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At least 1,657 records · Page 92Linked to original sources
PubMed1990

Shaker K+ channel subunits from heteromultimeric channels with novel functional properties.

A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes.

Animals↗
PubMed2006

Microarray analysis of cultured human brain aggregates following cortisol exposure: implications for cellular functions relevant to mood disorders.

Increased cortisol levels in humans are often observed in patients suffering from mood disorders. In this study human fetal brain aggregates were exposed to cortisol at 500 nM for 3 weeks, as an in-vitro model of chronic cortisol exposure. Microarray analysis on extracted mRNA using the Affymetrix U133A platform was then performed. Our results demonstrated a significant effect of cortisol on 1648 transcripts; 736 up-regulated and 912 down-regulated genes. The most differentially regulated biological categories were: RNA processing, protein metabolism, and cell growth. Within these categories we observed a down-regulation of fibroblast growth factor 2 (FGF2) (-1.5-fold) and aquaporin4 (AQP4) (-1.7-fold), alongside an up-regulation of fibroblast growth factor 9 (FGF9) (+1.7-fold) and vesicle associated membrane protein2 (VAMP2) (+1.7-fold). FGF2, FGF9, AQP4 and VAMP2 changes were confirmed at the protein level by immunohistochemistry. Alterations in FGF transcripts are in keeping with recent literature demonstrating such effects in patients with mood disorders.

Brain↗
PubMed1997

The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA.

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones. However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not. These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer.

DNA↗
PubMed1984

Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.

A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natural sequence. However, in the position of the serine residue, some alterations were compatible with activity. Assay of peptides containing the structure Arg-Gly-Asp-X (where X = another amino acid residue) showed that an Arg-Gly-Asp-Val sequence predicted to be present in some, but not all, fibronectin molecules as a result of alternative RNA splicings could potentially create a second cell attachment site in those fibronectin polypeptide chains carrying that sequence. Other proteins with potentially active Arg-Gly-Asp-X sequences include several proteins that are known to interact with the cell surface. Among these are various types of collagens, thrombin, and discoidin, a slime-mold protein that may be involved in cell aggregation. The result presented here show that the arginine, glycine, and aspartic acid residues are absolutely required for the cell recognition, and that the surrounding amino acids may play a role in the expression of cell attachment activity in fibronectin and other proteins having this sequence. We suggest, based on these data, that this recognition mechanism may be common to a number of biological systems.

Amino Acid Sequence↗
PubMed1999

Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.

ATP-Binding Cassette Transporters↗
PubMed2026

Spatial Transcriptomics Identifies Characteristic Immunological Niches in Atopic Dermatitis.

BACKGROUND: Atopic dermatitis (AD) is primarily driven by a Type 2 immune response, with T helper (TH2) cells producing IL-4 and IL-13, thereby promoting inflammation, itch, and a compromised skin barrier. Yet, the spatial organization of pathogenic immune cells and their interactions with stromal and epithelial compartments in human AD skin remain incompletely understood. METHODS: We performed 10× Genomics Visium spatial transcriptomics on FFPE skin biopsies from patients with AD (n = 6), psoriasis (n = 2), and healthy controls (n = 5). Data were integrated with AD single-cell RNA sequencing (scRNA-seq) datasets and complemented by imaging mass cytometry (IMC) and multiplex immunofluorescence (IF) to validate the spatial localization of immune cells. Cell-cell communication analysis revealed putative signaling interactions within immune niches. RESULTS: Spatial clustering resolved tissue compartments and demonstrated transcriptional dysregulation in keratinocytes in AD and psoriasis. AD lesions showed a conserved spatial organization of immune aggregates within the superficial dermis. Integration of scRNA-seq signatures revealed spatially organized co-localization of T cells and mature migratory dendritic cells (mmDCs). We developed a ring-based neighborhood analysis to characterize the cellular organization of the immune-stromal niches, revealing T cell-enriched regions surrounded by inflammatory fibroblasts and activated keratinocytes. Intercellular communication analysis further identified putative signaling within mmDC-T cell niches that may promote pathogenic T cell recruitment and activation. Application of tertiary lymphoid structure (TLS) signatures indicated the presence of TLS-like regions. IMC and IF validated the close spatial proximity between activated TH2 cells and mmDCs. CONCLUSION: AD lesions contain spatially organized TLS-like immune niches at the dermal-epidermal junction, characterized by the close association of T cells and mmDCs and coordinated interactions with surrounding stromal and epithelial compartments. These mmDC-T cell niches may represent potential targets for future therapeutic strategies aimed at disrupting persistent local inflammatory pathways and improving long-term disease control.

atopic dermatitis↗
PubMed2001

Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.

Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, islet-like organization.

Animals↗
PubMed2005

Three-dimensional in vitro reaggregates of embryonic cardiomyocytes: a potential model system for monitoring effects of bioactive agents.

To understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required. Therefore, the creation of 3-dimensional (3D) cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo. Here we have characterized a stable contracting in vitro tissue model, which consists of embryonic chicken cardiomyocytes. For establishing a cell-based test system, the 3D in vitro cardiomyocyte spheres were characterized according to messenger RNA expression of special cardiac cell types and protein expression pattern of functional markers such as connexin-43. Finally, the in vitro spheroid model was used for investigating the effect of isoproterenol, a *-adrenergic receptor agonist, on the contractibility mediated by the ligand receptor interaction.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Kuehneromycins A and B, two new biological active compounds from a Tasmanian Kuehneromyces sp. (Strophariaceae, Basidiomycetes).

In a search for new inhibitors of RNA-directed DNA-polymerases kuehneromycin A (1) was isolated from fermentations of a Tasmanian Kuehneromyces species. Its structure was elucidated by spectroscopic methods. Kuehneromycin A (1) is a non-competitive inhibitor of avian myeloblastosis virus (Ki 200 microM) and moloney murine leukemia virus (Ki 40 microM) reverse transcriptases. The second compound, kuehneromycin B (2) is a strong inhibitor of platelet aggregation stimulated with different inducers. In addition, both compounds exhibit cytotoxic and antimicrobial activities.

3T3 Cells↗
PubMed1987

The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript, and developmental regulation of expression.

A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (Mr 40,000) and D (Mr 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P] cAMP. These bands, labeled with the photoaffinity label or with 32 Pi, were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of Mr 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the Mr 37,000 polypeptide parallels the rate of receptor synthesis in vivo.

Affinity Labels↗
PubMed1996

Cell-substratum interactions mediate oncogene-induced phenotype of lung cancer cells.

In vivo and in vitro studies have linked small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells along a differentiation continuum. The transition of a SCLC toward a NSCLC phenotype is modeled in culture by the simultaneous overexpression of myc and ras genes in cultured SCLC cells. A major phenotypic distinction between SCLC and NSCLC in culture is that SCLC cells usually grow in floating aggregates, whereas NSCLC cells and myc- plus ras-expressing SCLC cells grow as adherent spreading monolayers like other epithelial cells. The present studies examine how myc, ras, cell aggregation, and attachment to laminin may interact to modulate transitions between the SCLC and NSCLC phenotypes. We find that myc-expressing SCLC cells, which normally grow as anchorage-independent cells in plastic flasks, will adhere to laminin and exhibit an epithelial morphology. In this setting, the cells express both NSCLC and SCLC markers, thus resembling a tumor type previously termed NSCLC with neuroendocrine features. Anchorage-dependent SCLC cells simultaneously expressing the myc family and an exogenous ras oncogene move further toward the NSCLC phenotype than the above myc-expressing cells. However, forced suspension of such cells restores the expression of neuroendocrine SCLC features. These studies indicate that cell environment, as much as gene expression events, profoundly affects aspects of the SCLC cell phenotype.

Basic Helix-Loop-Helix Proteins↗
PubMed2000

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.

Animals↗
PubMed1998

Engagement of CD99 induces up-regulation of TCR and MHC class I and II molecules on the surface of human thymocytes.

CD99 is a cell surface molecule involved in the aggregation of lymphocytes and apoptosis of immature cortical thymocytes. Despite its high level expression on immature cortical thymocytes, the functional roles of this molecule during thymic selection are only now being elucidated. Examination of the effects of CD99 engagement on the expression kinetics of the TCR and MHC class I and II molecules, which are involved primarily in thymic positive selection, revealed a marked up-regulation of these proteins on the surface of immature thymocytes. This increase was the result of accelerated mobilization of molecules stored in cytosolic compartments to the plasma membrane, rather than increased RNA and protein synthesis. Confocal microscopic analysis revealed the changes in subcellular distribution of these molecules. When CD99 was engaged, TCR and MHC class I and II molecules were concentrated at the plasma membrane, particularly at cell-cell contact sites. The TCRlow subpopulation of immature double positive thymocytes was much more responsive to CD99-mediated up-regulation than was the TCRhigh population. These findings suggest that CD99-dependent up-regulation may have possible implication in positive selection during thymocyte ontogeny.

12E7 Antigen↗
PubMed2006

Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA.

Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but few studies have been conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using primary culture of chicken embryonic myoblasts in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1) targeting GDF-8 mRNA was introduced into myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52-kDa GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)(10)-constructed luciferase reporter assay, a concomitant change in activity was initiated after 24 h. The activity rapidly decreased 30 h after siRNA-1 introduction, whereas high activity was maintained at 30-42 h in the control and siRNA-2-treated myoblasts. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and myotubes with aggregated nuclei were shorter and wider. These results strongly suggest that deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes.

Animals↗
PubMed2005

Schwann cell-autonomous role of neuropilin-2.

Neuropilins and group A plexins are components of receptor complexes for class 3 semaphorins, gradients of which help to guide migration of neural progenitor cells and axonal growth cones during development. We demonstrated previously that neuropilins and class 3 semaphorins are induced in sciatic nerve by crush or transection. We now report that in cultured rat Schwann cells, expression of mRNA encoding neuropilin-2 (NRP2) and plexin-A3 (PlexA3), proteins involved in semaphorin-3F (Sema3F) signal transduction, is diminished markedly by forskolin, an adenylate cyclase activator that, like axonal contact, induces Schwann cell synthesis of myelin lipids and proteins. Interestingly, Schwann cell expression of mRNA encoding NRP1, which participates in Sema3A signaling, is not downregulated by forskolin. Antibodies that recognize ectodomains of NRP2 but not control antibodies prevented cultured Schwann cells from aligning in parallel and forming columns. These results are consistent with the view that in nerves undergoing Wallerian degeneration, Schwann cell NRP2 facilitates assembly of Schwann cells into the tubular aggregates (bands of Büngner) that guide regenerating axons.

Animals↗
PubMed1993

Expression of cGMP-dependent protein kinase in Escherichia coli.

Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle. The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells. Three vectors were constructed for the expression of I alpha cGMP kinase in Escherichia coli. Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase. Most of the protein was in an insoluble and catalytic inactive form. Various solubilization and refolding conditions did not yield an active enzyme. A small fraction of the cGMP kinase was present in the soluble cell extract. This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity. To prevent aggregation two additional vectors were constructed. (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the amino-terminus of the cGMP kinase. (II) A gram/gram+ shuttle vector for expression under the control of the tac promotor was used. Both constructs directed the synthesis of an insoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed in E. coli, but mainly in an insoluble and inactive form. In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.

Amino Acid Sequence↗
PubMed1981

Phosphorescence and optical detection of magnetic resonance of cowpea chlorotic mottle virus.

Phosphorescence spectra of the tryptophan residues in cowpea chlorotic mottle virus were recorded at 77 K and the influence of the quaternary structure on the emission characteristics was investigated. The position of the phosphorescence maxima appeared to be invariant under changes in the aggregation state of the virus particle. In contrast to the results of fluorescence experiments, the phosphorescence probably originates from tryptophan residues, buried in the hydrophobic interior of the virus. Optical detection of magnetic resonance on the triplet state of the tryptophan residues at 1.2 K shows a slight shift in the zero-field transitions, when the interaction between the protein and the RNA is abolished. This shift is discussed in relation with changes in polarity and in polarizability of the environment of the phosphorescing tryptophan residues when the interaction between RNA and the protein subunits decreases. The zero-field transitions in the virus are further characterized by a large linewidth, when comparisons are made with similar transitions observed in other proteins. This shows great heterogeneity in the environment of tryptophan residues, and makes the recognition and interpretation of changes in the transitions very complicated.

Luminescent Measurements↗