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Results for “Functional characterization”

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At least 595 records · Page 33Linked to original sources

PubMed2002

Functional characterization of GABA(A) receptors in neonatal hypothalamic brain slice.

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.

Anesthetics↗

PubMed1992

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.

Animals↗

PubMed1988

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect.

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.

Animals↗

PubMed1985

[Functional characterization of microcirculation in tumors: chemotherapeutic implications].

This report describes some aspects of tumor vessels and the functional characteristics of tumor microcirculation with regard to selective increase in tumor blood flow. Elevation of the mean arterial blood pressure to about 150mmHg by angiotensin II resulted in a several-fold increase in blood flow in tumor tissues without increasing blood flow in normal tissues. Pressure elevation by angio-tensin II also selectively increased tumor blood flow in intra-corneally transplanted microfoci and influx of lymph flow from the primary lesion to lymph node metastatic lesions. New techniques for analyzing microhemodynamics of tumor vessels showed that the vascular level in tumor tissues and the hydrostatic pressure difference between the tumor vessels and extravascular tissue were enhanced. Thus a new approach to cancer chemotherapy (ATII-induced hypertension chemotherapy) has been demonstrated in which the delivery to tumor tissue of systemically administered anticancer drugs can be selectively enhanced.

Angiotensin II↗

PubMed1994

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.

Acetylation↗

PubMed1995

Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum.

1. The non-selective adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies. 2. Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7-11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 +/- 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues. 3. The rank order of agonist potency to reduce the rate of peristalsis was: N6-cyclopentyladenosine (CPA) > NECA > R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) > chloroadenosine (2-CADO) > S-PIA > 2-phenylaminoadenosine (CV-1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R-PIA = CHA = > NECA > 2-CADO > S-PIA > CV-1808). 4. The selective A1 adenosine antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and the nonselective adenosine antagonist 8-phenyltheophylline (8-PT) at their respective concentrations of 10 nM and 2 microM caused parallel rightward shifts in the concentration-response curve to the non-selective Al/A2 agonist NECA. DPCPX was significantly more potent at inhibiting NECA than 8-PT as revealed by their apparent pA2 values; DPCPX (9.5) and 8-PT (7.26). The high affinity of DPCPX relative to that of 8-PT suggests the presence of an Al and not an A2B receptor. In addition, the high affinity of DPCPX(pA2:9.37) against the selective Al agonist CPA, further confirms the presence of the Al receptor subtype.5. In this study we found that the Al adenosine receptor is involved in regulating in vitro peristalsis which is different from the adenosine receptor regulating inhibition of secretion (A2B) in the same region of intestine of the same species. We propose that A2B adenosine agonists could be of clinical value in the management of diarrhoea that is due to microbiological organisms where antimotility effects are not desired.

Adenosine↗

PubMed2005

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis.

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).

Amino Acid Sequence↗

PubMed1998

Cloning and functional characterization of a sigma receptor from rat brain.

We have cloned a sigma receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain sigma receptor is highly homologous to that of the sigma receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain sigma receptor cDNA leads to a two- to threefold increase in haloperidol binding, and this cDNA-induced binding is sensitive to inhibition by several sigma receptor-specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain sigma receptor interacts with haloperidol with an apparent dissociation constant (K(D)) of 3 nM. Functional expression of the cloned rat brain sigma receptor in HeLa cells also leads to an increase in the binding of two other sigma ligands, namely, (+)-pentazocine and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain sigma receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1 ,3-di(2-tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF-7 cells, which do not express type 1 sigma receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol-sensitive binding of (+)-pentazocine, a selective type 1 sigma receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 sigma receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the sigma receptor-specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.

Amino Acid Sequence↗

PubMed2001

Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.

WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.

Bacterial Proteins↗

PubMed1997

Functional characterization of choroid plexus epithelial cells in primary culture.

The objective of this study was to develop and evaluate a primary culture system for choroid plexus epithelial cells as an in vitro model for studying organic cation transport. Cells were dispersed from choroid plexus of neonatal rats by enzymatic digestion and grew as differentiated monolayers when plated on solid or permeable support. Electron microscopy showed that cultured cells were morphologically similar to intact choroid plexus epithelium, having apical tight junctions between cells, numerous mitochondria, basal nuclei and apical microvilli and cilia. As previously demonstrated for intact choroid plexus, immunocytochemistry showed that Na+,K+-ATPase was localized to the apical membrane, and GLUT-1, the facilitative glucose transporter, was localized to the basolateral membrane of cultured cells. Apical transport of L-proline by cultured cells was mediated by a sodium-dependent, electrogenic process, as in whole tissue. 14C-Tetraethylammonium (TEA), a prototypic organic cation, was accumulated by isolated choroid plexus in a time-dependent manner; uptake was inhibited by tetrapentylammonium (TePA). In cultured cells, apical TEA transport was mediated by a saturable process coupled to cellular metabolism. Unlabeled TEA and other organic cations (TePA, N1-methylnicotinamide and mepiperphenidol) inhibited TEA transport; the organic anion, p-aminohippurate, had no effect. Finally, TePA-sensitive transport of 14C-TEA was stimulated after preloading the cells with unlabeled TEA. Based on the morphological, biochemical and functional properties of these cultured cells, we conclude that this primary culture system should be an excellent in vitro model for experimental characterization of choroid plexus function.

Animals↗

PubMed1991

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.

Amino Acid Sequence↗

PubMed1996

Expression and functional characterization of the P-selectin glycoprotein ligand-1 in various cells.

We have examined the expression and function of P-selectin glycoprotein ligand-1 (PSGL-1), which is a high affinity ligand for P-selectin. Northern blot and flow cytometric analysis demonstrated that a variety of hematopoietic cells and cell lines expressed PSGL-1. However, P-selectin binding ability was dependent on the additional expression of a carbohydrate structure, sialyl Lewis x (sLex). All the peripheral lymphocytes expressed PSGL-1 and subpopulation expressed sLex. Two color analysis showed that the majority of the cells that bound P-selectin were sLex-negative I lymphocytes, and most of the sLex-positive cells were B lymphocytes that did not blind P-selectin, indicating that the carbohydrate on T lymphocytes recognized by P-selectin is not sLex, and that the sLex on B lymphocytes is not readily presented for P-selectin recognition. Transfected 293 cells detectably bound P-selectin only when the cells expressed both PSGL-1 and sLex. When cysteine 310 of PSGL-1 was mutated to alanine, P-selectin binding was markedly reduced, suggesting the importance of dimerization of PSGL-1. These findings indicate that a preferable conformation of both carbohydrate and protein structure is necessary for a functional P-selectin ligand.

B-Lymphocytes↗

PubMed2005

Phenotypical and functional characterization of non-human primate Aotus spp. dendritic cells and their use as a tool for characterizing immune response to protein antigens.

A population of cells exhibiting bona fide dendritic cell (DC) morphological and functional characteristics was obtained by treating Aotus spp. monocytes with human IL-4 and GM-CSF. Although the purity of mature DCs was relatively low IL-4/GM-CSF-treated monocytes (hereafter called Aotus spp. DCs) down-regulated CD14 and up-regulated discrete levels of CD80, MHC-Class II and CD1b molecules in response to different maturation stimuli. Aotus spp. DCs generated a potent allogeneic in vitro response evidenced in mixed lymphocyte reaction (MLR) where DCs were 2- to 10-fold more efficient than peripheral blood mononuclear cells (PBMCs). Aotus spp. DC ability to boost T-cells or priming naive T-cells in vivo was proved by vaccinating Aotus spp. with autologous DCs pulsed with tetanus toxoid (TT). A single dose of TT-pulsed DCs was sufficient to increase cellular response to TT in these experiments as assessed by lymphoproliferation and cytokine production. Since Aotus spp. represents a suitable animal model for evaluating anti-Plasmodium falciparum malaria vaccine, the results shown here suggest that using antigen-pulsed Aotus spp. DCs as vaccines might lead to identifying new prospects for malarial vaccines unidentified to date because they are being formulated in less efficient adjuvants.

Animals↗

PubMed2004

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis.

Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly participate in ATP synthesis, and have a very low activity of the tested marker enzymes. The presence of mitochondria in neutrophils was confirmed by quantification of mitochondrial DNA copy number, by detection of mitochondrial porin, and by JC-1 measurement of Deltapsi(m). During neutrophilic differentiation, HL-60 cells demonstrated a profound cytochrome c depletion and mitochondrial shape change reminiscent of neutrophils. However, blood neutrophils containing extremely low amounts of cytochrome c displayed strong caspase-9 activation during apoptosis, which was also observed in apoptotic neutrophil-derived cytoplasts lacking any detectable cytochrome c. We suggest that other proapoptotic factors such as Smac/DIABLO and HtrA2/Omi, which are massively released from the mitochondria, have an important role in neutrophil apoptosis.

Adenosine Triphosphate↗

PubMed2004

Structural and functional characterization of the dimerization region of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, alpha(1) beta(1), identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the beta(1) subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length alpha(1) in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of beta(1) to alpha(1) The same region of beta(1)[204-408] was found to promote beta /beta(1) homodimerization. Fusion of [204 beta(1)-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of beta(1)[204-408] with alpha(1) or beta(1) targeted the sGC subunits for proteasomal degradation, suggesting that beta(1)[204-408] forms structurally deficient complexes with alpha(1) and beta(1). Analysis of deletion constructs lacking portions of the beta(1) dimerization region identified two distinct segments contributing to alpha(1) binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of beta(1) extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of beta(1) to the alpha(1) subunit of sGC.

Amino Acid Sequence↗

PubMed1985

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus).

The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c(1)-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCl fractionation procedure, contained one Cyt of c type (formally c(1) with Em(7.0) of +240 millivolts), two b type Cyt with Em(7.0) values of +100 and -25 millivolts, ferredoxin-like centers presumably linked to succinic- and NADH-dehydrogenases, and a Rieske-type iron sulfur center (g(y) = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-1 showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1-hydroxyquinoline N-oxide with I(50) of 12 nanomolar, 30 nanomolar, and 0.1 micromolar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.

Journal Article↗

PubMed1987

Functional characterization of cell-to-cell coupling in cultured rat aortic smooth muscle.

Gap junction (GJ) occurrence and function was studied in cultured rat aortic smooth muscle cells, since cell-to-cell coupling is proposed to coordinate smooth muscle function but is difficult to study in the intact tissue. Cell proliferation in vitro formed a multilayered structure 10-15 cells thick. GJs connected cells to lateral and vertical neighbors, appearing in freeze fracture as P-face particles aggregated into circular plaques but also as linear arrays. The membrane potential was 58 +/- 3 mV. From quantification of the spread of electrotonic potentials according to a two-dimensional model, the intercellular resistivity was 900-1,400 omega X cm, whereas the nonjunctional membrane resistivity was 10(4) omega X cm2. Intercellular spread of 5(6)-carboxyfluorescein (CF; mol wt 376) in aortic cultures suggests that metabolic coupling is an important consequence of GJs in smooth muscle. CF transfer was not blocked by A23187 (10(-5) M), although rat fibroblasts became uncoupled by 10(-6) M. Ultimately uncoupled by the more potent ionophore ionomycin (10(-5) M), aortic cells seem more able to maintain GJ permeability during challenge from increased intracellular Ca than cells of noncontractile origin.

Animals↗

PubMed2000

Phenotypic and functional characterization of mouse hepatic CD8 alpha+ lymphoid-related dendritic cells.

Recently, attention has focussed on phenotypic and functional differences between classic myeloid dendritic cells (DC), and DC that reportedly develop from an early, committed lymphoid precursor. In mice, DC from these separate hemopoietic lineages differ by their surface expression of CD8 alpha. We undertook a comparative study of CD8 alpha+ (CD11blow; lymphoid-related) and CD8 alpha- (CD11bhigh; myeloid) DC isolated from mouse liver. CD8 alpha+ and CD8 alpha- DC each constituted </=1.0% of the freshly isolated, normal nonparenchymal cells (NPC). Both populations were enriched 10-15% by overnight culture and metrizamide density centrifugation. Flt3 ligand (Flt3L) potently induced equal expansion of both subsets in vivo. Tissue-resident CD8 alpha+ DC, freshly isolated from Flt3L-treated mice, existed primarily as immature cells (CD11c+, CD11blow, CD40-/low, CD80low, CD86low, MHC class IIlow), consistent with previous observations regarding bulk DC freshly isolated from nonlymphoid tissues. Following overnight culture in GM-CSF, CD8 alpha+ DC underwent phenotypic and functional maturation equivalent to that observed for CD8 alpha- DC. CD95 ligand (FasL) mRNA was detected in both immature and mature DC of each subset. In vitro analysis confirmed that flow-sorted, mature CD8 alpha+ and CD8 alpha- DC were strong and equally efficient stimulators of allogeneic T cell proliferation in primary MLR. Both immunohistochemical and genomic DNA analysis revealed that in vivo, sorted CD8 alpha+ DC trafficked from s.c. sites to T cell areas of allogeneic lymphoid tissue and were equally efficient at priming naive T cells compared with CD8 alpha- DC. This is the first comparative study of lymphoid-related DC isolated from nonlymphoid tissue.

Adoptive Transfer↗