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At least 235 records · Page 13Linked to original sources
PubMed1998

PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization.

We report the role of one member of a novel gene family, PACS-1, in the localization of trans-Golgi network (TGN) membrane proteins. PACS-1 directs the TGN localization of furin by binding to the protease's phosphorylated cytosolic domain. Antisense studies show TGN localization of furin and mannose-6-phosphate receptor, but not TGN46, is strictly dependent on PACS-1. Analyses in vitro and in vivo show PACS-1 has properties of a coat protein and connects furin to components of the clathrin-sorting machinery. Cell-free assays indicate TGN localization of furin is directed by a PACS-1-mediated retrieval step. Together, these findings explain a mechanism by which membrane proteins in mammalian cells are localized to the TGN.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon alpha 1 gene in Escherichia coli.

The human interferon (hIFN alpha 1) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems. The two AGG tandems (corresponding to Arg12 Arg13 and Arg163 Arg164) are known to inhibit the translation of hIFN alpha 1 mRNA and therefore they are considered to be responsible for the poor expression of hIFN alpha 1 gene in bacterial cells. To study the effect of these two tandems on the expression of hIFN alpha 1 in E. coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems. We found that, whereas the yield of hIFN alpha 1 protein per cell remained unchanged, the level of hIFN alpha 1 mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems. These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo. The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity. The most active was the product of gene hIFN alpha 1(c) in which the second AGG tandem (corresponding to Arg163, Arg164) was preserved while the least active was the protein of gene hIFN alpha 1(d) (devoid of both AGG clusters). The role of the AGG tandems in folding of the gene product is discussed.

Arginine↗
PubMed2001

Rat natural killer cell receptor systems and recognition of MHC class I molecules.

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

Animals↗
PubMed2000

Calcineurin Co-regulates contractile and metabolic components of slow muscle phenotype.

Activation of the transcription factor nuclear factor of activated T cells by the calcium-sensitive serine/threonine phosphatase calcineurin has been proposed as one of the molecular mechanisms by which motor nerve activity establishes the slow muscle phenotype. To investigate whether the calcineurin pathway can regulate the large spectrum of slow muscle characteristics in vivo, we treated rats for three weeks with cyclosporin A (an inhibitor of calcineurin). In soleus (slow muscle), but not in plantaris (fast muscle), the proportion of slow myosin heavy chain (MHC-1) and slow sarcoplasmic reticulum ATPase (SERCA2a) was decreased, whereas that of fast MHC (MHC-2A) and fast SERCA1 increased, indicating a slow to fast contractile phenotype transition. Cytosolic isoforms of creatine kinase and lactate dehydrogenase (most abundant in fast fibers), as well as mitochondrial creatine kinase and citrate synthase activities (elevated in fast/oxidative fibers) were dose dependently increased by cyclosporin A treatment in soleus muscle, with no change in plantaris. Calcineurin catalytic subunit was more abundant in soleus muscle fibers compared with plantaris. Taken together these results suggest that the calcineurin pathway co-regulates a set of multigenic protein families involved in the transition between slow oxidative (type I) to fast oxidative (type IIa) phenotype in soleus muscle.

Adenosine Triphosphatases↗
PubMed2003

A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links.

We describe a vertebrate hyaluronan and proteoglycan binding link protein gene family (HAPLN), consisting of four members including cartilage link protein. The encoded proteins share 45-52% overall amino acid identity. In contrast to the average sequence identity between family members, the sequence conservation between vertebrate species was very high. Human and mouse link proteins share 81-96% amino acid sequence identity. Two of the four link protein genes (HAPLN2 and HAPLN4) were restricted in expression to the brain/central nervous system, while one of the four genes (HAPLN3) was widely expressed. Genomic structures revealed that all four HAPLN genes were similar in exon-intron organization and were also similar in genomic organization to the 5' exons for the CSPG core protein genes. Strikingly, all four HAPLN genes were located immediately adjacent to the four CSPG core protein genes creating four pairs of CSPG-HAPLN genes within the mammalian genome. Furthermore, the two brain-specific HAPLN genes (HAPLN2 and HAPLN4) were physically linked to the brain-specific CSPG genes encoding brevican and neurocan, respectively. The tight physical association of the HAPLN and CSPG genes supports a hypothesis that the first HAPLN gene arose as a partial gene duplication event from an ancestral CSPG gene. There is some degree of coordinated expression of each gene pair. Collectively, the four HAPLN genes are expressed by most tissue types, reflecting the fundamental importance of the hyaluronan-dependent extracellular matrix to tissue architecture and function in vertebrate species. Comparison of the genomic structures for the HAPLN, CSPG genes and other members of the link module superfamily provide strong support for a common evolutionary origin from an ancestral gene containing one link module encoding exon.

Animals↗
PubMed2006

Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins.

The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.

Amino Acid Sequence↗
PubMed1998

The evolution of the type I interferons.

There are five recognized subtypes within the type I interferons (IFN), IFN-alpha, IFN-beta, IFN-delta, IFN-omega, and IFN-tau, although others may remain to be described, and the IFN-omega may have to be subdivided further because of their evident structural complexity. Together, they constitute an ancient family of intronless genes, possibly present in all vertebrates. THe IFNA/IFNB genes originated by duplication of a progenitor after the divergence of birds, most probably about 250 million years ago (MYA). The avian gene itself proceeded to duplicate to form a series of independent subtypes. The IFND, to date described only in the pig, arose from the IFNA lineage before the emergence of mammals about 180 MYA and might, therefore, be generally distributed in present day species. The IFNB, which occurs as a single gene in primates and rodents, have been duplicated in some other orders. Recent events have produced 10 or more genes in bovid species. The IFNA, which are clustered with the IFNW in humans and cattle, exist as multiple genes in all mammals so far examined as a result of a series of duplication events, some of which occurred recently and, therefore, independently in separate mammalian lineages. The IFNW diverged from the IFNA approximately 130 MYA, just prior to the emergence of mammals, and have continued to duplicate since then. The IFNT, which play a role in reproduction of ruminants, arose from an IFNW within the Artiodactyla suborder about 36 MYA and are found only in the suborder Ruminantia. These genes have also continued to duplicate to form an extensive family. Consequently, their involvement in early pregnancy is a feature of ruminants and not of other mammalian species.

Animals↗
PubMed2001

A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv. syringae strain B301D.

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.

Bacterial Proteins↗
PubMed1993

The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex.

Natural killer cells lyse tumor and virally infected cells in a specific manner that has not been molecularly characterized. Target cell expression of major histocompatibility complex (MHC) class I molecules is correlated with target cell resistance to natural killing. A mechanism to explain this observation is that NK cells may display two types of recognition and activation molecules that have opposing functions when bound to target cell ligands. One type of surface receptor such as the NKR-P1 molecule may activate NK activity whereas the other, represented by the mouse Ly-49 molecule, may engage target cell MHC molecules and inhibit cytotoxicity by transducing "negative" signals. NKR-P1 and Ly-49 are structurally related, and they are encoded by genetically linked loci in a chromosomal region, termed the NK gene complex (NKC), on distal mouse chromosome 6. Target cell susceptibility to natural killing may be dependent upon specific ligand-receptor interaction with these activating or inhibitory NKC-encoded molecules.

Amino Acid Sequence↗
PubMed2005

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study.

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

Base Sequence↗
PubMed2004

C-type lectin-like domains in Fugu rubripes.

BACKGROUND: Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps). RESULTS: We have performed an analysis of CTLD family composition in Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server http://anz.anu.edu.au:8080/Fugu_rubripes/ and their sequences as additional files with this paper. CONCLUSIONS: The vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates.

Amino Acid Sequence↗
PubMed1989

Family and population studies on the human pepsinogen A multigene family.

Human pepsinogen A (PGA) displays highly polymorphic isozymogen patterns after polyacrylamide gel electrophoresis and activity staining. The patterns differ with respect to the presence and the relative intensity of the individual fractions. Family studies strongly suggest that these isozymogen patterns are encoded by allelic haplotypes, encompassing different numbers and types of PGA genes. In this paper, we confirm the essential features of this multigene model. We establish the relationship between the haplotypes and the corresponding isozymogen patterns by determination of the PGA polymorphism at both the DNA and the protein level in 117 Dutch individuals, 60 of whom were unrelated. The combination of HindIII and EcoRI restriction fragment length polymorphisms (RFLPs) has enabled us to define different haplotypes, which are shown to segregate within families. Most genes are characterized by their specific EcoRI fragments. The HindIII RFLP is in strong linkage disequilibrium with PGA genes showing strong expression of the relevant isozymogen. Although a general picture of the relationship between genotypes and phenotypes is emerging, there are exceptions, suggesting that rare haplotypes evolve by unique crossover events.

DNA↗
PubMed2003

Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis.

The matrix metalloproteinases (MMPs) constitute a multigene family of over 23 secreted and cell-surface associated enzymes that cleave or degrade various pericellular substrates. In addition to virtually all extracellular matrix (ECM) compounds, their targets include other proteinases, chemotactic molecules, latent growth factors, growth factor-binding proteins and cell surface molecules. The MMP activity is controlled by the physiological tissue inhibitors of MMPs (TIMPs). There is much evidence that MMPs and their inhibitors play a key role during extracellular remodeling in physiological situations and in cancer progression. They have other functions that promoting tumor invasion. Indeed, they regulate early stages of tumor progression such as tumor growth and angiogenesis. Membrane type MMPs (MT-MMPs) constitute a new subset of cell surface-associated MMPs. The present review will focus on MT1-MMP which plays a major role at least, in the ECM remodeling, directly by degrading several of its components, and indirectly by activating pro-MMP2. As our knowledge on the field of MT1-MMP biology has grown, the unforeseen complexities of this enzyme and its interaction with its inhibitor TIMP-2 have emerged, often revealing unexpected mechanisms of action.

Animals↗
PubMed2000

Development of multigene and regulated lentivirus vectors.

Previously we described safe and efficient three-component human immunodeficiency virus type 1 (HIV-1)-based gene transfer systems for delivery of genes into nondividing cells (H. Mochizuki, J. P. Schwartz, K. Tanaka, R. O. Brady, and J. Reiser, J. Virol. 72:8873-8883, 1998). To apply such vectors in anti-HIV gene therapy strategies and to express multiple proteins in single target cells, we have engineered HIV-1 vectors for the concurrent expression of multiple transgenes. Single-gene vectors, bicistronic vectors, and multigene vectors expressing up to three exogenous genes under the control of two or three different transcriptional units, placed within the viral gag-pol coding region and/or the viral nef and env genes, were designed. The genes encoding the enhanced version of green fluorescent protein (EGFP), mouse heat-stable antigen (HSA), and bacterial neomycin phosphotransferase were used as models whose expression was detected by fluorescence-activated cell sorting, fluorescence microscopy, and G418 selection. Coexpression of these reporter genes in contact-inhibited primary human skin fibroblasts (HSFs) persisted for at least 6 weeks in culture. Coexpression of the HSA and EGFP reporter genes was also achieved following cotransduction of target cells using two separate lentivirus vectors encoding HSA and EGFP, respectively. For the regulated expression of transgenes, tetracycline (Tet)-regulatable lentivirus vectors encoding the reverse Tet transactivator (rtTA) and EGFP controlled by a Tet-responsive element (TRE) were constructed. A binary HIV-1-based vector system consisting of a lentivirus encoding rtTA and a second lentivirus harboring a TRE driving the EGFP reporter gene was also designed. Doxycycline-modulated expression of the EGFP transgene was confirmed in transduced primary HSFs. These versatile vectors can potentially be used in a wide range of gene therapy applications.

Animals↗
PubMed1998

Population structure of the relapsing fever spirochete Borrelia hermsii as indicated by polymorphism of two multigene families that encode immunogenic outer surface lipoproteins.

The tick-borne relapsing fever spirochete Borrelia hermsii evades the mammalian immune system by periodically switching expression among members of two multigene families that encode immunogenic, antigenically distinct outer surface proteins. The type strain, B. hermsii HS1, has at least 40 complete genes and pseudogenes that participate in this multiphasic antigenic variation. Originally termed vmp (for variable major protein) genes, they have been reclassified as vsp (for variable small protein) and vlp (for variable large protein) genes, based on size and amino acid sequence similarities. To date, antigenic variation in B. hermsii has been studied only in the type strain, HS1. Nucleotide sequence comparisons of 23 B. hermsii HS1 genes revealed five distinct groups, the vsp gene family and four subfamilies of vlp genes. We used PCR with family- and subfamily-specific primers, followed by restriction fragment length polymorphism analysis, to compare the vsp and vlp repertoires of HS1 and seven other B. hermsii isolates from Washington, Idaho, and California. This analysis, together with pulsed-field gel electrophoresis genome profiles, revealed that the eight isolates formed three distinct groups, which likely represent clonal lineages. Members of the three groups coexisted in the same geographic area, but they could also be isolated across large geographical distances. This population structure may result from immune selection by the host, as has been proposed for other pathogens with polymorphic antigens.

Borrelia↗
PubMed2003

Treatment of rat gliosarcoma brain tumors by HSV-based multigene therapy combined with radiosurgery.

Our laboratory has employed replication-defective herpes simplex virus type 1 gene transfer vectors for treatment of animal models of human malignant glioblastoma. The base vectors were defective for the immediate early (IE) genes ICP4, ICP27, and ICP22 but expressed the IE gene ICP0, which can arrest tumor cell division, and an IE thymidine kinase (alpha-tk) gene construct that mediates suicide gene therapy (SGT) in the presence of ganciclovir (GCV). Previously, we reported that SGT using ICP0/alpha-tk vectors in nude mouse models of glioblastoma was improved by coexpression of the gap-junction-forming protein connexin43 (Cx43) or human tumor necrosis factor alpha (TNF alpha). We also showed that further gains in therapeutic outcome could be achieved by combining TNF alpha-enhanced SGT with gamma-knife radiosurgery (GKR). To expand these observations, we have first repeated these studies in immunocompetent rats with brain tumors derived from implanted 9L gliosarcoma cells and second compared the most efficient vector from this study with a new recombinant vector, NUREL-C2, which expressed both TNF alpha and Cx43 along with ICP0 and alpha-tk. Results from the first part indicated that our ICP0/alpha-tk/TNF alpha vector in combination with GKR provides an effective therapy although this treatment was not statistically better than GKR combined with the ICP0/alpha-tk/Cx43 vector. Our observations in the second part suggested that NUREL-C2 may be more effective than the ICP0/alpha-tk/TNF alpha vector in combination treatments with GCV (P = 0.08) or GCV plus GKR (P = 0.10). GKR significantly enhanced the efficacy of NUREL-C2/GCV treatment (P = 0.02) as well as other virus/GCV treatments (P < or = 0.05). Conversely, the efficacy of GKR was significantly improved by both the ICP0/alpha-tk/TNF alpha vector and NUREL-C2 in combination with GCV (P = 0.02 and P < 0.01, respectively). Together these results indicate that NUREL-C2 may be an attractive candidate for Phase I gene-therapy safety studies in patients with recurrent malignant glioblastoma.

Animals↗