ABSTRACT
Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1ß and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.
Subject(s)
Inflammasomes , Toxoplasma , Mice , Animals , Humans , Female , Pregnancy , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Reactive Oxygen Species/metabolism , Pyroptosis , Trophoblasts/metabolism , Cathepsin B/metabolism , Cathepsin B/pharmacology , Placenta/metabolism , RNA, Small Interfering , Caspases/metabolism , Calcium-Binding Proteins/metabolism , CARD Signaling Adaptor Proteins/metabolism , NLR Proteins/metabolismABSTRACT
Cancer metastasis remains the most poorly understood process in cancer biology. It involves the degradation of extracellular matrix (ECM) proteins by a series of 'tumour-associated' proteases. Here we report the identification of a novel protease suppressor, NYD-SP8, which is located on human chromosome 19q13.2. NYD-SP8 encodes a 27 kD GPI-anchored cell surface protein, which shows structural homology to urokinase plasminogen activator receptor (uPAR). Co-immunoprecipitation experiments showed that NYD-SP8 binds to uPA/uPAR complexes and interfere with active uPA production. Overexpression of NYD-SP8 results in reducing activities of the three major classes of proteases known to be involved in ECM degradation, including uPA, matrix metalloproteinases (MMPs) and cathepsin B, leading to suppression of both in vitro and in vivo cancer cell invasion and metastasis. These data demonstrate an important role of NYD-SP8 in regulating ECM degradation, providing a novel mechanism that modulates urokinase signalling in the suppression of cancer progression.
Subject(s)
Extracellular Matrix/enzymology , Gene Expression Regulation, Neoplastic , Membrane Proteins/physiology , Phosphatidylinositols/metabolism , Receptors, Urokinase Plasminogen Activator/metabolism , Animals , Cathepsin B/metabolism , Cell Line, Tumor , Disease Progression , Humans , Membrane Proteins/metabolism , Mice , Mice, Nude , Neoplasm Metastasis , Protease Inhibitors/pharmacology , Recombinant Proteins/chemistryABSTRACT
AIM: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. METHODS: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR. RESULTS: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. CONCLUSION: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.
Subject(s)
Alternative Splicing , Cytoskeletal Proteins/genetics , RNA, Messenger/genetics , Testis/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary , Gene Expression Profiling , Humans , Male , Molecular Sequence Data , Open Reading Frames , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
AIM: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis. METHODS: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene's feature. RESULTS: A novel testis-specific gene, NYD-SP5, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved Ile and Gln residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. CONCLUSION: NYD-SP5 is a newly found testis-specific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.
Subject(s)
Proteins/genetics , Spermatogenesis/genetics , Testis/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , In Situ Hybridization , Male , Mice , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Proteins/chemistry , Proteins/physiology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Testis/growth & developmentABSTRACT
AIM: To identify a novel isoform of adaptin 2 beta subunit (named Ap2beta-NY) and to investigate its relationship with testicular development and spermatogenesis. METHODS: Using a human testis cDNA microarray, a clone (Ap2beta-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed. Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2beta-NY were determined. RESULTS: Ap2beta-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2beta-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2beta-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2beta-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2beta-NY was restrictively expressed in germ cells. CONCLUSION: Ap2beta-NY is an isoform of Ap2beta and may be involved in regulating the process of spermatogenesis and testis development.
Subject(s)
Adaptor Protein Complex beta Subunits/genetics , RNA Splicing , RNA, Messenger/genetics , Testis/metabolism , Adaptor Protein Complex beta Subunits/chemistry , Amino Acid Sequence , Base Sequence , DNA, Complementary , Humans , Male , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SpermatogenesisABSTRACT
AIM: To identify genes related to the human testis development by substrate hybridization technique. METHODS: A human testis cDNA microarray was constructed and hybridized with probes prepared from human adult and fetal testes and spermatozoa mRNAs by reverse transcription reactions. The differentially expressed genes were sequenced. And a newly identified cullin-3 (CUL-3) transcript variant (designated cul-3b) was bio-informatically analyzed with an online GenBank database. Multi-tissue reverse transcription polymerase chain reaction (RT-PCR) was used to determine the tissue expression profile of cul-3b. RESULTS: Cul-3b, a novel CUL-3 transcript variant, was identified. The expression level of cul-3b in adult testes was 3.79-fold higher than that in fetal ones. Cul-3b differed from cul-3 (including NM_003590 and AY337761) in the opening reading frame and had three internal ribosomal entry sites IRESes in the 5'-UTR. These led to a 24 amino acid (aa) truncation at N-terminus of CUL-3b as compared with CUL-3 and a more motivated expression pattern of cul-3b under some strict circumstances. Additionally, cul-3b expressed ubiquitously in human tissues according to multi-tissue RT-PCR. CONCLUSION: Cul-3b is a novel transcript variant of CUL-3, which may be important not only for the development of human testis but also for that of other organs.
Subject(s)
Cell Cycle Proteins/genetics , Cullin Proteins/genetics , RNA, Messenger/genetics , Base Sequence , Humans , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM: To investigate molecular mechanism of testis development and spermatogenesis. METHODS: A human testis cDNA microarray was hybridized with probes from human adult testis, embryo testis and human sperm, and the differential expressed clones were sequenced and analyzed. Expression of PIAS-NY gene was analyzed by RT-PCR. RESULT: A new isoform of PIAS family, named PIAS-NY, was isolated from human testis cDNA library. It was strongly expressed in adult testis and weakly expressed in both embryo testis and human sperm. Analysis of the open reading frame of PIAS-NY indicated that PIAS-NY was a polypeptide of 405 amino acid residues, and the sequence from the 15th amino acid to the end of PIAS-NY protein was the same as the N-terminal amino acids of PIASx-alpha and PIASx-beta protein. PIAS-NY protein contained two conserved putative LXXLL signature motifs and a zinc binding motif. Tissue distribution analysis revealed that PIAS-NY was predominantly expressed in testis, weakly in the pancreas, and almost imperceptibly in the other organs. CONCLUSION: PIAS-NY may play important role in testis development and/or spermatogenesis.
Subject(s)
DNA-Binding Proteins/genetics , Spermatogenesis/genetics , Testis/metabolism , Transcription Factors/genetics , Adult , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA-Binding Proteins/biosynthesis , Fetus , Gene Library , Humans , Male , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Pancreas/metabolism , Protein Inhibitors of Activated STAT , Protein Isoforms/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Spermatozoa/metabolism , Testis/embryology , Transcription Factors/biosynthesisABSTRACT
AIM: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa. METHODS: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. RESULTS: A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different developmental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients. CONCLUSION: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.
Subject(s)
Gene Expression Regulation, Enzymologic/genetics , Pyridoxal Kinase/genetics , RNA Splicing/genetics , RNA, Messenger/genetics , Testis/enzymology , Adult , Amino Acid Sequence , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Female , Humans , Infertility, Male/genetics , Isoenzymes/biosynthesis , Isoenzymes/genetics , Male , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Pregnancy , Pyridoxal Kinase/biosynthesis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Sertoli Cells/metabolism , Spermatogenesis/genetics , Spermatozoa/metabolism , Testis/embryology , Testis/growth & development , Tissue DistributionABSTRACT
This article reviews recent advances in apoptosis on the pathway inducing cancer cell to death, including Bcl-2 family pathway, NF-kappa B pathway, P13K/Akt pathway, Rb gene and p53 gene, especially the targets of anticancer drug in these pathways. It could be useful for the anticancer drug design and estimate. Furthermore, those cancer/testis antigen gene products are potential targets for antigen-specific immunotherapy of carcinoma.
Subject(s)
Antigens, Neoplasm/metabolism , Antineoplastic Agents/pharmacology , Antigens, Neoplasm/drug effects , Drug Delivery Systems , Drug Design , Humans , Male , NF-kappa B/drug effects , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinoblastoma Protein/drug effects , Retinoblastoma Protein/metabolism , Testis/drug effects , Testis/metabolismABSTRACT
AIM: To clone a new gene related to human spermatogenesis. METHODS: cDNA probes of embryo and adult testis were used to hybridize the cDNA microarray of adult testis, and the clones of differential hybridization were sequenced and analyzed. RESULTS: A novel isoform of calpastatin exclusively and highly expressed in human adult testis was found. CONCLUSION: A novel isoform of calpastatin expresses in human testis and it is related to spermatogenesis.
