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At least 145 records · Page 8Linked to original sources
PubMed1999

ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys.

Autosomal dominant polycystic kidney disease (ADPKD) cyst enlargement is exacerbated by accumulation of fluid within the lumen of the cyst. Extracellular nucleotides and nucleosides stimulate fluid and chloride (Cl-) secretion across epithelia and are potent autocrine and paracrine agonists within tissues. This study tests the hypothesis that ATP may be released by ADPKD epithelial cells. Once released, extracellular nucleotides and their metabolites may become "trapped" in the cyst lumen. As a consequence, extracellular ATP may augment ADPKD cyst enlargement through stimulation of salt and water secretion across ADPKD epithelia that encapsulate ADPKD cysts. To test this hypothesis, bioluminescence detection assays of ATP released from primary cultures of human ADPKD epithelial cells were compared with non-ADPKD human epithelial primary cultures. ADPKD cultures release comparable or greater amounts of ATP than non-ADPKD cultures derived from proximal tubule or cortex. ATP release in both ADPKD and non-ADPKD primary epithelial monolayers was directed largely into the apical medium; however, basolateral-directed ATP release under basal and stimulated conditions was also observed. Hypotonicity potentiated ATP release into the apical and basolateral medium in a reversible manner. Reconstitution of isotonic conditions with specific osmoles or inhibition with mechanosensitive ion channel blockers dampened hypotonicity-induced ATP release. "Flash-frozen" cyst fluids from ADPKD cysts, harvested from multiple donor kidneys, were screened by luminometry. A subset of cyst fluids contained as much as 0.5 to 10 microM ATP, doses sufficient to stimulate purinergic receptors. Taken together, these results show that ADPKD and non-ADPKD human epithelial primary cultures release ATP under basal and stimulated conditions and that ATP is released in vitro and into the cyst fluid by cystic epithelial cells in concentrations sufficient to stimulate ATP receptors. It is hypothesized that extracellular nucleotide release and signaling may contribute detrimentally to the gradual expansion of cyst fluid volume that is a hallmark of ADPKD.

Adenosine Triphosphate↗
PubMed2006

Complete protection against interleukin-1beta-induced functional suppression and cytokine-mediated cytotoxicity in rat pancreatic islets in vitro using an interleukin-1 cytokine trap.

Cytokines, particularly interleukin (IL)-1beta, have been postulated to cause beta-cell destruction in type 1 diabetes. We tested the efficacy of an IL-1 cytokine trap in counteraction of suppressive and toxic effects after exposure of rat pancreatic islets in vitro to IL-1beta. The IL-1 cytokine trap used herein comprised extracellular domains of the IL-1 receptor accessory protein and the human IL-1 receptor 1 arranged inline and fused to the Fc portion of human IgG1. Groups of isolated rat pancreatic islets were maintained in medium culture with or without IL-1beta (150 pmol/l) for 48 hours in the absence or presence of the IL-1 trap at 1-, 10-, or 100-fold excess the molar concentration of the cytokine. IL-1beta alone induced a strong inhibition of insulin secretion and glucose oxidation rate and a marked increase in medium nitrite accumulation as an indicator of nitric oxide generation. When the IL-1 trap was used at a ratio 10:1 or 100:1, a complete protection against these effects were observed. Moreover, the IL-1 trap (100:1) blocked the increased islet cell death seen in islets treated with a combination of IL-1beta + tumor necrosis factor-alpha + interferon-gamma as well as functional suppression induced by the cytokine combination. In conclusion, we show that addition of an IL-1 trap can protect rat pancreatic islets in vitro against noxious effects induced by IL-1beta. Exploring the IL-1 trap in relevant animal models of type 1 diabetes represents an interesting future intervention strategy.

Animals↗
PubMed2004

Expression of Btcl2, a novel member of Btcl gene family, during development of the central nervous system.

Cell-cell interactions are primarily mediated by secreted and transmembrane proteins which play essential roles in the neuronal circuit formation. However, molecular mechanisms underlying neuronal circuit formation, which is mediated by the cell-cell interactions, remain largely elusive. We isolated and characterized a novel gene, Btcl2 (brain-specific transmembrane protein containing CUB [complement subcomponent C1r/C1s, sea urchin protein Uegf, and BMP-1] and LDLa [low-density lipoprotein receptor domain class A] domains 2), using the signal sequence trap (SST) method. The extracellular domain of BTCL2 contains two CUB domains and an LDLa domain. BTCL2 and BTCL1 have similar domain structures, sharing 51% overall identity. The CUB1, CUB2, and LDLa domains of these two proteins share 63%, 72%, and 84% identity, respectively. The CUB domains of BTCL1 and BTCL2 share significant identity with those of neuropilins. Btcl2 mRNA was detected as a single 6-kb transcript in Northern blot analysis. In situ hybridization (ISH) analysis revealed that both Btcl1 and Btcl2 mRNAs were observed restrictively in brain throughout embryonic and postnatal stages. Btcl1 and Btcl2 mRNAs were expressed uniquely in the pontine nucleus and subplate, which are required for establishing the neuronal circuit formation. These results will aid in resolving the mechanisms underlying neuronal circuit formations (e.g., pontocerebellar and thalamocortical axon guidance) and permit more precise studies aimed at understanding the role of BTCL1 and BTCL2 in the central nervous system.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular mechanisms of neurotoxin action on voltage-gated sodium channels.

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.

Animals↗
PubMed2004

The bone morphogenetic protein type Ib receptor is a major mediator of glial differentiation and cell survival in adult hippocampal progenitor cell culture.

Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture.

Adenoviridae↗
PubMed1990

Serum apolipoprotein A-I in alcoholic patients with chronic calcifying pancreatitis.

Serum apolipoprotein A-I measurement was compared in alcoholic patients according to presence or absence of chronic pancreatitis and liver fibrosis. Among alcoholic patients without liver disease, apolipoprotein A-I was significantly lower in patients with chronic pancreatitis (157 +/- 70 mg/dl) than in patients without pancreatitis (209 +/- 74 mg/dl, p less than 0.001). In cirrhotic patients, apolipoprotein A-I was lower in patients with chronic pancreatitis (82 +/- 35 mg/dl) than in patients without pancreatitis (102 +/- 45 mg/dl), but this difference was not significant. The decrease of serum apolipoprotein A-I was independent of nutritional parameters whether or not there was cirrhosis. Immunohistochemical study of pancreatic samples with chronic pancreatitis showed that apolipoprotein A-I was located in the pancreatic fibrosis whereas lobules were unstained. This study suggests that apolipoprotein A-I is trapped by the pancreatic extracellular matrix and that this sequestration might explain, in part, the decrease of the serum apolipoprotein A-I.

Adult↗
PubMedDate not supplied

Interaction of native and cell-modified low density lipoprotein with collagen gel.

We have examined the binding of native and cell-modified low density lipoprotein (LDL) to gels of Type I collagen. Diffusion of native 125I-LDL into the collagen gel was slow, reaching equilibrium after 24 to 48 hours, while L-3H-glucose, a low molecular weight marker, equilibrated in 6 hours. Binding of 125I-LDL was measured at 48 hours as the amount associated with the collagen after extensive washing. Binding was saturable with an increasing concentration of LDL. Prior incubation with cell-free culture medium resulted in modest, but progressive, increases in electrophoretic mobility and binding to collagen. Incubation with cells produced a marked increase in electrophoretic mobility and a 5- to 10-fold increase in collagen binding; the presence of butylated hydroxytoluene during incubation prevented both effects. These changes in LDL were induced by porcine aortic endothelial cells, smooth muscle cells, human skin fibroblasts, and a variety of cell lines, as well as by acetylation. There was a curvilinear relationship between the amount of LDL protein bound and the net negative charge of the LDL; increasing net charge was associated with progressively greater increases in binding. These results suggest a potential role for collagen in trapping lipid in the extracellular matrix of arterial intima by slowing the diffusion of and by binding LDL. The data also demonstrate that binding of LDL to collagen is enhanced by modifications that increase its net negative charge.

Animals↗
PubMedDate not supplied

Biosynthesis of chondroitin sulfate proteoglycan by P388D1 macrophage-like cell line.

Macrophages are in large part responsible for the subendothelial deposition of lipid within the artery wall during the early stages of atherogenesis. Proteoglycans secreted by these cells may play a role in this pathological process either by trapping lipoproteins in the extracellular matrix or by enhancing the formation of lipid-laden foam cells. The synthesis and secretion of proteoglycan was studied in the P388D1 macrophage-like cell line cultured in the presence of 35S-sulfate. The radiolabeled proteoglycan had a Kd of 0.69 on Sepharose CL-2B corresponding to an Mr of 2.8 x 10(5). It consisted of approximately 13 chondroitin sulfate chains of Mr 20,000 attached to a core protein with an Mr of 18,000. The chondroitin sulfate chains contained both N-acetylgalactosamine 6-sulfate and N-acetylgalactosamine 4-sulfate residues. No disulfated N-acetylgalactosamine residues were present. The P388D1 proteoglycan bound specifically to immobilized human low density lipoprotein. These results suggest that, in the focal regions of the arterial wall in which macrophages are found during the development of fatty streaks, proteoglycans secreted by these cells may affect the transport and cellular metabolism of plasma-derived lipids.

Acetylgalactosamine↗
PubMed1998

CD44 expression and growth factors.

Soluble factors such as growth factors and cytokines present in the tumor microenvironment regulate a variety of genes associated with malignant properties of tumor cells such as growth, migration, invasion, and metastatic capacities. CD44 is a multi-functional adhesion molecule involved in cell to cell and cell to extracellular matrix interaction, the trapping of growth factors and cytokines, and the regulation of cell traffic. Growth factors and cytokines modify the expression, selective isoform splicing and functions of CD44, resulting in changes in the biological properties of the cells. These include adhesion of circulating tumor cells to endothelium and body cavities, and survival in response to growth factors presented by the CD44 molecule. The modification of CD44 on both tumor and host cells by growth factors may play an important role in tumor progression.

Animals↗
PubMed1992

In vitro growth of human multiple myeloma: implications for biology and therapy.

In vitro data allow presentation of a plausible scenario for the in vivo growth, progression, and dissemination of human multiple myeloma (MM) that involves the interactions between the monoclonal B-cell clone and the bone marrow (BM) microenvironment. A large series of adhesion and extracellular matrix molecules allow trapping of circulating plasma cell precursors within the BM, and a battery of locally released cytokines promote their growth and final differentiation. Malignant B cells establish close contacts with BM stromal cells and release a host of cytokines that recruit and activate BM stromal cells and also T lymphocytes to produce other cytokines. All these cytokines might conceivably act in concert in a self-perpetuating mechanism of mutual help between malignant plasma cells and BM stromal cells to favor the progressive expansion of the malignant clone through a sort of an "avalanche effect." Also, most cytokines produced by malignant B cells, stromal cells, and activated T lymphocytes, including IL-1 beta, TNF-beta, M-CSF, IL-3, and IL-6, have osteoclast-activating properties, thus explaining why the expansion of the B-cell clone is matched by the activation and numeric increase of osteoclasts.

B-Lymphocytes↗
PubMed1997

Nitric oxide production in rat thalamus changes with behavioral state, local depolarization, and brainstem stimulation.

Since its discovery as a putative neurotransmitter in the CNS, several functional roles have been suggested for nitric oxide (NO). However, few studies have investigated the role of NO in natural physiology. Because NO synthase (NOS) has been localized in regions believed to be important for attention and arousal, we hypothesized that NO production would be state-dependent. To test this hypothesis, we used in vivo microdialysis, coupled with the hemoglobin-trapping technique, to monitor extracellular NO concentrations in rat thalamus during wake, slow-wave sleep (SWS), and rapid eye movement (REM) sleep. The thalamus is known to receive a massive innervation from the NOS/cholinergic neurons in the mesopontine brainstem, which have been suggested to play a key role in EEG desynchronized states. To test whether thalamic NO output was sensitive to neuronal-dependent changes in the mesopontine brainstem, we measured thalamic NO concentration in response to electrical stimulation in the laterodorsal tegmentum (LDT) of anesthetized rats. Finally, the calcium dependence of NO release was tested by local depolarization with a high potassium dialysate or by addition of a calcium chelator. The results showed that (1) extracellular NO concentrations in the thalamus were high during wake and REM sleep and significantly lower during SWS, (2) thalamic NO release increased in response to LDT stimulation in both a site-specific and tetrodotoxin (TTX)-dependent manner, and (3) NO production was calcium-dependent. These data suggest that thalamic NO production may play a role in arousal.

Animals↗
PubMed1988

A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite.

As a consequence of gene cloning and DNA sequencing several gene families are emerging in the field of cell-cell recognition. These include immunoglobulins, integrins, certain extracellular glycoproteins and a family of functionally unrelated proteins which include factor B. We report here the cloning and sequencing of a gene from Plasmodium falciparum, coding for a protein we call thrombospondin related anonymous protein (TRAP), which shares certain sequence motifs common to other well-characterized proteins. The most significant homology is based around the sequence Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), present in three copies in region I of thrombospondin (TSP), six copies in properdin (P) and one copy in all the circumsporozoite (CS) proteins sequenced so far. TRAP also shares with certain extracellular glycoproteins, including TSP, the cell-recognition signal Arg-Gly-Asp (RGD), which has been shown to be crucial in the interaction of several extracellular glycoproteins with members of the integrin superfamily. Unlike the CS protein, TRAP is expressed during the erythrocytic stage of the parasite life cycle.

Amino Acid Sequence↗
PubMed2004

Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium.

Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.

Blood Platelets↗
PubMed1989

Modulation of effect of extracellular calcium buffering in cardiac muscle.

The buffering of extracellular calcium by citrate, with identical free calcium levels in the buffered and unbuffered medium, was previously found to markedly reduce tension in frog and guinea pig atria. We now report the following results. 1) In guinea pig, postest contractions are not reduced by citrate. 2) In frog the negative inotropic effect of citrate is greatly attenuated by partially replacing extracellular sodium concentration ([Na+]o) with lithium or sucrose. In contrast, in low [Na+]o, sodium salts of weak acids (which cause intracellular acidosis) still reduce tension. These findings strongly support the suggestion that citrate does not reduce tension directly, e.g., by causing intracellular acidosis or by reducing voltage-dependent calcium currents. 3) Higher stimulation rates also decrease the effect of citrate. 4) Treatment with neuraminidase or phospholipase D, both of which alter sarcolemmal calcium binding, dose not change the effect of citrate. 5) The positive inotropic effect of strophanthidin is reversibly lost in the presence of citrate. Our results provide a different, novel approach to support earlier suggestions that the extracellular matrix and/or the sarcolemma contain abundant calcium-binding sites that supply some of the calcium for contraction. Citrate presumably binds calcium more strongly, thereby "trapping" calcium released from the extracellular "stores," so that less calcium is made available for contraction. The inotropic effect of cardiac glycosides may also depend on the calcium that is bound to these stores.

Animals↗
PubMed1997

Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.

To move forward, migrating cells must generate traction forces through surface receptors bound to extracellular matrix molecules coupled to a rigid structure. We investigated whether cells sample and respond to the rigidity of the anchoring matrix. Movement of beads coated with fibronectin or an anti-integrin antibody was restrained with an optical trap on fibroblasts to mimic extracellular attachment sites of different resistance. Cells precisely sense the restraining force on fibronectin beads and respond by a localized, proportional strengthening of the cytoskeleton linkages, allowing stronger force to be exerted on the integrins. This strengthening was absent or transient with antibody beads, but restored with soluble fibronectin. Hence, ligand binding site occupancy was required. Finally, phenylarsine oxide inhibited strengthening of cytoskeletal linkages, indicating a role for dephosphorylation. Thus, the strength of integrin-cytoskeleton linkages is dependent on matrix rigidity and on its biochemical composition. Matrix rigidity may, therefore, serve as a guidance cue in a process of mechanotaxis.

3T3 Cells↗
PubMed2002

Sandwich immunoassay for the measurement of murine syndecan-4.

A sensitive and specific immunoassay for mouse syndecan-4 is described. The assay is a non-competitive direct sandwich enzyme-linked immunosorbent assay based on the production of an affinity-purified polyclonal antibody directed against syndecan-4 extracellular domain, used both as a trapping and detecting antibody. The standard curve is constructed with recombinant 6-His-tagged syndecan-4 extracellular domain. The assay allowed quantification of syndecan-4 core protein in the partially purified proteoglycan fraction from adipocyte 3T3-F442A cells, as well as in the cellular whole and clarified lysates. Removal of glycosaminoglycan chains on syndecan-4 core protein is required for maximal epitope exposure. The standard curve ranged between 7 and 70 fmol per well. For the 14 standard curves run on different days, the absorbance at 490 nm for 35.5 fmol of recombinant syndecan-4 was 0.610 +/- 0.110 (n = 14) with a corresponding blank absorbance of 0.089 +/- 0.030. At low (5.7 fmol) and high (42.8 fmol) levels of whole cell syndecan-4, the intra-assay and inter-assay coefficients of variation were 4.9 and 15.3 percent, and 3.3 and 9.7 percent, respectively. A survey of the syndecan-4 expression in various mouse tissues shows that syndecan-4 is highly expressed in the kidney, brain, testes and liver, but can also be measured in several different adipose tissue sites.

3T3 Cells↗
PubMed2000

Extracellular mutations of protease-activated receptor-1 result in differential activation by thrombin and thrombin receptor agonist peptide.

The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide having the tethered ligand sequence (thrombin receptor agonist peptide or TRAP). We conducted a mutational analysis of extracellular residues of the receptor potentially involved in interaction with both the tethered ligand and the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laevis oocytes or inositol phosphate accumulation in COS-7 cells was used to assess receptor activation. We have also examined the binding of a radiolabeled TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses without significantly altering thrombin-induced responses or TRAP binding. Several point mutations and deletion of extracellular domains (DeltaEC3, DeltaNH3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second extracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of thrombin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants with the ability to dissociate receptor-specific binding from functional activity.

Amino Acid Sequence↗