Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.
Aggrecans ; chemistry ; genetics ; metabolism ; Animals ; Biomarkers ; metabolism ; Cartilage, Articular ; chemistry ; metabolism ; pathology ; Collagen ; chemistry ; classification ; genetics ; metabolism ; Extracellular Matrix Proteins ; chemistry ; genetics ; metabolism ; Gene Expression ; Humans ; Osteoarthritis ; diagnosis ; genetics ; metabolism ; pathology ; Protein Isoforms ; chemistry ; classification ; genetics ; metabolism

OBJECTIVE: To investigate the expression of myosin heavy chain (MHC) in normal laryngeal muscle and the difference between the adductor and abductor. METHOD: Seven patients with total laryngectomy were enrolled in this study. The adductor muscles were acquired from the lateral cricoarytenoid (LCA) muscle and the abductor muscles were acquired from the posterior cricoarytenoid (PCA) muscle. The expression of myosin heavy chain were detected with fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining respectively. RESULT: (1) MHC-II b was expressed in laryngeal muscles at mRNA levels, and not expressed at the protein level; (2) At both mRNA level and protein level, the expression of MHC-I was higher in the PCA muscles than in the LCA muscles while MHC-II level was higher in the LCA muscles than in the PCA muscles. CONCLUSION (1) MHC-II b protein was not expressed in human laryngeal muscles; (2) Phenotypic differences were significant in laryngeal adductor and abductor muscles based on their different functions. PCA contained larger percentage of MHC-I fibers, while LCA contained more MHC-II fibers.
Aged ; Female ; Humans ; Laryngeal Muscles ; chemistry ; metabolism ; Male ; Middle Aged ; Myosin Heavy Chains ; genetics ; metabolism ; Phenotype ; Protein Isoforms

Crystallography, X-Ray ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; chemistry ; genetics ; metabolism ; Heparin ; metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Isoforms ; chemistry ; metabolism ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Sulfates ; chemistry ; metabolism

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.
Amino Acid Sequence ; Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Chromosomal Proteins, Non-Histone ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Crystallography, X-Ray ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Histones ; chemistry ; genetics ; metabolism ; Models, Molecular ; Oryza ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Isoforms ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Viridiplantae ; genetics ; metabolism

Hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1beta subunit and one of three subunits (HIF-1alpha, HIF-2alpha or HIF-3alpha). The stability and activity of HIF-1alpha are regulated by various post-translational modifications, hydroxylation, acetylation, and phosphorylation. Therefore, HIF-1alpha interacts with several protein factors including PHD, pVHL, ARD-1, and p300/CBP. Under normoxia, the HIF-1alpha subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)- mediated ubiquitin-proteasome pathway. The association of pVHL and HIF-1alpha under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, in the hypoxia condition, HIF-1alpha subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Eventually, HIF-1 acts as a master regulator of numerous hypoxia-inducible genes under hypoxic conditions. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation/survival, and glucose/iron metabolism. Moreover, it was reported that the activation of HIF-1alpha is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1a itself or HIF-1alpha interacting proteins inhibit tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. This review summarizes the molecular mechanism of HIF-1a stability, the biological functions of HIF-1 and its potential applications of cancer therapies.
Alternative Splicing ; Gene Expression Regulation ; Gene Therapy ; Growth Substances/metabolism ; Humans ; Protein Isoforms/chemistry/genetics/*metabolism ; Protein Subunits/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Transcription Factors/chemistry/genetics/*metabolism ; Transcription, Genetic

No abstract available.
Aged, 80 and over ; Base Sequence ; Bone Marrow/pathology ; DNA/chemistry/metabolism ; Female ; Fusion Proteins, bcr-abl/*genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Multiplex Polymerase Chain Reaction ; Protein Isoforms/genetics ; Sequence Analysis, DNA

Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus family, replicates robustly in permissive cell lines and is able to infect laboratory mice. MHV-68 has emerged as a model for studying the basic aspects of viral replication and host-virus interactions of its human counterparts. Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication (oriLyt). A family of transcription factors, CCAAT/enhancer binding proteins (C/EBPs), assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation. In this study, we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection, using MHV-68 as a model. We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay, indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication. Collectively, our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.
Animals ; Base Sequence ; CCAAT-Enhancer-Binding Proteins ; genetics ; metabolism ; Cell Line ; Chromatin Immunoprecipitation ; Cricetinae ; DNA Replication ; DNA, Viral ; chemistry ; genetics ; metabolism ; Electrophoretic Mobility Shift Assay ; Genome, Viral ; Herpesviridae Infections ; genetics ; metabolism ; virology ; Humans ; Mice ; Molecular Sequence Data ; Plasmids ; Promoter Regions, Genetic ; Protein Isoforms ; genetics ; metabolism ; Replication Origin ; Rhadinovirus ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Latency ; genetics

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADH1 and PeBADH2) were cloned from Populus euphratica. Both PeBADH1 and PeBADH2 genes encode the proteins of 503 amino acid residues, with a calculated molecular mass of 54.93 kDa and 54.90 kDa, respectively. Reverse transcription PCR showed the divergence of expression pattern between the PeBADH1 and PeBADH2 genes in P. euphratica. The recombinant PeBADH1 and PeBADH2 proteins were overexpressed in E. coli, and purified by Ni-affinity chromatography. The PeBADH2 protein had 1.5-fold higher enzymatic activity towards the substrate aldehyde than PeBADH1 protein. The PeBADH1 protein revealed higher thermal stability than PeBADH2 protein. These results indicated obvious functional divergence between the PeBADH1 and PeBADH2 genes.
Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; physiology ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Populus ; genetics ; Protein Isoforms ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Substrate Specificity

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
Acanthamoeba castellanii/cytology/*genetics/physiology ; Amebiasis/*parasitology ; Amino Acid Sequence ; Autophagy ; Cell Membrane/metabolism ; DNA, Protozoan/chemistry/genetics ; Gene Dosage ; Gene Silencing ; Genes, Reporter ; Humans ; Molecular Sequence Data ; Phagosomes/metabolism ; Protein Isoforms ; Protozoan Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; RNA, Small Interfering/chemical synthesis/genetics ; Recombinant Fusion Proteins ; Sequence Alignment

We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolatioin and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.
Sequence Homology, Amino Acid ; Sequence Analysis, DNA ; Rats ; Protein Isoforms/genetics ; Phosphorylation ; Phosphoproteins/genetics/*metabolism ; Molecular Sequence Data ; Mice ; Male ; Humans ; Gene Expression Profiling ; Female ; DNA, Complementary/chemistry/genetics ; Cyclic AMP-Dependent Protein Kinases/*physiology ; Cloning, Molecular ; Cell Line ; Base Sequence ; Animals ; Amino Acid Sequence ; Adult