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Results for “regulatory module”

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PubMed1996

Interleukin-1 increases vacuolar-type H+-ATPase activity in murine peritoneal macrophages.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pHi tends to be acid loaded. We have previously reported that a vacuolar-type H(+)-ATPase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pHi regulation in peritoneal macrophages. IL-1 caused a time- and dose-dependent increase in macrophage pHi recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on V-ATPase activity. Thus, cytokines present within the inflammatory milieu are able to modulate pHi regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.

Animals↗

PubMed2006

Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities.

The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO(2) tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO(2), the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.

Amino Acid Sequence↗

PubMed1995

Effects of CapG overexpression on agonist-induced motility and second messenger generation.

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control-transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.

3T3 Cells↗

PubMed2003

Global regulation in Pseudomonas aeruginosa: the regulatory protein AlgR2 (AlgQ) acts as a modulator of quorum sensing.

The Pseudomonas aeruginosa protein AlgR2 (AlgQ) was originally identified as a regulatory protein implicated in alginate production. It also regulates the synthesis of polyphosphate as well as of a variety of secretable virulence factors, upregulating neuraminidase and siderophore synthesis and downregulating rhamnolipid biosurfactant and extracellular protease synthesis. In this study, we show that the regulatory effect of AlgR2 on elastase protease synthesis is exerted at transcriptional level on the lasB gene. We also demonstrate that AlgR2 negatively modulates the expression of quorum sensing regulatory genes lasR and rhlR. Finally, results obtained from DNA retardation assays provide evidence that AlgR2 can bind specifically to the lasR and rhlR promoters. Altogether, these data provide strong support for the hypothesis that AlgR2 is a global transcriptional regulator in P. aeruginosa.

Alginates↗

PubMed2006

Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways.

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.

2-Methoxyestradiol↗

PubMed1990

lcrR, a low-Ca2(+)-response locus with dual Ca2(+)-dependent functions in Yersinia pestis.

The low-Ca2+ response (Lcr) of Yersinia includes a regulatory cascade and a set of virulence-related proteins, one of which is the V antigen. The regulatory genes modulate both bacterial growth and expression of the virulence-related proteins in response to temperature and the presence of Ca2+ and nucleotides. In this study we defined a new Lcr locus, lcrR, in Yersinia pestis KIM. An lcrR mutant, obtained by insertion mutagenesis, failed to grow at 37 degrees C whether Ca2+ was present or not. However, it grew normally in the presence of ATP, showing that the Ca2(+)- and nucleotide-responsive mechanisms are separate in Y. pestis. The lcrR mutant was avirulent in mice, probably due to its compromised growth at 37 degrees C. beta-Galactosidase measurements and Northern (RNA blot) analysis revealed that lcrR transcription was regulated primarily by temperature. The DNA sequence of the lcrR locus contained a single open reading frame of 441 bases that could encode a protein with a molecular weight of 16,470 and a pI of 10.73. Expression of an lcrR-containing clone in Escherichia coli yielded a 16,000-molecular-weight protein. At 37 degrees C, the lcrR mutant strongly expressed V antigen and initiated lcrGVH transcription whether Ca2+ was present or not, indicating that this mutant had lost the transcriptional downregulation of lcrGVH shown by the parent in the presence of Ca2+. In the absence of Ca2+, the mutant failed to express LcrG, even though lcrGVH mRNA initiated upstream of lcrG at the normal sites. These data suggest that the lcrR locus is necessary for the regulation of LcrG expression in the absence of Ca2+. Therefore, this locus has a dual regulatory role in the low-Ca2+ response.

Amino Acid Sequence↗

PubMed2001

The recruitment of SOX/OCT complexes and the differential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatory enhancer function.

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain. We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo. We now report that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

Animals↗

PubMed1987

Functional analysis of the regulatory region adjacent to the cargB gene of Saccharomyces cerevisiae. Nucleotide sequence, gene fusion experiments and cis-dominant regulatory mutation analysis.

In Saccharomyces cerevisiae the expression of the cargB gene (coding for ornithine aminotransferase) is submitted to dual regulation: an induction by allophanate and a specific induction process by arginine. We have determined the nucleotide sequence of the cargB gene along with its 5' region. The coding portion of the gene encodes a protein of 423 amino acid residues with a calculated Mr value of 46049. To characterize further the regulatory mechanisms modulating the expression of the gene we have analyzed fusions of several fragments of the 5' non-coding region to lacZ, compared the 5' sequences of the cargA (coding for arginase) and cargB coregulated genes and determined the nature of two constitutive cis-dominant mutations affecting the arginine control. These approaches allowed us to define three domains in the 5' non-coding region. The upstream one is implicated in the induction by allophanate. The two other domains are involved in the specific control by arginine; the target of the ARGR gene products, that mediate a positive regulation by arginine, lies upstream of another site where a repression by the CARGRI molecule occurs. The constitutive cargB+O- mutations are located in this repressor domain. The 5' non-coding region of cargA presents the same two-domain organization. These two domains contain three sequences homologous to the cargA and cargB 5' regions.

Amino Acid Sequence↗

PubMed1989

Beta-adrenergic modulation of transient inward current in guinea-pig cardiac myocytes. Evidence for regulation of Ca2(+)-release from sarcoplasmic reticulum by a cyclic AMP dependent mechanism.

Transient inward current (Iti) indicating Ca2(+)-release from the sarcoplasmic reticulum and L-type Ca2(+)-current (ICa) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of ICa caused by beta-adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskolin (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of Iti were used to manifest changes of sarcoplasmic Ca2(+)-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 microM EGTA spontaneous transient inward currents were recorded at a constant holding potential of -50 mV in the majority of myocytes. Superfusion with a solution containing ISO (greater than or equal to 5 x 10(-8) M) increased the amplitude of spontaneous Iti and reduced its time-to-peak. The effects of ISO on Iti developed in parallel to stimulation of ICa. In myocytes which did not show spontaneous cyclic Ca2(+)-release in the above condition, this could be evoked de novo by ISO. Spontaneous Iti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 microM. Brief (50 ms) activation of ICa by voltage steps from -50 to +10 mV usually failed to trigger Ca2(+)-release from the SR. The increase of ICa-amplitude upon administration of ISO went ahead with the induction of Ca2(+)-release by brief activation of ICa. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 3'5'-cyclic adenosine monophosphate. The effects on ICa and SR Ca2(+)-release were dependent o the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca2(+)-release events per number of Ca2(+)-currents elicited and a shortening of time-to-peak of Iti's. The stimulating effects of ISO or FORSK on Ca2(+)-release were only partially due to an increase of the triggering ICa. Ca2(+)-currents too small to trigger Ca2(+)-release before beta-adrenergic stimulation could evoke Ca2(+)-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on ICa were reversible, the stimulatory effects on Ca2(+)-release persisted after washing out the substances. The results give support to the hypothesis that beta-adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca2(+)-release.

Animals↗

PubMed1999

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Adult↗

PubMed2004

Geomagnetic field effect on cardiovascular regulation.

The goal of the present research was try to explain the physiological mechanism for the influence of the geomagnetic field (GMF) disturbance, reflected by the indices of the geomagnetic activity (K, K(p), A(k), and A(p) indices), on cardiovascular regulation. One hundred forty three experimental runs (one daily) comprising 50 min hemodynamic monitoring sequences were carried out in rabbits sedated by pentobarbital infusion (5 mg/kg/h). We examined the arterial baroreflex effects on the short term blood pressure and heart rate (HR) variabilities reflected by the standard deviation (SD) of the average values of the mean femoral arterial blood pressure (MAP) and the HR. Baroreflex sensitivity (BRS) was estimated from blood pressure/HR response to intravenous (i.v.) bolus injections of vasoconstrictor (phenylephrine) and vasodilator (nitroprusside) drugs. We found a significant negative correlation of increasing GMF disturbance (K(p)) with BRS (P = 0.008), HR SD (P =0.022), and MAP SD (P = 0.002) signifying the involvement of the arterial baroreflex mechanism. The abrupt change in geomagnetic disturbance from low (K = 0) to high (K = 4-5) values was associated with a significant increase in MAP (83 +/- 5 vs. 99 +/- 5 mm Hg, P = 0.045) and myocardial oxygen consumption, measured by MAP and HR product (24100 +/- 1800 vs. 31000 +/- 2500 mm Hg. bpm, P = 0.034), comprising an additional cardiovascular risk. Most likely, GMF affects brainstem and higher neural cardiovascular regulatory centers modulating blood pressure and HR variabilities associated with the arterial baroreflex.

Animals↗

PubMed1999

Nitric oxide reduces nontransferrin-bound iron transport in HepG2 cells.

Nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) modulate iron regulatory protein (IRP) activity and may, therefore, affect iron uptake through transferrin receptor expression. However, iron also enters the cell as nontransferrin-bound iron (NTBI), and the aim of this study was to evaluate the effects of NO donors on NTBI transport in HepG2 cells, a model of liver physiology. Incubation with SNP and SNAP led to a time- and concentration-dependent reduction in Fe3+ and Fe2+ uptake, thus indicating an effect on the transporter rather than on the reductase. In terms of Fe2+ uptake, no variations in the Michaelis-Menten constant (Km) and a reduction in maximum uptake (Vmax) (50, 33, and 16.6 fmol/microgram protein/min in control, SNP-, and SNAP-treated cells, respectively) were detected, which suggested a decrease in the number of putative NTBI transport protein(s). Gel shift assays showed that IRP activity was reduced by SNP and slightly increased by SNAP. Northern blot analysis of transferrin receptor messenger RNA (mRNA) levels showed variations similar to those observed for IRPs, but both NO donors increased L-ferritin mRNA levels and had no effect on the stimulator of Fe transport (SFT) mRNA. In conclusion, NO donors significantly reduce NTBI transport in HepG2 cells, an effect that seems to be IRP and SFT independent. Moreover, the reduction in NTBI uptake after NO treatment suggests that this form of iron may play a minor role in the increased hepatic iron stores observed in inflammation or that other liver cells are more involved in this pathological condition.

Apoptosis↗

PubMed1988

Effect of pertussis toxin on parathyroid hormone-stimulated cyclic AMP production in cultured kidney cells.

The effect of pertussis toxin, which inactivates the guanine nucleotide binding regulatory proteins Gi and Go on cAMP production in response to parathyroid hormone PGE2 or forskolin, was examined in confluent opossum kidney (OK) cells. This effect was compared with that caused by dexamethasone. The response to PTH was increased in cells preincubated with either agent. The effect of pertussis toxin was selective for PTH, since cAMP production in response to neither PGE2 nor forskolin was increased. In contrast, the response to forskolin was enhanced in dexamethasone-treated cells. These results indicate that both stimulatory and inhibitory guanine nucleotides binding regulatory proteins modulate PTH-induced cAMP production in OK cells. Moreover, pertussis toxin and dexamethasone appear to affect different levels of the PTH-receptor-adenylate cyclase complex.

Animals↗

PubMed1998

Exercise stimulates c-Jun NH2 kinase activity and c-Jun transcriptional activity in human skeletal muscle.

Exercise causes selective changes in gene expression leading to alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling pathways that enable exercise to modulate gene regulatory events. We determined the effects of exercise on c-Jun NH2-terminal kinase (JNK) activity, a signaling molecule involved in the regulation of transcription. Biopsies of vastus lateralis muscle were taken from eight subjects at rest and after 60 min of cycle ergometer exercise. Exercise increased JNK activity in all subjects (5.9 +/- 1.8 fold above basal). JNK activation was associated with an increased expression of its downstream nuclear target c-Jun mRNA. When two additional subjects were studied using a one-legged exercise protocol, JNK activity increased only in the exercising leg, indicating that exercise-induced JNK signaling represents an intrinsic response of the contracting muscle, rather than a systemic response to exercise. These studies demonstrate that the JNK pathway may serve as a link between contractile activity and transcriptional responses in human skeletal muscle.

Adult↗

PubMed1999

Cloning and partial characterization of the proteasome S4 ATPase from Plasmodium falciparum.

Certad, G., Abrahem, A., and Georges, E. 1999. Cloning and Partial characterization of the proteasome S4 ATPase from Plasmodium falciparum. Experimental Parasitology 93, 123-131. The ATP-ubiquitin-proteasome pathway mediates the nonlysosomal degradation of cytosolic proteins in eukaryotic cells. The activities of this pathway have been shown to regulate cell growth and differentiation through modulation of regulatory proteins. The proteasome is a large complex consisting of two multisubunit structures, the 20S and 19S(PA700) or P28 complexes, that combine to form the 26S particles. In this study, we describe the cloning of a cDNA encoding the proteasome subunit 4 ATPase homologue from Plasmodium falciparum (PFS4). Analysis of the PFS4 cDNA sequence shows an open reading frame encoding a deduced protein of 455 amino acids. Moreover, comparison of PFS4 cDNA sequence to that of genomic fragments encoding PFS4 showed identical sequences with no detectable introns. Database searches revealed a high sequence identity to those of rice, yeast, mouse, Drosophila, and human S4 ATPases. However, PFS4 contains two unique inserts of nine and seven amino acid residues in the N-terminal domain. Interestingly, only the rice S4 contains the latter (seven amino acids) insert with four identical amino acids. In vitro expression of the full-length cDNA encoding the PFS4, using a transcription-translation-coupled reticulocyte lysate, shows a 50-kDa [(35)S]methionine-labeled protein which was immunoprecipitated with PFS4 anti-peptide antiserum. Southern blot analysis of genomic DNA digests shows a single gene copy of PFS4 in P. falciparum. Of interest was the effect of the proteasome-specific natural product, lactacystin, on the growth of the parasite, with IC(50) values of 0.6-0.92 microM. The latter IC(50) values of lactacystin for different clones of P. falciparum are comparable to those obtained for mammalian cell lines (0.65 microM), suggesting the presence of a conserved proteasome complex. Moreover, lactacystin was equally toxic to drug-sensitive and resistant parasites.

Acetylcysteine↗

PubMed1996

Autophosphorylation of a bacterial protein at tyrosine.

Autophosphorylation at tyrosine is a common process in eukaryotic kinases, which is generally modulated by regulatory ligands and affects the properties of these enzymes. We report that this type of modification occurs also in bacteria, namely in an 81 kDa protein from Acinetobacter johnsonii. This protein is phosphorylated at the expense of ATP exclusively at tyrosine residues. It is located in the inner-membrane fraction of cells and can be totally solubilized by detergents. It has been purified to homogeneity by antiphosphotyrosine immunochromatography. Analysis of the peptides released under trypsin proteolysis of the protein has shown that it autophosphorylates at several tyrosine residues. The discovery of protein autophosphorylation in bacteria seems of special interest for studying the regulatory aspects of this modification when considering the relative simplicity of the bacterial systems, as compared with most eukaryotic systems, namely in terms of physiology and genetics.

Acinetobacter↗

PubMed1997

Protein kinase C inhibits epidermal growth factor receptor phosphorylation in enterocytes.

Epidermal growth factor (EGF) is an important proliferative signal in the gastrointestinal tract. The EGF receptor (EGFr), which transduces the mitogenic stimulus to the cell, may be regulated by a number of factors including extracellular matrix, cell-cell contact, and other peptides. As protein kinase C (PK-C) has been shown to phosphorylate and down-regulate the EGFr in certain tumor cell lines, we propose that PK-C, an important regulatory enzyme, modulates the phosphorylation of the EGFr in the IEC 6 rat enterocyte cell line. IEC 6 cells were cultured in dishes with Dulbecco's modified Eagle's medium, (DMEM)/5% fetal bovine serum (FBS), which was changed to DMEM/1% FBS 24 hr prior to all experiments. Cells (three dishes per group) were treated with the PK-C activating phorbol ester phorbol-12-myristate-13-acetate (PMA) (100 nM) or vehicle for 1 hr and challenged with EGF (50 ng/ml) or vehicle for 15 min. Cell lysates were then prepared. EGFr tyrosine phosphorylation was determined by immunoprecipitating the EGFr and immunoblotting with an antibody against phosphotyrosine. EGFr apparent molecular weight was assessed in the same lysates by Western blot with an anti-EGFr antibody. Blots were analyzed by computer densitometry. Data are expressed as mean +/- SEM; n = 3 with P value determined by t test. Exposure of cells to PMA resulted in a decrease in the EGF-stimulated EGFr phosphotyrosine content from 96 +/- 5 U in control to 66 +/- 6 U in PMA (P < 0.01). The amount of receptor did not change, 43 +/- 3 U in control vs 44 +/- 3 U in PMA (P = 0.44). Further, exposure to PMA in the absence of EGF caused a gel shift of the EGFr band consistent with a nontyrosine phosphorylation of the protein. We demonstrate that activation of PK-C results in a modification of the EGFr coincident with inhibition of EGF-stimulated receptor tyrosine kinase activity. These data support a role for PK-C in the regulation of EGFr function and hence modulation of mitogenic signals in enterocytes.

Animals↗

PubMed1993

The cellular immunology of bovine paratuberculosis: immunity may be regulated by CD4+ helper and CD8+ immunoregulatory T lymphocytes which down-regulate gamma/delta+ T-cell cytotoxicity.

In a previous investigation, we obtained evidence that the major histocompatibility complex (MHC)-restricted proliferative response of CD4+ lymphocytes to Mycobacterium paratuberculosis antigens was depressed in naturally infected and immunized animals. Findings suggested that depression of the response was attributable to an abrogation in the ability of CD4+ cells to respond to specific antigens and/or the actual loss of antigen-reactive cells. In vitro cell experiments indicated that the depression was associated with the presence of gamma/delta+ T cells that modulated CD4+ cell function. Examination of additional animals confirmed and extended these observations and showed that the ability of gamma/delta+ T cells to regulate CD4+ responses were blocked by the presence of CD8+ cells. CD4+ T cells from some exposed animals incorporated [3H]-thymidine in the presence of CD8+, gamma/delta+ cells and/or antigen and antigen-presenting cells, but CD4+ cell proliferation was abrogated when CD8+ were excluded from the assays. Likewise, gamma/delta+ T-cell proliferation was abrogated when CD8+ cells were present. The mechanism by which CD8+ cells blocked gamma/delta+ T-cell responses could not be determined, however, the observed effect resembled the veto cell phenomenon. The data suggest that the development of protective immunity against M. paratuberculosis may be dependent on the capacity of CD8+ cells to modulate the regulatory activity of gamma/delta+ T populations.

Animals↗