Receptor kinetics differ for endothelin-1 and endothelin-2 binding to Swiss 3T3 fibroblasts

The equilibrium binding, kinetics of ligand-receptor interactions, and biological activity of endothelin-1 and -2 have been studied in Swiss 3T3 fibroblasts. Scatchard analyses of saturation binding data for ET-1 and -2, performed at 4 degrees C to prevent internalization of the occupied receptor, revealed similar affinity constants and numbers of binding sites for endothelin-1 and -2. Experiments designed to determine ligand-induced effects on 45Ca efflux demonstrated no qualitative or quantitative differences between the two endothelin isoforms. In contrast, kinetic studies resulted in different rates of dissociation for the two isoforms and different extents of dissociation. Specifically, only 40% of the bound [125I]endothelin-1 was dissociated at 4 h following the addition of excess unlabeled ligand, whereas 85-90% of the bound [125I]endothelin-2 was dissociated under the same conditions. Endothelin-1 and -2 also differed in the percent of specific cell-associated ligand bound after a 2 h incubation at 37 degrees C following an initial equilibration at 4 degrees C. The differences in dissociation rates and association or internalization rates at 37 degrees C are the first data that differentiate between the two isoforms. It is suggested that isoform-specific differences in the rate of dissociation from cell surface endothelin receptors influence the level of cell-associated endothelin and may be important in determining physiologic responses in vivo.     

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance

Platelet-derived growth factor (PDGF)-BB elicits a migratory response including reorganization of the actin cytoskeleton in different cell types. Here we have investigated the effects of PDGF-BB stimulation on beta(1) integrin containing focal adhesions in human diploid fibroblasts adhered to collagen type I. Stimulation with PDGF-BB dissociated focal adhesions and relocated beta(1) integrins from focal adhesions to the periphery of the cells. These changes were rapid and transient in character. Relocation of beta(1) integrins was prevented by inhibitors of phosphoinositide-3-kinase and protein kinase C. PDGF-BB stimulated fibroblasts exhibited an increased diffusion coefficient of cell surface beta(1) integrins as determined by fluorescence recovery of photobleaching. The cell surface expression of beta(1) integrins was not changed after stimulation with PDGF-BB. Our data suggest that PDGF-BB increases the dynamic properties of cell-surface beta(1) integrins, which most likely are important for the migratory response elicited by PDGF-BB.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

In vitro probe acylcarnitine profiling assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry predicts severity of patients with glutaric aciduria type 2

Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency, MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase. The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M). In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate their clinical severity or not. Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines; C14, C12, C10 or C8 as well as C2, were extremely low. In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S. The ratio of C16/C14, C16/C12, or C16/C10, in the culture medium was significantly higher in MAD-S compared with that in MAD-M. Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S, while their effects were less pronounced in MAD-M. In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates. This strategy may be applicable for other metabolic disorders.     

猪耳皮肤成纤维细胞的培养

本研究以猪耳皮组织为材料,采用胰蛋白酶冷热处理结合法成功地分离和培养了猪耳皮肤成纤维细胞。此方法的细胞存活率相对于胰蛋白酶热处理法较高。传代细胞与原代细胞的形态和生长速度均相似。传代细胞未检测到凋亡现象。细胞已传至15代以上,染色体倍性正常,说明我们所建立的胰蛋白酶冷热处理结合法可以快速有效的分离和培养猪耳皮肤成纤维细胞,并且能稳定的进行传代培养。     

Strukturanomalie (inv (p-q+)?) eines Chromosoms der Gruppe C+X bei einem jungen mit multiplen dysplasien und dissoziationen der psychischen entwicklung

Zusammenfassung Bei einem elfjährigen Jungen mit dissoziierter psychischer Entwicklung, mit Hinweisen auf einen diffusen, linksseitig betonten Hirnschaden sowie mit dysplastischen Stigmata fand sich in Leukocyten und Fibroblasten ein subtelozentrisches Chromosom in der Gruppe C+X. Die Karyotypen der Eltern sind normal.

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Structural anomaly (inv(p-q+)?) of a C+X group chromosome in a dysplastic boy with dissociated mental development

Summary A boy with dissociation of mental development, slight diffuse encephalopathy, and few dysplasias is reported. Cytogenetic investigation of leukocytes and fibroblasts revealed a subtelocentric chromosome in group C+X whereas the caryotypes of the parents are normal.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Ke 128/1).     

Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression

Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues. Under constant conditions in vitro, however, rhythms of peripheral tissue explants or immortalized cells damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2(Luciferase-SV40) knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.     

Culture substrate dependence of mouse fibroblasts survival at 4° C

Summary When L-929 mouse fibroblasts grown in Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) were stored in a monodisperse suspension at 4° C, the viability decreased rapidly from the beginning of storage. The viability in this study was determined by counting electronically the number of cells with the capacity to attach to glass substrate and with the membrane boundary resistant to a proteolytic digestion. When, however, the dissociated cells were preincubated briefly at 37° C, and subsequently stored at 4° C as they were attaching on a glass substrate, the rapid loss of viability could be reduced effectively. A biphasic survival profile consisting of an initial phase of slowly decreasing viability and the subsequent phase of rapidly decreasing viability were then observed. The rapid viability loss occurred not only when the cell suspension was prepared by mechanical dislodging but also after trypsinization or dispase treatment. Such viability loss was also observed when the dissociated cells were not stored at 4° C directly but preincubated in a monodisperse suspension at 37° C in a siliconized plate and then stored at 4° C. The above results show that the rapid loss of viability is associated closely with the fact that the cells were not attached to the substrate but in suspension. This work was supported in part by grants-in-aids from the Institute of Physical and Chemical Research and from the Mitsubishi Foundation.     

Different aggregation properties of sea urchin embryonic cells at different developmental stages. II. Stage specific response to fibronectin and collagen

Fibronectin and collagen were added to cells dissociated from embryos at the blastula and at the 16 cell stages. Both molecules stimulate aggregation of cells dissociated from blastula but they do not affect aggregation of blastomeres dissociated from the 16 cell stage. The stage-specific response to fibronectin and collagen appears to be related to the onset of new functional role(s) of the two molecules at the blastula stage.     

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.     

The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.     

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside G_M1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A–C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein populations associated with detergent-resistant membranes, and their potential interactions in cell signaling.     

Normal human fibroblasts are resistant to RAS-induced senescence

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated beta-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS.     

Role of peroxisomal oxidation in the conversion of arachidonic acid to eicosatrienoic acid in human skin fibroblasts.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1-(14)C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [3H]20:4 to [3H]20:3. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [3H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [3H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [3H]16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal beta-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.     

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellular phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity (Bmax) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5 alpha-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium (Kd) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4 degrees C elute from DEAE-Sephacel at approximately 0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37 degrees C, extracted with 0.4 M KCl in a buffer containing 10 mM Na2MoO4, and desalted by G-25, they elute at less than or equal to 0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37 degrees C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociated more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly-an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.     

A membrane glycoprotein-containing fraction which promotes all aggregation.

Glycoproteins have been extracted from 16C rat fibroblasts using acetone/lithium di-iodosalicylate/phenol purification. This fraction increases the aggregation of the fibroblasts $\text{in vitro}$.     

Stress and matrix‐responsive cytoskeletal remodeling in fibroblasts

In areolar “loose” connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross‐sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large “sheet‐like” morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch‐induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues. J. Cell. Physiol. 228: 50–57, 2013. © 2012 Wiley Periodicals, Inc.     

Affinity separation of messenger RNA by thermo-responsive polymer carrying oligo(dT).

The conjugate between oligo(dT)16 and thermo-responsive polymer, poly(N-isopropylacrylamide), was prepared for isolation of poly(A)+ RNA from total RNA. The hybridization reaction between the conjugate and poly(A) (average length: 320 base) was equilibrated in 10 min, and all the poly(A) (16 nmol base for 24 nmol base of conjugate) was precipitated when raising the solution temperature to 35 degrees C. The precipitate was dissolved in water, and poly(A) was dissociated from the conjugate by heating to 65 degrees C. This separation system was successfully applied to the isolation of poly(A)+ RNA from total RNA.