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Results for “regulatory module”

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PubMed2000

Role of CD28/B7 costimulation in the dexamethasone-induced suppression of IFN-gamma.

In vitro exposure of peripheral blood mononuclear cells (PBMC) to glucocorticoids (GC), at concentrations observed during psychologic stress, induces a shift in the human type 1/type 2 cytokine balance toward a type 2 cytokine response. The mechanisms involved in these cytokine alterations are unknown but likely include modulation of regulatory cytokines or the interaction between the antigen-presenting cell (APC) and T lymphocyte or both. The CD28/B7 costimulation pathway has been reported to modulate the type 1/type 2 cytokine balance and may contribute to the GC-associated cytokine alterations. Therefore, we sought to determine the effect of dexamethasone (Dex) on the expression and function of the human CD28/B7 costimulatory pathway and whether these alterations contribute to the Dex-induced type 1/type 2 cytokine alterations. Dex inhibited the expression of both CD80 and CD86 on THP-1 cells, a human acute monocytic leukemia cell line, as determined by flow cytometry. Dex also inhibited the expression of CD28 and CTLA-4 on phytohemagglutinin (PHA)-stimulated CD3+ T lymphocytes, which was attenuated by the addition of interleukin-12 (IL-12). Lastly, activation of CD28 with anti-CD28 antibody attenuated the Dex-induced decrease in interferon-gamma (IFN-gamma) production by anti-CD3 antibody-stimulated PBMC. These data suggest that Dex induces a modulation of the CD28/B7 costimulatory pathway that contributes to the shift in the type 1/type 2 cytokine balance toward a predominant type 2 cytokine response.

Abatacept↗

PubMed1995

Pathogenesis of psoriasis.

The information summarized in this article supports the "framework hypothesis." The details of cells of the immune system in the pathogenesis are covered elsewhere in this issue. We believe the observations reported herein allow the following conclusions. The entire epidermis of any individual with the psoriatic phenotype has the capacity to express overt clinical disease. Control of expression is linked to a complex interaction of cells of the epidermis, cells of the dermis, cells of the immune system, and possibly other noncellular humoral elements. The keratinocytes of psoriatic patients are unique in that the inherent phenotype has a capacity for hyperproliferation and altered differentiation. Proliferation and differentiation are controlled at the level of the gene. Thus, it is important to consider not only cytokines and growth factors released by the various cell types, but also the role of regulators of transcription, translation, and the modification of the cytokines and growth factors. The large number of alterations of cytokine and growth factor profiles within psoriasis cause us to postulate that the genetic aberration in psoriasis is quite basic, that is, it is proximal to the common element in the cascade of inflammatory events that lead to a lesion of psoriasis. We speculate that such a defect may be in transcription regulatory elements associated with one or more cytokines (or growth factors). This mutation could occur (1) in the regulatory element itself; (2) in the receptor, which binds both ligand and regulatory element; (3) in the ligand (cytokine or growth factor); or (4) in a gene responsible for the control of proliferation that is under the influence of the sites mentioned in 1, 2, and 3. A mutation within a key regulatory element for a gene controlling proliferation could produce the following scenario of lesional expression. Such a mutation will probably result in less affinity for a receptor/ligand complex. Because this regulatory element in interaction with the receptor/ligand complex normally suppresses gene expression of a promoter of proliferation, the result is a phenotype that tends to be more proliferative and less differentiative. Conversely, if such a mutation produced a regulatory element that has increased affinity for a promoter receptor/ligand complex, then the same phenotype can evolve. Because a wide variety of receptor/ligand complexes can modulate each regulatory element, such a genetic defect could have wide-ranging effects on phenotypic expression. Moreover, as this change in affinity for binding the receptor/ligand complexes only represents a change in sensitivity, not total unresponsiveness, one can expand on the scenario to conclude that the clinical phenotype is only expressed once the system is overwhelmed. This scenario fits the information presented. Both keratinocytes and fibroblasts from psoriatic subjects are hyperproliferative, but only under specific culture conditions. Furthermore, psoriatic keratinocytes appear to be relatively resistant to the antiproliferative and prodifferentiative effects of retinoids or the vitamin D analogues that have antipsoriatic activity. If a genetic defect decreases the affinity of a suppressive receptor/ligand complex for the regulatory element of IL-6 or TGF-alpha, for example, then these cells could be relatively resistant to homeostatic regulation by indigenous corticosteroids, vitamin D, and retinoids. This would result in a situation in which any trigger phenomenon that releases a cytokine, whose receptor/ligand complex upregulates this regulatory element, would overwhelm the defective counterregulatory signal. Subsequently, cells in the skin generate additional cytokines that invoke an inflammatory cascade that eventuates in the egress of immune cells into the skin and the evolution of a lesion of psoriasis. Similar scenarios could be derived from mutations in the other pathways described in this article.

Cytokines↗

PubMed1996

The regulatory light chains of myosin modulate cross-bridge cycling in skeletal muscle.

We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' -tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling.

Animals↗

PubMed1995

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures.

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary long-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-beta 1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified.

Bone Marrow↗

PubMed1985

Regulation of the expression of iso 2-cytochrome c gene in S. cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3.

CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis. Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element. The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site. Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function. This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions. To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate+ phenotype on an iso 1-cytochrome c-deficient strain. Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate- recipient strain. The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.

Alleles↗

PubMed2003

Biphasic effects of leptin in porcine granulosa cells.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

Animals↗

PubMed2004

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin.

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.

Actomyosin↗

PubMed1999

Galanin and galanin receptors.

The development of a strain of galanin knockout mice has provided confirmation of a neuroendocrine role for galanin, as well as supporting results of previous physiological investigations indicating a role for galanin in analgesia and neuropathic pain, and potentially in neuronal growth and regeneration processes. Whether elevation of galanin expression in neurodegenerative disorders such as Alzheimer's disease represents a survival response or exacerbates functional deficit in afflicted individuals remains to be determined. More detailed analysis of the phenotype of the galanin knockout mouse should provide insights into the physiological role of galanin in memory and learning processes, as well as in hypothalamic function and other aspects of neuroendocrine regulation. Biochemical and molecular cloning efforts have demonstrated that the multiplicity of actions of galanin is matched by complexity in the distribution and regulation of galanin and its receptors. A focus on characterisation of galanin receptors has resulted in the molecular cloning of three receptor subtypes to date. The distribution and functional properties of these receptors have not yet been fully elucidated, currently precluding assignment of discrete functions of galanin to any one receptor subtype. It is not currently possible to reconcile available pharmacological data using analogs of galanin and chimeric peptides in functional assay systems with the pharmacological properties of cloned receptor subtypes. This highlights the value of further knockout approaches targeting galanin receptor subtypes, but also raises the possibility of the existence of additional receptor subtypes that have yet to be cloned, or that receptor activity may be modulated by regulatory molecules that remain to be identified. The development of receptor subtype-specific compounds remains a high priority to advance work in this area. The ability to selectively modulate the many different actions of galanin, through a clearer understanding of receptor structure-function relationships and neuronal distribution, promises to provide important insights into the molecular and cellular basis of galanin action in normal physiology, and may provide lead compounds with therapeutic application in the prevention and treatment of a range of disorders.

Amino Acid Sequence↗

PubMed1997

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi.

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kdapp for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.

Amino Acid Sequence↗

PubMed1995

Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer.

Most eukaryotic genes are controlled by a complex array of cis-acting regulatory elements that modulate transcriptional activity. Two major regulatory elements reside in the chicken ovalbumin gene, a steroid-dependent regulatory element (SDRE, -892 to -780) and a negative regulatory element (NRE, -308 to -88). The SDRE is required for responsiveness to estrogen and glucocorticoid. The NRE appears to have the dual role of repressing transcription in the absence of steroids and of cooperating with the SDRE to activate transcription in the presence of steroids. The experiments described herein were designed to investigate the role of the NRE in repressing gene expression. Transfection of OvCAT fusion genes containing deletions in the NRE into primary oviduct cell cultures identified three elements (-308 to -256, -239 to -220, and -174 to -88) that repress transcription. Oligomers corresponding to portions of these elements also independently repress the viral thymidine kinase promoter. Interestingly, the element from -239 to -220 functions mechanistically as a silencer and shares sequence identity with silencers in other genes (TCTCTCCNA). Mobility shift studies indicated that all of the negative elements bind specific protein complexes from oviduct, none of which is appreciably affected by treatment with steroid hormones. However, oviduct-specific proteins bind to the regions from -280 to -252 and from -134 to -88, providing the first identification of potential tissue-specific elements in the ovalbumin gene. These results demonstrate that the region of DNA originally called the NRE is a multifunctional regulatory element that may be involved in several diverse regulatory activities.

Animals↗

PubMed2003

Potential use of drugs that target neural-immune pathways in the treatment of rheumatoid arthritis and other autoimmune diseases.

Many autoimmune disorders share two common features, dysregulation of the immune system and stress pathways. Two stress pathways, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS), regulate immune system responses, through release of corticosteroids and norepinephrine (NE), respectively. These neuromediators act on immune cells via specific receptors on their surface to modulate the production of key regulatory cytokines. Glucocorticoids modulate immune responses by glucocorticoid binding to cytoplasmic glucocorticoid receptors within target cells. NE regulates immune responses through interaction with plasma membrane beta- or alpha-adrenergic receptors (AR). Both NE and glucocorticoids promote humoral immunity by altering macrophages and T cell cytokine production after an antigen challenge. Glucocorticoids and NE do this by inhibiting interleukin (IL)-12, and interferon (IFN)-gamma, which drives cell-mediated immunity. Additionally, catecholamines drive humoral immunity by stimulating macrophage IL-10 production. These catecholamine effects are mediated largely via beta(2)-AR activation. Both glucocorticoids and NE inhibit inflammation. However, under some circumstances NE promotes inflammation through interaction with macrophage alpha1-AR and subsequent increases in tumor necrosis factor alpha (TNFalpha production. Although macrophages do not normally express alpha(1)-AR, expression of this receptor on macrophages and monocytes occurs in some disease states, including rheumatoid arthritis (RA). Through these mechanisms the HPA axis and the SNS influence the course and progression of RA. Thus, the HPA axis and the SNS are likely to play key roles in the pathology of RA. Furthermore, therapeutic agents targeting the neural pathways that normally regulate immune system homeostasis may prove beneficial for treating RA and other autoimmune diseases.

Adjuvants, Immunologic↗

PubMed2005

Biochemical properties of peptides encrypted in bovine milk proteins.

Milk proteins are precursors of many different biologically active peptides. These peptides are inactive within the protein sequence, requiring enzymatic proteolysis for release of the bioactive fragment from the proteins precursor. It is evident that activated peptides originating from milk proteins should be taken into account as potential modulators of various regulatory processes in the body. Activated peptides are potential modulators of various regulatory processes in the living system: immuno-modulatory peptides stimulate the activities of cells of the immune system and several cytomodulatory peptides inhibit cancer cell growth, antimicrobial peptides kill sensitive microorganisms, angiotensin-I-converting enzyme (ACE)-inhibitory peptides exert an hypotensive effect, opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, mineral binding peptides may function as carriers for different minerals, especially calcium, antithrombotic peptides inhibit fibrinogen binding to a specific receptor region on the platelet surface and inhibit aggregation of platelets. Moreover, many milk-derived peptides reveal multifunctional properties, i.e. specific peptide sequences having two or more different biological activities have been reported. Bioactive peptides can interact with target sites (e.g. receptors, enzymes) at the luminal side of the intestinal tract, or they could be absorbed and reach any potential site of action in the system to elicit physiological effects. Bioactive peptides encrypted in bovine milk proteins can be produced on an industrial-scale and are claimed to be health enhancing components for functional foods, nutraceuticals and pharmaceutical preparations that are used to reduce risk of disease or to enhance certain physiological functions.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2005

Gene therapy targeted at calcium handling as an approach to the treatment of heart failure.

Chronic congestive heart failure primarily of ischemic origin remains a leading cause of morbidity and mortality in the United States and other leading countries. The current main stream of therapy is, however, palliative and uses a complex regimen of drugs, the actions of which are not understood completely. On the other hand, unfavorable remodeling after cardiac injuries of multiple causes has been thought to lead to cardiac contractile dysfunction in heart failure, and a body of scientific evidence points to a central role of intrinsic defects in intracellular calcium handling in cardiomyocytes that arise from the distorted functions of several key regulatory molecules on plasma membrane or sarcoplasmic reticulum (SR), a muscle-specific intracellular membrane complex that stores calcium at high concentration. Accordingly, the initial appetite to use gene transfer strategies to modulate calcium regulatory proteins was to validate molecular targets for the development of new pharmaceuticals; however, remarkable therapeutic efficacies found in an initial series of studies using various heart failure animal models immediately promoted us to seek ways to directly apply gene transfer to cure clinical heart failure. The first part of this article reviews our up-to-date knowledge of various functional components to regulate calcium handling in cardiomyocytes, including beta-adrenergic receptor, L-type calcium channel, ryanodine receptor (RyR) and its associated proteins, sarco-endoplasmic reticulum calcium ATPase (SERCA), and phospholamban (PLN), and their abnormalities in failing hearts. A series of new somatic gene transfer attempts targeting calcium handling in cardiomyocytes are discussed thereafter.

Calcium↗

PubMed2006

The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent.

This study aimed to determine the effect of insulin-like growth factor-I (IGF-I) on early antral bovine follicular development, and the expression of insulin-like growth factor-binding protein-2 (IGFBP-2). Antral follicles separated into three different size groups were cultured for 6 days in medium supplemented with either a low (10 ng/ml) or high (1 microg/ml) dose of human recombinant IGF-I. Oestradiol production by follicles in all size ranges, cultured in the presence of the high concentration of IGF-I, significantly increased by day 6 (P < 0.05). Follicles in the smallest size range, 165-215 microm, cultured in a high dose of IGF-I, were found to be significantly increased in size (P < 0.01). Oocyte health of the largest follicles (281-380 microm) was significantly improved by the addition of IGF-I to the culture medium. mRNA expression of IGFBP-2 was decreased in the granulosa cells of follicles, size range 216-280 microm, cultured with a high dose of IGF-I (P < 0.05). Granulosa cells (P < 0.05) and oocytes (P < 0.01) of the largest follicles (281-380 microm) showed a decrease in IGFBP-2 expression (protein) when cultured in the control and low-IGF-I treatment groups. Therefore, the response of a bovine follicle to IGF-I is both dose and stage dependent. This work supports a role for IGF-I in modulating somatic and germ-cell maturation and development in early antral follicles. Furthermore, the inverse relationship between the level of IGF-I stimulation and IGFBP-2 expression suggests a local regulatory system modulating IGF-I availability.

Animals↗

PubMed2001

TspO as a modulator of the repressor/antirepressor (PpsR/AppA) regulatory system in Rhodobacter sphaeroides 2.4.1.

The TspO outer membrane protein of Rhodobacter sphaeroides has been shown to be involved in controlling the transcription of a number of genes which encode enzymes involved in photopigment biosynthesis and the puc operon. The display of regulated genes appears identical to those genes encompassing the PpsR/AppA repressor/antirepressor regulon, although the effect of TspO is modest relative to that of PpsR/AppA. To directly address the hypothesis that TspO is effective through the PpsR/AppA system, we constructed mutant strains with mutations in both tspO and appA. In all cases, the phenotypes examined resembled those of the appA lesion by itself, leading us to conclude that TspO works through or modulates the PpsR/AppA system and acts upstream of the site of action of these regulatory proteins. In earlier publications, we had suggested that TspO is involved in the efflux of a certain intermediate(s) of the porphyrin biosynthesis pathway and that transcriptional regulation of target gene expression could be explained by the accumulation of a coactivator of AppA function. Although the data reported here do not precisely identify this coactivator, they lend support to this hypothesis. We discuss the importance of this form of gene control as the result of the recent extension of the TspO system to Sinorhizobium meliloti, as described by Davey and de Bruijn (M. E. Davey and F. J. de Bruijn, Appl. Environ. Microbiol. 66:5353-5359, 2000). It is therefore possible that this system constitutes a more widely, although not universally, demonstrated form of gene regulation.

Bacterial Proteins↗

PubMed2000

Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome.

The virion-associated protein of human immunodeficiency virus, type 1 (HIV-1), Vpr, is a small protein with 96 amino acid residues that has the ability to modulate transcription of HIV-1 long terminal repeat (LTR) promoter activity and affects several cellular functions. In this study we have employed molecular approaches to further investigate the mechanism by which Vpr exerts its regulatory effect upon the LTR. We show that by structural and functional interaction with Tat, a potent viral regulatory protein, Vpr synergistically enhances the transcriptional activity of the HIV-1 LTR. Because Tat utilizes cyclin T1 and its partner, CDK9 to elevate the level of transcription from the LTR, we examined the cooperativity between Vpr, Tat, and cyclin T1/CDK9 on viral gene transcription. Results from co-transfection studies indicated superactivation of LTR by Tat and cyclin T1/CDK9 in the presence of wild type Vpr. This activation was not observed with the R73S mutant of Vpr, which contains arginine to serine transition at residue 73. Interestingly, expression of R73S mutant in cells exerts a negative effect on the observed superactivation of the LTR by Tat, cyclin T1/CDK9, and wild type Vpr. Results from protein-protein interaction studies indicated that Vpr is associated with both Tat and cyclin T1 in cells expressing these proteins. Use of deletion mutant proteins in binding studies revealed that the binding sites for Tat and Vpr within cyclin T1 are distinct and that association of these two viral proteins with cyclin T1 is independent from each other. These observations suggest a working model on the cooperative interaction of Vpr with viral and cellular proteins and its involvement in control of viral gene transcription and replication. Moreover identification of R73S mutant of Vpr provides a new therapeutic avenue for controlling HIV-1 gene transcription and replication in the infected cells.

Arginine↗

PubMed2006

Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and modulate airway inflammation in a murine model of asthma.

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4(+) CD25(+) T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4(+) CD25(+) T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25(+) depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25(+) T cells from these mice to suppress T-cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4(+) CD25(+) T cells of OVA-treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0.05). Depletion of CD25(+) cells accelerated OVA-induced airway inflammation and increased the expression of interleukin (IL)-5 and IL-4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4(+) CD25(+) T cells, which made IL-10, but little IL-4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen-induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4(+) CD25(+) T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.

Animals↗

PubMed2005

Na+/H+ exchanger regulatory factor isoform 1 overexpression modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and activity in human airway 16HBE14o- cells and rescues DeltaF508 CFTR functional expression in cystic fibrosis cells.

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Binding Sites↗