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101.
Tatiana V. Byzova Wes Kim Ronald J. Midura Edward F. Plow 《Experimental cell research》2000,254(2):299
αVβ3, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of αVβ3, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for αVβ3 in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of αVβ3 activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn2+ markedly enhanced αVβ3-dependent adhesion to BSP. αVβ3-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that αVβ3 activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by αVβ3-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of αVβ3 can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of αVβ3 on neoplastic cells may contribute to tumor growth and metastatic potential. 相似文献
102.
The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew
distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury
could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together
with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and
zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles
in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure. 相似文献
103.
Microinjection of plant protoplasts and cells has been recently reported, however a system that combines simplicity of design, harmless immobilization, high resolution visibility and ability to monitor individual target cells is lacking. This report describes a system which combines these features. It consists of a microinjection-microculture dish containing immobilized protoplasts and a simple chamber that maintains sterility and humidity during injection. Highly purified protoplast preparations are plated at low population density as a thin monolayer of widely separated cells embedded in agarose layered over a thicker (0.2 mm at center to 1 mm at edge) layer of agarose-solidified medium. This physical arrangement allows for rapid location, mapping and injection of the immobilized protoplasts and also their subsequent location for growth monitoring. The double layers of agarose provide adequate nutrition for culturing injected cells to the microcalli stage. In addition to protoplast injection, this system was also used to inject 3- to 4-day old nonspherical cells derived from protoplasts. Colony formation rates from injected protoplasts and cells with regenerated walls were equivalent to those of uninjected controls. Furthermore, tobacco protoplasts stored at 4°C in liquid medium for up to two weeks remained fully competent for plating and injection. These cold-stored protoplasts, when injected, formed colonies at rates similar to those from fresh preparations. The ability to store protoplasts without loss of viability considerably increases the ease and convenience of cell injection experiments.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of the other products that may also be suitable. 相似文献
104.
A. Coulombe H. Duclohier E. Coraboeuf N. Touzet 《European biophysics journal : EBJ》1987,14(3):155-162
Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios P
Na/P
Cl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for P
Ba/P
Cl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes. 相似文献
105.
The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8×10-8 cm2 s-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C14 was characterized by D=0.8 – 1.0 × 10–8 cm2 s-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane. 相似文献
106.
Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species 总被引:1,自引:1,他引:0
Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met5 ]-enkephalin-Arg6 -Gly7 -Leu8 . The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu5 ]- enkephalin at the C-terminus. 相似文献
107.
Eiichi Tachikawa A. William Tank David H. Weiner Werner F. Mosimann Nobuyuki Yanagihara Norman Weiner 《Journal of neurochemistry》1987,48(5):1366-1376
Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca2+. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca2+ and are not inhibited by TIT5. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca2+ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca2+/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells. 相似文献
108.
Carboxylmethylation of Calmodulin in Cultured Pituitary Cells 总被引:3,自引:1,他引:2
We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells. 相似文献
109.
Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site 总被引:1,自引:0,他引:1
Robert W. Newcomb 《Journal of neurochemistry》1987,49(2):574-583
The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified. 相似文献
110.
Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells 总被引:4,自引:2,他引:2
A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献