Beta Gene Transcription Research Articles

Dietary microalgal-fabricated selenium nanoparticles improve Nile tilapia biochemical indices, immune-related gene expression, and intestinal immunity

BackgroundFeed supplements, including essential trace elements are believed to play an important role in augmenting fish immune response. In this context, selenium nanoparticles (SeNPs) in fish diets via a green biosynthesis strategy have attracted considerable interest. In this investigation, selenium nanoparticles (SeNPs, 79.26 nm) synthesized from the green microalga Pediastrum boryanum were incorporated into Nile tilapia diets to explore its beneficial effects on the immune defense and intestinal integrity, in comparison with control basal diets containing inorganic Se source. Nile tilapia (No. 180, 54–57 g) were fed on three formulated diets at concentrations of 0, 0.75, and 1.5 mg/kg of SeNPs for 8 weeks. After the trial completion, tissue bioaccumulation, biochemical indices, antioxidant and pro-inflammatory cytokine-related genes, and intestinal histological examination were analyzed.ResultsOur finding revealed that dietary SeNPs significantly decreased (P < 0.05) serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and cholesterol, while increasing (P < 0.05) high-density lipoproteins (HDL). The Se concentration in the muscle tissues showed a dose-dependent increase. SeNPs at a dose of 1.5 mg/kg significantly upregulated intestinal interleukin 8 (IL-8) and interleukin 1 beta (IL-1β) gene transcription compared with the control diet. Glutathione reductase (GSR) and glutathione synthetase (GSS) genes were significantly upregulated in both SeNPs-supplemented groups compared with the control. No apoptotic changes or cell damages were observed as indicated by proliferating cell nuclear antigen (PCNA) and caspase-3 gene expression and evidenced histopathologically. SeNPs supplementation positively affects mucin-producing goblet cells (GCs), particularly at dose of 1.5 mg/kg.ConclusionTherefore, these results suggest that Green synthesized SeNPs supplementation has promising effects on enhancing Nile tilapia immunity and maintaining their intestinal health.

Effects of Treatment with Lactobacilli on Necrotic Enteritis in Broiler Chickens.

Growth promoter antibiotics have been commonly used for the control of necrotic enteritis (NE) in broilers for decades. However, due to a ban on the use of these antibiotics, alternatives such as probiotics have been tested widely for NE control. The present study tested the efficacy of four different species of lactobacilli (two isolates of Lactobacillus johnsonii and one of Ligilactobacillus (L.) salivarius, Limosilactobacillus (L.) reuteri, and L. crispatus) against NE. Day-old male broiler chickens were divided into six groups and orally inoculated with 1 × 107 or 1 × 108 colony-forming units (CFU) of lactobacilli on 1, 7, 14, and 20days of age. While negative and positive control groups did not receive lactobacilli, the latter was challenged with Clostridium perfringens (CP). Chickens, at 21days old, were challenged for 3days with 3 × 108CFU of a virulent strain of CP. Tissues were collected for immune system gene expression, immunophenotyping, intestinal histomorphometry, and microbiota analysis. Lactobacilli inoculation conferred partial protection in chickens against NE, marked by lowered lesion scores and improved villus:crypt ratio. Immunomodulatory effects were demonstrated by the significant alteration of interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-12p35, IL-17, and transforming growth factor beta (TGF-β) gene transcription in the duodenum and jejunum as well as subtle changes in the frequency of CD8 + T cells and B cells in the cecal tonsil of the treated chickens. Microbiota analysis showed increased levels of some bacterial phyla including Actinobacteria, Lactobacillaceae, and Firmicutes. In conclusion, these findings suggest that the use of certain lactobacilli can reduce NE severity and modulate immune responses and intestinal microbiota composition in chickens.

Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110β in the regulation of insulin secretion from rodent and human beta cells

Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and β (p110α and -β) in insulin granule recruitment and exocytosis in rodent and human islets. The p110α and p110β subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85β) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110β blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kβ (p110β/p85β) even when p110β catalytic activity was inhibited. Conversely, both the wild-type p110β and a catalytically inactive mutant directly facilitated exocytosis. Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110β is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.

Fine-scale quantification of HCG beta gene transcription in human trophoblastic and non-malignant non-trophoblastic tissues

Human chorionic gonadotropin (HCG) is produced by syncytiotrophoblast of placenta. It delays the apoptosis of corpus luteum and functions in implantation. Its possible role in male reproduction has been raised. HCG beta subunit is encoded by CGB, CGB5, CGB7 and CGB8 genes located at 19q13.3 in a common genome cluster with beta subunit non-coding CGB1 and CGB2. We conducted a sensitive quantification and comparison of CGB gene expression in human trophoblastic (blastocysts, n = 6; normal/failed pregnancy, n = 51) and non-malignant non-trophoblastic tissues (15 different tissue types, samples n = 241), by real-time RT-PCR. We showed a wide transcriptional window of CGB genes in normal pregnancy, a significant reduction in recurrent miscarriages, and a high expression (especially CGB1/CGB2) in ectopic and molar pregnancies. Expression was several orders of magnitude lower in the non-placental tissues, with the highest CGB levels being seen in testis, prostate, thymus, skeletal muscle and lung samples. The contribution of CGB1/CGB2 to the summarized expression of six CGB genes was not proportional to their gene dosage: 1/1000 to 1/10,000. An interesting exception was the testis exhibiting a much higher CGB1/CGB2 to total CGB mRNA ratio of approximately one-third, corresponding to gene dosage. In conclusion, the expressional profile of CGB genes, activated already in blastocyst stage, is associated with the status of pregnancy. The presence of CGB transcripts in testes, and in particular CGB1/CGB2 transcripts, may indicate a role in male reproductive tract.

Luteinizing Hormone Beta Promoter Stimulation by Adenylyl Cyclase and Cooperation with Gonadotropin-Releasing Hormone 1 in Transgenic Mice and LBetaT2 Cells1

Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GnRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GnRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotroph cells. Both cAMP and pituitary adenylyl cyclase-activating peptide increase GnRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GnRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GnRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotroph cells, alone and with GnRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of protein kinase A (PKA); GnRH1 stimulation was partially suppressed by either PKA or protein kinase C inhibitors. Only the proximal GnRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3' NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GnRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GnRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GnRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.

Protective effect of CCR5 delta 32 heterozygosity is restricted by SDF-1 genotype in children with HIV-1 infection.

To determine the influences on pediatric AIDS of a heterozygous 32 base pair deletion in the CC-chemokine receptor 5 gene (CCR5 wt/Delta 32) and a common polymorphism in the 3' untranslated region of stromal cell-derived factor-1 beta gene transcript (SDF1-3'A). The rate of HIV-1 disease progression and viral burden were compared according to the CCR5 and SDF-1 genotypes in 127 (58 Caucasians, 60 African-Americans and nine Hispanics) perinatally HIV-1-infected children. Regardless of ethnic background, the CCR5 wt/Delta 32 genotype was associated with a delayed onset of AIDS-defining infectious complications during the first 5 years of infection [relative hazard (RH) = 0.22; 95% confidence interval (CI), 0.012--1.02; P = 0.053]. Similarly, CCR5 wt/Delta 32 conferred an early protection against severe immune suppression and HIV-1 encephalopathy, but only in those without SDF1-3'A (RH = 0; 95% CI, 0--0.70; P = 0.020, and RH = 0; 95% CI, 0--0.71; P = 0.021, respectively). When examined before 5 years of age (n = 81), the children with CCR5 wt/Delta 32 had significantly lower levels of cell-associated HIV-1 DNA than wild-type homozygotes (P = 0.016, adjusted by race), while SDF1-3'A carriers had relatively higher levels (P = 0.047, adjusted by race). Although the disease-retarding effect of CCR5 wt/Delta 32 subsequently disappeared, time to death was still significantly delayed in the CCR5 Delta 32 heterozygotes without SDF1-3'A (RH = 0; 95% CI, 0--0.53; P = 0.008). In pediatric AIDS, the protective effect of CCR5 wt/Delta 32 is more pronounced in early years of infection and appears to be abrogated by the SDF1-3'A genotype.

A Nuclear Matrix Attachment Region Upstream of the T Cell Receptor β Gene Enhancer Binds Cux/CDP and SATB1 and Modulates Enhancer-dependent Reporter Gene Expression but Not Endogenous Gene Expression

We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.

Sp1 Binds to the Rat Luteinizing Hormone β (LHβ) Gene Promoter and Mediates Gonadotropin-releasing Hormone-stimulated Expression of the LHβ Subunit Gene

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive function by regulating the biosynthesis and secretion of the pituitary gonadotropins. Although it is known that GnRH induces luteinizing hormone beta (LHbeta) gene transcription, the mechanisms by which this occurs remain to be elucidated. We have shown previously that GH3 cells transfected with the rat GnRH receptor cDNA (GGH3-1' cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LHbeta gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (-797/+5LHbetaLUC) and respond to a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we have shown that DNA sequences at -490/-352 confer GnRH responsiveness to the rat LHbeta gene. We have now identified two putative binding sites for Sp1, a three-zinc-finger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein complexes formed using GGH3-1' and alphaT3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LHbeta gene promoter by GnRH. These data define GnRH-responsive elements in the LHbeta 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LHbeta subunit gene.

CREB controls LAP/C/EBP beta transcription.

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.

Polarized secretion of fibrinogen by lung epithelial cells.

The lung epithelium has recently been identified as a novel site of fibrinogen (FBG) biosynthesis. A coordinated upregulation of A alpha, B beta, and gamma chain FBG gene transcription occurs upon stimulation of A549 lung epithelial cells with dexamethasone (DEX) and the proinflammatory mediator interleukin-6 (IL-6). Subsequently, the cells synthesize and secrete fully assembled FBG. This study addresses the polarity of such FBG secretion by A549 cells cultured on polycarbonate membrane filters. After induction with IL-6 and DEX, cells were metabolically labeled, and FBG was immunopurified from the apical and basolateral chambers. Analysis by gel electrophoresis revealed that A549 cells secreted newly synthesized FBG in a polarized manner, with the majority (80%) of FBG secreted basolaterally. Consistent with this observation, immunoelectron microscopy using Protein A-gold labeling showed FBG within secretory vesicles in close proximity to the basolateral aspect of the A549 cell membrane. Polarized secretion was microtubule-dependent since depolymerization using colchicine significantly reduced the basolateral component of secretion, causing intracellular retention of FBG. These data provide evidence that FBG is secreted by lung alveolar epithelial cells vectorially toward the basement membrane, which may reflect in vivo processes associated with local injury, inflammation, and repair mechanisms.

Induction of inflammatory cytokines in placental monocytes of gravidae infected with the human immunodeficiency virus type 1.

Placental mononuclear cells (PMC) are susceptible to infection with the human immunodeficiency virus (HIV). PMC secreted tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta), and IL-6 among other factors, which, in turn, regulate HIV replication in latently infected cells. We assessed the induction of these cytokines in PMC from HIV-infected (HIV+) and uninfected (control) gravidae following exposure to lipopolysaccharide (LPS), HIV lysate (iHIV), recombinant HIV env (GP160) and HIV gag (gag55), and synthetic HIV p17 (HGP30) antigens. In comparison to control PMC, HIV+ PMC constitutively secreted higher levels of IL-1beta and IL-6 and were refractory to stimulation by iHIV, GP160, gag55, and HGP30. Control PMC IL-1 beta levels were boosted by LPS; gag55 and HGP30 augmented IL-6 but not IL-1 beta. Both groups exhibited low basal TNF-alpha production that was augmented by LPS. HIV+ PMC exhibited higher constitutive levels of IL-1 beta, IL-6, and TNF-alpha gene transcription than control PMC. These levels could be further augmented by LPS, yet the incremental levels were lower than those obtained from PMC of uninfected women. The high basal constitutive secretion of cytokines by HIV+ PMC and their refractoriness to activation may reflect a virus-mediated dysregulation of cytokine expression culminating in compromised host defenses against secondary opportunistic infections associated with AIDS.

Biosynthesis of gonadotropins in vivo.

GnRH is potent stimulator of gonadotropin's alpha and beta chains synthesis in vivo. Stimulation of LH beta gene transcription requires pulsatile GnRH administration but the transcription of alpha subunit can be stimulated independently of GnRH mode of administration. Castration increases whereas in vivo estradiol and testosterone replacement decreases the rate of gene transcription of pituitary gonadotropin subunits. Thyroid hormones can enhance or diminish the pituitary levels of LH beta and FSH beta subunit mRNAs in female rats. Inhibin, activin and follistatin were shown to be potent regulators of FSH beta gene expression.

Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone.

Physiological gonadotropin levels are modulated by complex interrelationships between the sex steroids and the hypothalamic GnRH pulse generator, and the steroids and GnRH individually regulate gonadotropin subunit (alpha, LH beta, FSH beta) gene expression. Steroids may act directly at the pituitary level or indirectly at the hypothalamus to alter GnRH pulses, and they can have positive or negative actions, depending on the model system and physiological state. GnRH pulse frequency and amplitude have subunit-specific effects on the gonadotropin genes, and alteration of pulse frequency during the reproductive cycle can selectively favor LH or FSH synthesis. The cloning of the gonadotropin subunit genes and sensitive molecular approaches to the study of transcriptional regulation have permitted insights into the sites of steroid and GnRH action and into the mechanisms by which such hormonal effects occur. This review describes several such approaches including the measurement of endogenous gene transcription by nuclear run-off assays, definition of hormone-sensitive gene regions by transient transfection analysis, and the use of transgenic animal technology to verify hormonal and tissue-specific control of gene expression. Recent studies in the rat model suggest that some steroid actions, such as estrogen stimulation of LH beta gene transcription and alteration of estrogen and GnRH receptor number, occur directly at the level of the pituitary, while suppressive effects of estrogen on gonadotropins may occur at least partly if not primarily via hypothalamic effects. Changes in GnRH pulses may also alter GnRH receptor number, thus modifying the potential signal received by the gonadotroph. Current and emerging molecular technologies will probably identify additional targets of steroid and GnRH action and allow greater insight into gonadotropin regulation and reproductive function.

Heterogeneity of rearranged T cell receptor V alpha and V beta gene transcripts in synovial fluid T cells of HLA-B27 positive reactive arthritis patients.

To analyze the T cell receptor (TCR) V-alpha/beta gene usage by synovial fluid (SF) and peripheral blood (PB) T cells of HLA-B27+ reactive arthritis (ReA) patients. The TCR V-alpha/beta gene usage was determined by the polymerase chain reaction on freshly isolated SF and PB mononuclear cells (MNC) of five HLA-B27+ ReA patients. A total of 30 TCR V alpha and 23 V beta (sub)family specific primers in combination with a C alpha or C beta specific primer, respectively, were used. In five patients most of the TCR V alpha and V beta gene segments expressed by PB T cells were also detected in the paired SF samples. Although one patient showed an increased expression of TCR V alpha2 in SF when compared to PB, the SF samples showed a heterogeneous TCR V-gene repertoire similar to PB. Although this study was limited to a small group of patients, the apparent lack of a restricted TCR V-gene repertoire in SF does not support the involvement of a single or limited number of T cell subsets in the disease process of HLA-B27+ ReA patients.

Thyrotropin (TSH)-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C.

TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.

Messenger RNAs for the multifunctional cytokines interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha are present in adnexal tissues and in dermis of normal human skin.

Interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are 3 cytokines that play a key rôle in cutaneous homeostasis and in the immunopathogenesis of a number of dermatologic diseases. Most studies have focused on their production by keratinocytes and Langerhans cells. To determine whether there are non-epidermal sites of cytokine transcription, biopsy specimens of normal human skin were probed for IL-1 alpha, IL-1 beta and TNF-alpha messenger RNAs using the method of in situ hybridization. The results demonstrate that each cytokine mRNA is present at multiple sites within the skin, including epidermal appendages and adnexal structures (hair follicles, sebaceous glands), the dermal microvasculature, arrectores pilorum smooth muscle, and the dermal connective tissue. These data provide evidence that in vivo there are multiple sites other than the epidermis of constitutive IL-1 alpha, IL-1 beta, and TNF-alpha gene transcription in normal human skin.

CD34-positive early human thymocytes: T cell receptor and cytokine receptor gene expression.

CD34, a stem cell marker, has been shown to be expressed on human CD3-CD4-CD8- (triple-negative; TN) thymocytes. Phenotypic and functional analyses suggest the following differentiation sequence: CD34+1-3-4-8(-)--> CD34+1+3-4 +/- 8(-)-->CD34-1+3-4+8(+/-)-->CD34-1++3-4+8+. In this report, we examined cytokine receptor gene expression on these subsets by reverse transcription-polymerase chain reaction analysis (RT-PCR). We were able to detect interleukin-7 receptor (IL-7R), c-kit and IL-2R gamma in all CD34+ thymocyte subsets, consistent with previous functional studies. We found IL-1R, granulocyte/macrophage colony-stimulating factor receptor-alpha and IL-4R transcripts in CD3- and CD34+ subsets. Secondly, we investigated T cell receptor (TCR)-delta and -beta gene rearrangement and transcription in CD34+ thymocytes. Our results show that a full-length TCR-delta transcript and the recombination activating genes RAG-1 and RAG-2 mRNA were already expressed in the CD34+1- subset. Mature V beta-containing TCR transcripts were also detected in the CD34+1+ subset, but not in the CD1- fraction. Furthermore, PCR analysis of D-J beta gene rearrangements showed that > or = 70% of CD34+1- cells are in a TCR beta germ-line configuration, although D-J beta recombination had already started in this population.

Restricted usage of T cell receptor V alpha-V beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy.

Prolonged myocardial cell damage initiated by acute myocarditis is thought to be one of the most important etiology of dilated cardiomyopathy. To investigate the immunological mechanisms involved in the pathogenesis of dilated cardiomyopathy, we analyzed the phenotypes of infiltrating cells and examined the expression of perforin in infiltrating cells in the hearts of patients with dilated cardiomyopathy as well as acute myocarditis. We also examined the expression of HLA and intercellular adhesion molecule-1 (ICAM-1) in myocardial tissue of these patients. Furthermore, to evaluate the antigen specificity of infiltrating T cells and persistence of viral genomes in the myocardial tissue, we analyzed the expression of T cell receptor (TCR) V alpha and V beta genes as well as enterovirus genomes by PCR. We found infiltration of perforin-expressing killer cells and enhanced expression of HLA class I and ICAM-1 in the myocardial tissue. We also found that the repertoires of TCR V alpha as well as V beta gene transcripts were restricted, indicating that a specific antigen in the hearts was targeted. Because no enterovirus genomes were detected in all patients, it is strongly suggested that a cell-mediated autoimmune mechanism triggered by virus infection may play a critical role in the pathogenesis of dilated cardiomyopathy. However, we could not exclude the possibility that viruses other than enteroviruses could be pathogenic in these patients.

Functional analysis of estrogen-responsive elements in chinook salmon (Oncorhynchus tschawytscha) gonadotropin II beta subunit gene

The salmon gonadotropin II gene regulates ovulation and spawning. Analysis of the 5' flanking sequence of the hormone-specific beta-subunit of salmon gonadotropin II (sGTHII beta) gene reveals the presence of several presumptive estrogen-responsive elements (ERE). The participation of ERE in the control of sGTHII beta gene transcription was examined by the transient expression of sGTHII beta gene promoter-chloramphenicol acetyltransferase chimeric DNA constructs in HeLa cells, with the cotransfection of a rainbow trout estrogen receptor expression vector. Three ERE have been identified: the proximal ERE [pERE, at -267 base pair (bp) from the transcription start site], the distal ERE (dERE, at -2698 bp, three GGTCA motifs each separated by exactly 31 bp), and the half-ERE (1/2ERE, at -157 bp as a GGTCA motif), respectively. The pERE (TGTCAATCTGACC) represents a novel but less effective variation of the consensus ERE (cERE). The dERE is a unique estrogen-induced enhancer. It requires the participation of the pERE to be functional and the enhancer activity of pERE and dERE is promoter specific. The contribution of 1/2ERE is minor and is not cell-type specific. The activation of the ERE in the sGTHII beta gene and the synergistic cooperation between the dERE and pERE by estradiol-17 beta is dose dependent. DNA sequences in the vicinity of the ERE decreases their hormone responsiveness and the synergism between dERE and pERE. These negative regions may contribute to the quiescent endocrine state of the sGTHII beta gene during the regressive phase of the reproductive cycle in teleost.

Functional analysis of estrogen-responsive elements in chinook salmon (Oncorhynchus tschawytscha) gonadotropin II beta subunit gene.

The salmon gonadotropin II gene regulates ovulation and spawning. Analysis of the 5' flanking sequence of the hormone-specific beta-subunit of salmon gonadotropin II (sGTHII beta) gene reveals the presence of several presumptive estrogen-responsive elements (ERE). The participation of ERE in the control of sGTHII beta gene transcription was examined by the transient expression of sGTHII beta gene promoter-chloramphenicol acetyltransferase chimeric DNA constructs in HeLa cells, with the cotransfection of a rainbow trout estrogen receptor expression vector. Three ERE have been identified: the proximal ERE [pERE, at -267 base pair (bp) from the transcription start site], the distal ERE (dERE, at -2698 bp, three GGTCA motifs each separated by exactly 31 bp), and the half-ERE (1/2ERE, at -157 bp as a GGTCA motif), respectively. The pERE (TGTCAATCTGACC) represents a novel but less effective variation of the consensus ERE (cERE). The dERE is a unique estrogen-induced enhancer. It requires the participation of the pERE to be functional and the enhancer activity of pERE and dERE is promoter specific. The contribution of 1/2ERE is minor and is not cell-type specific. The activation of the ERE in the sGTHII beta gene and the synergistic cooperation between the dERE and pERE by estradiol-17 beta is dose dependent. DNA sequences in the vicinity of the ERE decreases their hormone responsiveness and the synergism between dERE and pERE. These negative regions may contribute to the quiescent endocrine state of the sGTHII beta gene during the regressive phase of the reproductive cycle in teleost.