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PubMed2001

Histological and functional analysis of vascular smooth muscle cells in a novel culture system with honeycomb-like structure.

Vascular smooth muscle cells (SMCs) undergo phenotype change with the development of atherosclerosis. The phenotype changes of SMCs have been observed in various culture conditions, such as collagen-coated dishes. Here, we report the morphological and functional features of SMCs in a novel culture system using type I-collagen in a characteristic three-dimensional structure designated as honeycombs. The number of ribosome and mitochondria in SMCs cultured in honeycombs was one half or third of those cultured on collagen-coated plastic plates. DNA and protein synthesis of SMCs cultured in honeycombs were less than 1 and 30-40%, respectively, of those cultured on plastic plates. In addition, PDGF-BB did not increase the amount of DNA synthesis in SMCs in honeycombs. SMCs in honeycombs were shown to express several proteins, which are known to express in SMCs in medial layers of arteries. Particularly, caldesmon heavy chain was expressed in SMCs cultured in honeycombs, whereas not in those on plastic plates. Although focal adhesion kinase (FAK) was clearly detected in SMCs in honeycomb, the phosphotyrosine content of focal adhesion kin ase decreased in the process of culture. Immunoblot analysis showed dear different expression of ERK1 and ERK2 of mitogen-activated protein kinase in SMCs. SMCs in honeycombs expressed ERK2, more abundantly compared to ERK1, whereas SMCs in plates show the same levels of expressions for both proteins. Thus, the histological and functional feature of SMCs in the novel culture system is different from SMCs in plastic plates. The three-dimensional culture system described here may be indicating that cultured SMCs are able to express different proteins responding to the surrounding structures.

Animals↗
PubMed2005

Functional analysis of CYP2D6.31 variant: homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution.

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.

Amino Acid Substitution↗
PubMed1997

Functional analysis in vivo of the double mutant mouse deficient in both proteolipid protein (PLP) and myelin basic protein (MBP) in the central nervous system.

Myelination is an important developmental process of the central (CNS) and peripheral nervous system (PNS). To unravel the functions of the two dominant myelin proteins in the CNS, proteolipid protein (PLP) and myelin basic protein (MBP), we generated and characterized the homozygous double mutant mouse line (plp-/-, mbp-/-), which is viable and fertile. Plasma membrane processes of oligodendrocytes deficient in PLP and MBP, but not in myelin-associated glycoprotein (MAG), spirally wrap large diameter axons, tightly adhering at their extracytosolic surfaces and forming a pseudo-compacted myelin. Neuromotor activity and coordination are considerably improved compared to the shiverer trait.

Animals↗
PubMed2003

Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2.

Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn(48) and Asn(57). Our results show that although the apparent affinities for [3H]uridine and [3H]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC(50) for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC(50) for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2.

Amino Acid Substitution↗
PubMed1991

Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

The LAT1 gene encoding the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae was disrupted, and the lat1 null mutant was used to analyze the structure and function of the domains of E2. Disruption of LAT1 did not affect the viability of the cells. Apparently, flux through the PDH complex is not required for growth of S. cerevisiae under the conditions tested. The wild-type and mutant PDH complexes were purified to near-homogeneity and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme assays. Mutant cells transformed with LAT1 on a unit-copy plasmid produced a PDH complex very similar to that of the wild-type PDH complex. Deletion of most of the putative lipoyl domain (residues 8-84) resulted in loss of about 85% of the overall activity, but did not affect the acetyltransferase activity of E2 or the binding of pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), and protein X to the truncated E2. Similar results were obtained by deleting the lipoyl domain plus the first hinge region (residues 8-145) and by replacing lysine-47, the putative site of covalent attachment of the lipoyl moiety, by arginine. Although the lipoyl domain of E2 and/or its covalently bound lipoyl moiety were removed, the mutant complexes retained 12-15% of the overall activity of the wild-type PDH complex. Replacement of both lysine-47 in E2 and the equivalent lysine-43 in protein X by arginine resulted in complete loss of overall activity of the mutant PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed2003

Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.

A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.

Animals↗
PubMed2002

Characterization and functional analysis of granulocyte concentrates collected from donors after repeated G-CSF stimulation.

BACKGROUND: Neutropenic patients often develop bacterial or fungal infections not responding to broad-spectrum antibacterial or antifungal agents. Clinical efforts were made with transfusion of granulocyte concentrates; however, functions of granulocytes after multiple G-CSF stimulations and after apheresis are not yet investigated and described sufficiently. STUDY DESIGN AND METHODS: The aim of this study was to characterize functional and immunologic variables of granulocytes in blood samples drawn from donors before and after each stimulation episode with G-CSF, in the resulting granulocyte concentrates and in the patients 8 hours after transfusion. RESULTS: Chemotaxis was not influenced, neither by G-CSF application nor by apheresis. Multiple G-CSF stimulations enhanced oxidative burst and phagocytosis of Escherichia coli in donor granulocytes. These values returned to basal levels in granulocyte concentrates. Expression of granulocytic surface antigens was downregulated after application of G-CSF but returned to normal and in part enhanced values in concentrates. A clinically relevant increase of proinflammatory cytokines could not be detected. Leukotriene B4 production was reduced after the fourth G-CSF stimulation in the donor blood and enhanced in the granulocyte concentrate after apheresis. Results in recipients indicate that changes of granulocyte function noted in concentrates were only transient. CONCLUSION: Stimulation of healthy donors with repeated G-CSF injections and subsequent granulocyte apheresis does not dramatically change decisive functions of granulocytes.

Adult↗
PubMed1993

Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.

Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.

Adenosine Triphosphatases↗
PubMed1990

Characterization and functional analysis of interferon-gamma-induced intercellular adhesion molecule-1 expression in human keratinocytes and A-431 cells.

Human intercellular adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein that serves as one of the major ligands for lymphocyte function-associated antigen-1 (LFA-1), a member of the integrin supergene family of adhesion molecules that is involved in cell-cell adhesion. Homotypic and heterotypic conjugate formation between leukocytes and between leukocytes and target cells via the LFA-1/ICAM-1 interaction has been demonstrated to be a critical event in numerous immunologic and inflammatory processes. While LFA-1 is expressed by all leukocytes, ICAM-1 is not normally expressed by all tissues with which leukocytes interact, but ICAM-1 may be induced de novo by various cytokines, including interferon-gamma (IFN-gamma). The constitutive and IFN-gamma-induced expression and function of ICAM-1 in human keratinocytes (HK) and A-431 cells in culture has been analyzed. While A-431 cells constitutively express ICAM-1 when assessed by northern blotting, by biosynthetic labeling and immunoprecipitation, and by flow cytometry, HK do not. When these two cell types are exposed to recombinant human (rh-) IFN-gamma at 1000 U/ml for 24 h, A-431 cells upregulate ICAM-1 and HK express ICAM-1 to an equal degree when assessed by these same parameters. Furthermore, in an in vitro adhesion assay, rh-IFN-gamma treatment of the HK or A-431 cells greatly increases the specific adherence of radiolabeled T cells to these cells. These data provide further evidence for the potential role of the regulated expression of ICAM-1 by keratinocytes in immunologic and inflammatory responses occurring in the skin.

Cell Adhesion↗
PubMed2006

Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.

An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells.

Animals↗
PubMed1991

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.

Amino Acid Sequence↗
PubMed2001

Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Amino Acid Sequence↗
PubMed2003

Optical bioimaging: from living tissue to a single molecule: imaging and functional analysis of blood flow in organic microcirculation.

Activity of blood cells, erythrocytes, leucocytes, and platelets, in microcirculation was observed by using an intravital microscope and confocal laser scanning microscope connected with an image processing system including fluorescence and phosphorescence emission methods. Dynamic functions of the blood flow were mainly observed in mesentery, brain, and liver tissues of rats. The results are summarized as follows: Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, particularly at high shear rate. The spring constant and Young's modulus of diabetic erythrocytes obviously stiffened, making them hard to deform in the capillary. During hemorrhagic shock and thrombosis, flow velocity and oxygen partial pressure of blood decreased in the brain and liver tissues that can be visualized by using FITC stained erythrocytes and Pd-porphyrin derivative as a pO(2) probe. Platelet adhesion and thrombus formation in the micro-vessels accelerated under the photodynamic reaction; diabetic platelets showed augmented adhesion and aggregation on the vessel wall which was followed by acute thromboembolism. Active oxygen radicals take part in thrombus formation, accompanied with adhesion of the activated leucocytes. Fluorescent dye probes, rhodamine G and acridine orange, are quite useful for visualization of the flow behavior of platelets and leucocytes, respectively.

Animals↗
PubMed1985

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo.

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell clones here studied were restimulated either with IPSm or with schistosomulum Ag, it appears that such IPSm-specific T cell clones could be involved in the concomitant immunity mechanisms.

Animals↗
PubMed1993

Functional analysis of the carboxy-terminal transforming region of v-Myc: binding to Max is necessary, but not sufficient, for cellular transformation.

To understand the mechanism by which the Myc protein contributes to cell growth and development, our laboratory is studying functions of the avian myelocytomatosis virus 29 (MC29) Gag-Myc protein (v-Myc) in the mouse fibroblast cell line C3H10T1/2. Previously, we identified two specific regions in v-Myc which are required for co-transformation with activated H-ras. One maps to the amino-terminal portion of v-Myc (amino acids 1-137) and has the potential to activate transcription of a basal promoter. The second region spans the carboxy-terminal region of v-Myc (amino acids 244-410), contains a basic/helix-loop-helix/leucine zipper motif and specifies the nuclear location of the protein. In this study, we have generated a series of deletion mutations within the MC29 gag-myc gene to define precisely the carboxy-terminal transforming region using the co-transformation of C3H10T1/2 cells as an assay. v-Myc proteins encoded by selected deletion mutations were also examined for their intracellular location, the ability to interact with the Max protein and the potential to bind specifically to DNA. Our results demonstrate that integrity of both the basic/helix-loop-helix and the leucine zipper motifs of v-Myc is required for co-transforming activity, but that the major nuclear localization signal sequence of v-Myc can be deleted without compromising the ability of v-Myc to cooperate with activated H-Ras p21 to transform C3H10T1/2 cells. In addition, while the binding of v-Myc to Max is necessary for ras/myc co-transformation, it is not sufficient, and also requires the integrity of Myc sequences specifying site-specific DNA binding.

Amino Acid Sequence↗
PubMed1997

[Modification of gene targeting method for functional analysis of the target gene in vivo].

Gene targeting in ES cells is a powerful tool to generate mice bearing predesigned mutations in the germ line. However, these mice carrying such constitutional mutations are often lethal and, therefore, we cannot study other functional aspects of the gene at later stages or in particular tissues. To inactivate the target gene in particular tissues or at particular stages of development, conditional gene inactivation based on the Cre-loxP recombination system of bacteriophage P1 is considered one of the applicable techniques. To express the Cre gene in a cell-type or developmental stage specific manner, either transgenic or adenoviral technology is most considerable technique in vivo.

Animals↗
PubMed1985

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus.

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.

DNA, Viral↗
PubMed1995

Functional analysis of the human pro-opiomelanocortin promoter in the small cell lung carcinoma cell line DMS-79.

DMS-79 is a human cell line derived from a small cell lung carcinoma (SCLC), which expresses the pro-opiomelanocortin (POMC) gene. We took it as a model in which to study the mechanism of POMC gene expression in these tumors: precursor processing is altered and gene expression is essentially unresponsive to glucocorticoids. POMC gene structure appeared normal by Southern blot analysis, indicating that gene rearrangement was not responsible for its expression in DMS-79. Indeed, using transient expression of human POMC-luciferase fusion genes in DMS-79, we showed that (1) the normal human POMC promoter was functional in DMS-79, and (2) the same proximal promoter region (-417; + 21) produced the full transcriptional activity in DMS-79 and in the mouse pituitary cell line AtT-20. Progressive 5' deletion analysis revealed differences between AtT-20 and DMS-79: region (-611; -376) was active in AtT-20 and not in DMS-79, whereas region (-95; -161) was active in both cell lines and (-376; -417) was only active in DMS-79. The latter partially overlaps a motif homologous to the DE-2 rat element which confers the tissue-specific expression of POMC in AtT-20 cells; however, this motif had no effect in DMS-79. These data suggest that POMC gene transcription is achieved through a different set of transacting factors in DMS-79 and AtT-20. Altogether, our results provide evidence that DMS-79 is a valid model of tumors responsible for the ectopic ACTH syndrome and that the mechanism of POMC gene expression in these SCLC cells is different from that in pituitary cells.

Animals↗