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PubMed1975

Diagnosis and prognosis in colon cancer based on a profile in immune reactivity.

An immunologic profile consisting of measurements of circulating carcinoembryonic antigen (CEA), tumor antigen-induced inhibition of monomuclear cell migration (IMM) and skin reactivity to purified protein derivative, streptokinase-streptodornase, and mumps was assessed as a diagnostic and prognostic tool in 16 patients with colon cancer. Preoperatively, 10 of 14 patients tested had elevated CEA, 12 of 12 showed tumor antigen-induced IMM, and 10 of 11 failed to react to 2 or more recall antigens. Potential surgical cure (7 patients) was accompanied by normal CEA in 4, absent tumor antigen-induced IMM in all 7, and increased skin-test reactivity in 6. Disseminated cancer (9 patients) was associated with elevated CEA in all 9, with absent IMM in all 7 and with suppressed skin-test reactivity in 6 of 9.

Adult↗
PubMed1988

Pyridine induction of cytochrome P-450 in the rat: role of P-450j (alcohol-inducible form) in pyridine N-oxidation.

Previous research has shown that pyridine (PY) pretreatment of rabbits elevates total hepatic microsomal cytochrome P-450 content in association with the appearance of intense bands in the region of P-450LM4 and LM3 in the electrophoretic pattern of the microsomes and increased rates of PY N-oxide production and N-nitrosodimethylamine, alcohol and aniline metabolism. In the present study, PY administration (100 mg/kg i.p., 4 days) to rats was found to elevate hepatic microsomal cytochrome P-450 content approximately 2.5-fold as compared to controls. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a protein band of enhanced intensity migrating in the region of P-450j, the major ethanol-inducible form of rat liver cytochrome P-450. Immunochemical analysis confirmed that the level of immunoreactive P-450j protein was increased in PY-induced microsomes as compared to preparations from control, phenobarbital- or beta-naphthoflavone-induced animals. The rate of PY N-oxide production was enhanced 4-fold in PY-induced microsomes relative to untreated controls and was approximately 1.5- to 2-fold greater than that monitored for either phenobarbital- or beta-naphthoflavone-induced microsomes. When data were normalized for increased P-450 content, an altered substrate specificity was suggested for the PY-induced microsomes. para-Nitrophenol hydroxylase activity, which is exhibited primarily by the ethanol-inducible form of P-450, was elevated approximately 4-fold in PY-induced microsomes relative to uninduced microsomes and, when expressed per nanomol of P-450, was approximately 2- to 3-fold greater in PY-induced preparations as compared to phenobarbital-, beta-naphthoflavone-induced, or uninduced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Hydroxylase↗
PubMed1989

Screening for lipoprotein[a] elevations in plasma and assessment of size heterogeneity using gradient gel electrophoresis.

Plasma was screened for the presence of lipoprotein[a] using 2-16% nondenaturing, polyacrylamide gradient gel electrophoresis. Gels were scanned with a densitometer after staining with Sudan black B. Bands that migrated above low density lipoprotein bands were identified as lipoprotein[a] by immunoblotting with polyclonal and monoclonal antibodies to apolipoprotein[a]. Lipoprotein[a] was measured by gradient gel electrophoresis and by radioimmunoassay in 115 male patients with premature coronary artery disease and 132 control subjects. Lipoprotein[a] bands were detected in 96.7% of subjects with lipoprotein[a] values above 40 mg/dl; in 31.3% with values between 21 and 40 mg/dl, and in 6.5% with values below 20 mg/dl. This gel methodology is a simple and effective procedure for detecting elevated plasma lipoprotein[a] levels and for investigating size heterogeneity, but does not replace immunoassay for quantitation.

Coronary Disease↗
PubMed1992

Maturing thymocytes in accelerated rejection of cardiac allografts in presensitized rats.

LBNF1 cardiac allografts are rejected within 36 hr in LEW rats sensitized with BN skin grafts 7 days earlier (acute rejection in unmodified hosts = 8 days). We have studied and compared the function and migration patterns of thymocytes one day after engraftment in sensitized recipients, unmodified hosts, and normal naive rats. Thymocytes from animals experiencing accelerated rejection were more mature and functionally active, as shown by a significant elevation in percentage of OX-44+ (CD37+) cells, increased alloreactivity to BN and WF antigens, and proliferative responses to Con A and exogenous IL-2. However, the cells could neither lyse BN targets in vitro nor trigger rejection of otherwise indefinitely functioning test cardiac allografts in immunologically unresponsive T cell-deficient (B) rats after adoptive transfer. The traffic of 111In-labeled thymocytes was then evaluated. The migration index increased significantly during accelerated graft rejection, with thymocytes preferentially circulating in the blood, penetrating peripheral lymph nodes--and, interestingly, migrating back to the thymus. Thus, immunoresponsive and functionally active thymocytes, which lack the ability to recognize primed specific antigen, appear during accelerated rejection of cardiac allografts in sensitized rats. These cells migrate to the periphery, and then return in large numbers to their site of origin, the thymus. Hence, this study describes a novel behavior of thymocytes in the state of host alloreactivity that is distinct from the physiological one in otherwise normal thymus.

Animals↗
PubMed2006

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells.

OBJECTIVE: Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. METHODS: Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. RESULTS: Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells. CONCLUSIONS: Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Blotting, Western↗
PubMed2003

The requirement of the LC8 dynein light chain for nuclear migration and septum positioning is temperature dependent in Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, the multisubunit motor complex cytoplasmic dynein plays essential roles in nuclear migration and septum positioning. The 8 kDa light chain, LC8, the smallest subunit, is conserved among eukaryotic organisms. Besides being a component in the dynein complex, LC8 also interacts with a wide spectrum of mammalian and viral proteins. To date, the function of this small polypeptide is not well understood. To address this issue, we have created a deletion mutation (DeltanudG) at the nudG locus encoding LC8 in A. nidulans. At 42 degrees C, the DeltanudG mutant forms minute colonies lacking asexual reproduction: this phenotype resembles the phenotype of the dynein heavy chain null mutant. The mutant nuclei largely clustered in the spore body after conidial germination, and the septum was often assembled distally toward the hyphal apex, whereas a control germling has its nuclei distributed along the hypha and the septum formed near the spore body. When the mutant was grown at 23 degrees C, however, its colony resembled a control one, and so did the patterns of nuclear distribution and septum positioning. Elevation of the growth temperature gradually reduced colony size and abolished asexual sporulation. After a period of growth at 23 degrees C that allowed the nuclei to move out of the spore end, a temperature shift to 42 degrees C prevented newly divided nuclei from migrating apart, suggesting that LC8/NUDG was required for both initiating and maintaining dynein motor functions at elevated temperatures. A functional GFP-NUDA fusion was used to test whether LC8/NUDG is required for DHC (dynein heavy chain)/NUDA localization. We found that at 23 degrees C GFP-NUDA localized to the hyphal apex and the septation site in DeltanudG cells as in control cells. Such localizations were absent at 42 degrees C in mutant cells, but not in control cells. We conclude that LC8 plays a role in DHC localization/function, and the requirement for such a role in A. nidulans cells is temperature dependent.

Aspergillus nidulans↗
PubMed1983

Interferon-induced increase in neuraminidase-releasable sialic acid and glycosphingolipid metabolism in mouse lymphoma and L1210 leukemic cell lines: correlation with susceptibility to natural killer cell-mediated lysis.

Changes in sialoglycoconjugates and glycosphingolipid (GSL)5 metabolism were demonstrated in mouse EL4, P52 and YAC-1 lymphoma and L1210 leukemia cell lines treated with beta-interferon (IFN). Expression of cell surface (neuraminidase-releasable) sialic acid on IFN-treated cells was markedly elevated (three- to six-fold). The increase in neuraminidase-releasable sialic acid is contributed by sialoglycoproteins and particularly by cell-surface gangliosides in IFN-treated cells. Incorporation of [3H]-galactose into all GSL was elevated in IFN-treated cells. Thin-layer chromatographic analysis of GSL of IFN-treated cells showed an increase in several GSL homologues with striking changes in ganglioside with chromatographic migration of GM2, GM1, and GD1a relative to control cells. IFN-treated tumor-cell lines became resistant to lysis by virus-induced IFN-activated natural killer (NK) cells, as shown previously, but addition of neuraminidase to IFN-treated and untreated cells caused only a moderate increase in NK-sensitivity. This suggests that IFN-mediated protection of target cells from NK lysis was not due to a preferential masking of target structure by elevated levels of sialic acid. These membrane-associated changes in GSL and sialic acid in IFN-treated cells may be potentially significant, because a correlation between certain GSL expression, sialic acid phenotype and susceptibility of target cells to NK-cell-mediated lysis have been found in several other systems.

Animals↗
PubMed1993

[Immunologic aspects of allogenic valve implantation].

Immunological response against myocardial antigens was assessed in 12 patients (pts) (9 males, 3 females, aged 24-62 years) with aortic homografts (AH) and 36 pts (29 males, 7 females, aged 26-61 years) with aortic valve prostheses (AVP). In all pts a titre of anti-myocardial antibodies, a concentration of free-circulating immune complexes and leucocytes migration inhibition test with myocardial antigen were assessed before operation and 1, 12, 24 months after operation. In 20 pts with AH immunological humoral response against HLA antigens was evaluated. After the operation pathological indexes of immunological response against myocardial antigens were observed both in pts with AH as in pts with AVP. The mean titre of anti-myocardial antibodies before operation was in both groups between 1:4 and 1:8, and 1 month after operation between 1:32 and 1:64. In 12 and 24 months after operation the mean titre in AH and AVP group was respectively 1:8 and 1:4. Before operation pathological concentration of immune complexes (> 0.084 mg/ml) was observed in 17% AH and 11% AVP pts. Pathological elevation of immune complexes 1, 12 and 24 months after operation was observed respectively in AH and AVP group in 58%, 42%, 33% and 67%, 50%, 42%. Leucocyte migration inhibition index was pathological (< 0.8) before operation in 58% in both groups, and 1, 12, 24 months after operation was pathological in AH and AVP group respectively in 67%, 17%, 17% and 61%, 33%, 31%. In most pts of both groups immunological response against myocardial antigens was transient and was terminated up to 12-24 months after operation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1997

Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior.

PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production. METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment. The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels. The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment. RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF. Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls. Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment. Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period. The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period. The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period. CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate. These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.

Antimetabolites↗
PubMed2005

Increased plasma levels of angiogenin and the risk of bladder carcinoma: from initiation ot recurrence.

BACKGROUND: Angiogenesis is a well known prerequisite for tumor growth and metastasis. It is believed that angiogenin initiates cell migration and aids cell proliferation. Based on this, the authors hypothesized that individuals who had increased plasma levels of angiogenin were at an elevated risk for carcinoma of the urinary bladder. METHODS: In this ongoing case-control study, the authors used an enzyme-linked immunosorbent assay to compare plasma levels of angiogenin in 209 patients with bladder carcinoma and in 208 healthy control participants who were matched according to age (+/- 5 years), gender, and ethnicity. RESULTS: The mean plasma angiogenin concentration was significantly higher in patients compared with controls (343.2 ng/mL vs. 308.0 ng/mL, respectively; P < 0.01). High plasma angiogenin levels were associated with a two-fold increased risk for bladder carcinoma. Moreover, in patients who had superficial bladder carcinoma, plasma angiogenin levels were significantly higher among those who had recurrent disease than in those who were without recurrence (P < 0.01). Similarly, patients who had superficial bladder carcinoma with higher angiogenin levels had a shorter recurrence-free survival than patients who had lower angiogenin levels (P < 0.01). Finally, elevated angiogenin levels were associated with an increased recurrence risk, with hazard ratio of 2.85. CONCLUSIONS: The results of this study demonstrated that the plasma levels of angiogenin were significantly higher in patients who had bladder carcinoma compared with healthy control participants and in patients with superficial bladder carcinoma who had recurrent disease compared with patients who were without recurrence. Therefore, an elevated plasma level of angiogenin may serve as a novel predictor for the risk of bladder carcinoma.

Biomarkers, Tumor↗
PubMed1990

Cancer in migrants to Uruguay.

Mortality rates from different cancers in the principal groups of migrants to Uruguay are compared with those in their countries of origin (Argentina, Brazil, Italy and Spain) and in the Uruguay-born population. Oesophageal cancer is very common in Uruguay and European-born populations, initially at low risk, appear to acquire rather higher rates after migration. For most migrants, the mortality from cancers of the breast, colon-rectum, corpus uteri and prostate is closer to the moderately elevated rates of Uruguay than those in their countries of origin. The results suggest that migrants to Uruguay undergo changes in some important environmental exposures, probably dietary, which give rise to substantial alterations in cancer risk within their lifespan.

Age Factors↗
PubMed2007

Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages.

The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2-EGFP. In serum-free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2-EGFP to the plasma membrane. Serum-induced translocation was blocked by transfection of short-form TRPV2 (s-TRPV2) lacking a pore-forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met-Leu-Phe (fMLP) also induced translocation of TRPV2-EGFP to the plasma membrane. The fMLP-induced translocation was blocked by an inhibitor of PI 3-kinase, LY294002, and pertussis toxin. Whole-cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s-TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s-TRPV2 or siRNA. Finally, fMLP-induced migration of macrophage was blocked by ruthenium red or transfection of s-TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP-induced calcium entry.

Amino Acid Sequence↗
PubMed1989

Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates.

The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Amino Acids↗
PubMed2006

Changes in gap junctional connexin isoforms during prostate cancer progression.

BACKGROUND: Connexins have their traditional function as part of gap junction (GJ) structures, but have recently been shown to have GJ-independent roles. Although GJs and their connexin subunits are thought to be down-regulated in cancer, depending on the connexin examined, many times the expression level is preserved or even increased. This is further apparent by the importance of GJs in "bystander effects" of radiation and viral targeting treatments. METHODS: We surveyed connexin isoforms in prostate cancer cell lines and tissue with RT-PCR and immunohistochemistry. Upon modulating GJ function, we observed prostate epithelial cell behaviors. RESULTS: Advanced cells within PC-3 and LNCaP prostate cancer progression models exhibit elevated connexin 26 (Cx26) levels-a trend validated in clinical samples. When GJs were inhibited, adhesion was not affected, but invasion and migration were strikingly decreased. A link between the expression of Cx26 and integrin adhesion-linked functions are suggested by Cx26's direct interaction with focal adhesion kinase (FAK). CONCLUSIONS: These results suggest a novel mechanism for adhesion regulation by a GJ-independent Cx26 function that correlates with prostate disease progression. The increased Cx26 expression during prostate cancer progression plays a role in adhesion regulation possibly through its interaction with FAK.

Cell Adhesion↗
PubMed1991

Induction of hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes.

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.

Antibiotics, Antineoplastic↗
PubMed2003

Crucial role of local peroxynitrite formation in neutrophil-induced endothelial cell activation.

INTRODUCTION AND METHODS: The reaction of superoxide anions and NO not only results in a decreased availability of NO, but also leads to the formation of peroxynitrite, the role of which in the cardiovascular system is still discussed controversially. In cultured human endothelial cells, we studied whether there is a significant interaction between endothelial NO and neutrophil-derived superoxide anions in terms of endothelial peroxynitrite formation. We particularly studied whether a significantly higher redox-stress can be found in those endothelial cells directly adjacent to an activated neutrophil. RESULTS: A considerable part of the 2,7-dihydrodichlorofluoresceine signal in endothelial cells was due to oxidation by peroxynitrite. Providing superoxide radicals by enzymatic source or by the neutrophil respiratory burst increased the fluorescence, which was attenuated by blockade of endothelial NO-synthase, suggesting that peroxynitrite was formed from neutrophil- or extracellular enzyme-derived superoxide and endothelial NO. Considerably higher fluorescence intensity was observed in endothelial cells in direct neighborhood to a neutrophil. This was particularly pronounced in the presence of a NO-donor and was accompanied by a strong activation of NF-kappaB and increased expression of E-selectin in these cells. CONCLUSION: Endothelial cells adjacent to neutrophils may have elevated levels of peroxynitrite that result in an increased expression of adhesion molecules. Such cells might represent a preferential site for adhesion and migration of additional neutrophils when simultaneously high concentrations of NO and neutrophil-derived superoxide are present.

E-Selectin↗
PubMed1998

Sodium azide enhances neutrophil migration and exocytosis: involvement of nitric oxide, cyclic GMP and calcium.

Azide, in the absence of other stimuli, enhanced neutrophil migration in a chemotactic way. The effect of azide on migration was significant at concentrations > or = 1 microM and maximal at 10 microM azide. Although azide itself could not induce exocytosis, at concentrations > or = 10 microM azide enhanced exocytosis induced by a combination of the chemotactic peptide f-methionyl-leucyl-phenylalanine (fMLP) and cytochalasin B (CB). Azide can be oxidized by catalase and myeloperoxidase in the presence of H2O2, resulting in the generation of nitric oxide (NO). Formation of NO from azide was detected by ESR spectroscopy with carboxy-PTIO as a NO-selective probe, and by measurement of nitrite formation. Azide-induced migration, and the enhancement by azide of fMLP/CB-induced exocytosis, were blocked by pre-incubating cells with aminotriazole, an inhibitor of catalase and myeloperoxidase, suggesting that the effect of azide was mediated by NO. Azide-induced migration, but not the enhancement by azide of fMLP/CB-induced exocytosis, was inhibited to a large extent by inhibitors of soluble guanylate cyclase and by inhibitors of cGMP-dependent protein kinase. These observations suggest that azide-induced migration is mediated via cGMP and cGMP-dependent protein kinase, while the enhancement of fMLP/CB-induced exocytosis is not. Azide caused a sustained elevation of the intracellular Ca2+-concentration of neutrophils stimulated with fMLP/CB, which was not affected by inhibitors of the cGMP-signalling cascade. Since neutrophil exocytosis has been shown to be closely correlated with increases in intracellular Ca2+, a further increase by azide of the intracellular Ca2+-level of cells stimulated with fMLP/CB provides a likely mechanism for the enhancement of fMLP/CB-induced exocytosis by azide.

Animals↗
PubMed1995

Phosphorylation of tau in situ: inhibition of calcium-dependent proteolysis.

In this study, the in situ phosphorylation and subsequent calcium-activated proteolysis of tau protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of tau was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated tau migrated slightly slower on sodium dodecyl sulfate-polyacrylamide gels than tau from untreated cells. Immunostaining with the phosphate-sensitive monoclonal antibody Tau-1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser-Pro motifs within the molecule. Calcium-dependent, in situ proteolysis of tau protein was induced by treating the cells with the calcium ionophore A23187. Tau protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified tau protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with tau isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of tau metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.

Calcimycin↗