the cells.

Remarkable Expression in the Colon Adenocarcinoma ofHmat-Xa, a Human Mannosyltransferase-Like Gene, That Is Homologous to Drosophila Gene GC15914

We cloned a novel human mannosyltransferase-like gene, designated Hmat-Xa, as a gene homologous to the Drosophila GC15914 gene encoding the 9QVXN0 protein: see "Project Report for FY2002 on the 'Construction of Libraries of Human Genes Participating in Glycosylation' project" 43-45 (2003), New Energy and Industrial Technology Development Organization (NEDO), NEDO and Research Association for Biotechnology, Tokyo, Japan (in Japanese). After that, the GTDC1 gene, as reported by Zhao et al., DNA Cell Biol., 23, 183-187 (2004), was found to be the same as the Hmat-Xa gene. Domain EXFGI/L/VX(2)L/VE in the Hmat-Xa protein, also present in both human mannosyltransferase II/III and mannosyltransferase IV/V, which are involved in the synthesis of lipid-linked oligosaccharides, and some bacterial mannosyltransferases. A real-time PCR study of Hmat-Xa mRNA expression in human normal and tumor multiple tissue cDNA identified its tissue-specific expression and its remarkable expression in colon adenocarcinoma as compared to the normal counterpart. Thus the elevated expression of Hmat-Xa might serve as a candidate marker for colon adenocarcinoma.

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

Downregulation of chromobox protein homolog 7 expression in multiple human cancer tissues

To investigate the relationship between chromobox protein homolog 7 (cbx7) expression and the occurrence and development of colorectal carcinoma (CRC), gastric carcinoma (GC) and hepatocarcinoma (HCC) tissues. The samples of neoplastic tissues and the corresponding cutting-edge normal tissues from 22 cases of CRC, 20 cases of GC, 30 cases of HCC were surgically collected. Level of cbx7 mRNA was detected with a fluorescent quantitative RT-PCR assay, and the correlationship among expression of cbx7 mRNA, the patients' clinicopathologic features and the surviving time after surgery was analyzed. The relative copy number of cbx7 mRNA in carcinomas and the normal tissues was 0.010 ± 0.015 vs 0.053 ± 0.042 for CRCs, 0.197 ± 0.195 vs 1.891 ± 1.254 for GCs, and 0.008 ± 0.008 vs 0.030 ± 0.021 for HCCs, respectively. Compared with the corresponding normal tissues, cbx7 expression was significantly downregulated in CRCs, GCs, and HCCs (t = -7.351, -5.417 and -6.680, respectively, P < 0.01). The expression of cbx7 mRNA in CRCs had significant differences not only between two age groups (the relative copy number of cbx7 mRNA in age > 55 group was 0.007 ± 0.015, but 0.017 ± 0.012 in age ≤ 55 group, t = -2.586, P = 0.022); but also between vascular embolus-positive and negative groups (the level of cbx7 mRNA in positive and negative group was 0.022 ± 0.021 vs 0.006 ± 0.011, t = -3.175, P = 0.010). The area under the receiver operating characteristics (ROC) curve is 0.769 (P = 0.033). when the Cut-off value of the relative copy number of cbx7 mRNA was 0.002 in CRCs. The values less-than 0.002 were defined as low expression. The CRC patients with low expression of cbx7 had a shorter overall survival time; whose 5 years survival rate was only 30.8% (4/13); while the rate was 77.8% (7/9) in high expression of cbx7 group. The difference had statistical significance (χ(2) = 4.329, P = 0.037). The similar differences could not be found among GC and HCC patients. Downregulation of cbx7 expression was very common among multiple carcinomas cases, and the downregulation influenced the prognosis of CRC patients.

FBN1 isoform expression varies in a tissue and development-specific fashion

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39±3 (%, mean±SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19±2 (%, mean±SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.