A multicenter study on anaphylaxis caused by peanut, tree nuts, and seeds in children and adolescents.

Peanut (PN) and tree nuts (TNs) are common causes of anaphylaxis in Western countries, but no information is available in Korea. To feature clinical characteristics of anaphylaxis caused by PN, TNs, and seeds, a retrospective medical record review was performed in 14 university hospitals in Korea (2009-2013). One hundred and twenty-six cases were identified, with the mean age of 4.9 years. PN, walnut (WN), and pine nut accounted for 32.5%, 41.3%, and 7.1%, respectively. The median values of specific IgE (sIgE) to PN, WN, and pine nut were 10.50, 8.74, and 4.61 kUA /l, respectively. Among 50 cases managed in the emergency department, 52.0% were treated with epinephrine, 66.0% with steroid, 94.0% with antihistamines, 36.0% with oxygen, and 48.0% with bronchodilator. In conclusion, WN, PN, and pine nut were the three most common triggers of anaphylaxis in Korean children, and anaphylaxis could occur at remarkably low levels of sIgE.

Increased sputum levels of thymus and activation-regulated chemokine in children with asthma not eosinophilic bronchitis.

BACKGROUND: Thymus and activation-regulated chemokine (TARC), a member of the CC chemokine family, plays a crucial role in Th2-specific inflammation. We aimed to determine the concentration of sputum TARC in children with asthma and eosinophilic bronchitis (EB) and its relation with eosinophilic inflammation, pulmonary function, and bronchial hyper-responsiveness. METHODS: In total, 90 children with asthma, 38 with EB, and 45 control subjects were enrolled. TARC levels were measured in sputum supernatants using an ELISA. We performed pulmonary function tests and measured exhaled fractional nitric oxide, eosinophil counts in blood, and sputum and serum levels of total IgE in all children. RESULTS: Sputum TARC levels were significantly higher in children with asthma than in either children with EB (p=0.004) or the control subjects (p=0.014). Among patients with asthma, sputum TARC concentration was higher in children with sputum eosinophilia than in those without sputum eosinophilia (p=0.035). Sputum TARC levels positively correlated with eosinophil counts in sputum, serum total IgE levels, exhaled fractional nitric, and the bronchodilator response. Negative significant correlations were found between sputum TARC and FEV1/FVC (the ratio of forced expiratory volume in one second and forced expiratory vital capacity) or PC20 (the provocative concentration of methacholine causing a 20% decrease in the FEV1). CONCLUSION: Elevated TARC levels in sputum were detected in children with asthma but not in children with EB. Sputum TARC could be a supportive marker for discrimination of asthma from EB in children showing characteristics of eosinophilic airway inflammation.

Relationship between sputum clusterin levels and childhood asthma.

BACKGROUND: Clusterin is a sensitive cellular biosensor of oxidative stress and has been studied as a biomarker for inflammation-associated diseases. Clusterin levels in childhood asthma have not been evaluated. OBJECTIVES: (1) To evaluate sputum clusterin levels in children with asthma compared to a control group. (2) To assess the relationships between sputum clusterin levels and airway inflammation, pulmonary function, and bronchial hyperresponsiveness. METHODS: This study included 170 children aged 5-18 years with stable asthma (n = 91), asthma exacerbation (n = 29), or no asthma (healthy controls; n = 50). Induced sputum, pulmonary function, and methacholine challenge tests were performed. Stable asthma was classified into two groups according to the severity. Clusterin levels in sputum were measured using an enzyme-linked immunosorbent assay. RESULTS: Children with stable asthma had a higher clusterin level than healthy controls [4540 (3872-5651) pg/mL vs. 3857 (1054-4369) pg/mL, P < 0.001]. The clusterin level was also more elevated in eosinophil-dominant sputum than in non-eosinophilic sputum in stable asthma [5094 (4243-6257) pg/mL vs. 4110 (1871-4839) pg/mL, P = 0.0017]. Clusterin levels were associated with asthma severity. Paradoxically, clusterin levels were lower during asthma exacerbation than in stable asthma [1838 (350-4790] pg/mL vs. 4540 (3872-5651) pg/mL, P < 0.001]. Clusterin levels were strongly correlated with the methacholine concentration that caused a 20% decrease in the forced expiratory volume in 1 s (r = -0.617, P < 0.001); there was no significant correlation between clusterin levels and other pulmonary function parameters. CONCLUSIONS AND CLINICAL RELEVANCE: Clusterin levels were altered in children with stable asthma and asthma exacerbation because of its antioxidant and anti-inflammatory effects. Clusterin may be a marker that reflects airway inflammation and severity of symptoms, and it can be used in the assessment and management of childhood asthma.

IgE cross-reactivity of peanut with walnut and soybean in children with food allergy.

BACKGROUND: Peanut allergies are common and can be life-threating for sensitised individuals. Peanut allergens share significant amino acid homology with those of other legumes and tree nuts, but their cross-reactivity still remains unclear. OBJECTIVE: We sought to determine the clinical significance of the cross-reactivity of peanut allergens with those of walnut and soybean. METHODS: Pooled sera from eight subjects with both peanut and walnut specific IgE were investigated in an inhibition test. After the sera were incubated with either peanut or walnut protein extracts, the quantity of IgE antibodies against the peanut and walnut was measured using an immunoCAP test. Likewise, pooled sera from 18 subjects with both peanut and soybean specific IgE antibodies were incubated with either peanut or soybean protein extracts and evaluated with a peanut and soybean immunoCAP test. SDS-PAGE and immunoblotting were also performed with peanut, walnut and soybean protein extracts and relevant sera. RESULTS: Peanut specific IgE was inhibited up to 20% and 26% by walnut and soybean protein extracts, respectively. In reverse, walnut and soybean specific IgE were inhibited up to 21% and 23% by peanut protein extracts, respectively. In the immunoblot analysis, pooled serum from the subjects with peanut specific IgE antibodies reacted with walnut protein extracts significantly. CONCLUSION: Although the clinical significance of the cross-reactivity of peanut specific IgE with walnut and soybean protein extracts has not been established, we believe that individuals who are allergic to peanuts need to be cautious about consuming walnuts and soybeans.

The role of interleukin-17 in mouse models of atopic dermatitis and contact dermatitis.

BACKGROUND: Atopic dermatitis (AD) and contact dermatitis (CD) are both T cell-mediated eczematous disorders. Interleukin (IL)-17, expressed by T helper (Th)17 cells, is involved in recruitment of inflammatory cells into AD and CD skin. AIM: In this study, we investigated whether IL-17 regulates immune dysregulation and affects skin barrier in oxazolone (OXA)-induced AD-like and CD-like disease models in mice, by comparing IL-17 null mutant (IL-17(-/-) ) vs. wild-type (WT) mouse strains in the models. METHODS: IL-17(-/-) and WT Balb/c mice were used for OXA induction of AD-like and CD-like skin diseases. Ear swelling was measured by a micrometer. Skin biopsies were obtained for RNA isolation and histology. Real-time quantitative PCR analysis was performed to quantify mRNA expression of Th2 cytokines. Skin permeability was measured by a vapometer, and structural changes in the skin were evaluated by electron and confocal microscopy. RESULTS: Both OXA-induced AD and CD responses were alleviated in IL-17(-/-) mice relative to WT, as demonstrated by reductions in ear swelling, inflammatory cell infiltration and levels of Th2 cytokines. These endpoints were used to characterize inflammatory dysregulation in both AD and CD models. Skin-barrier dysfunction, measured by increases in transepidermal water loss and dysfunction of lamellar bodies, and reductions in lipid distribution, were seen in both AD and CD in WT mice. In IL-17(-/-) mice, however, these responses were significantly diminished. CONCLUSIONS: The results indicate that the IL-17 gene may play a role in modulating immune dysregulation and affecting skin barrier in OXA-induced AD-like and CD-like skin disease models in the Balb/c mouse.

Genomic instability in Gadd45a-deficient mice.

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

Chitinase induce the release of IL-8 in human airway epithelial cells, via Ca2+-dependent PKC and ERK pathways.

Chitinases are produced in significant quantities by hosts defending against infections with chitin-containing organisms. However, little is known about the immune response of exogenous chitinase in human epithelial cells. IL-8 has been suggested to have a role in the pathogenesis of the allergenic inflammation of bronchial asthma. We examined whether Streptomyces griseus (S. griseus) chitinase-induced IL-8 on airway epithelium and identified the involvement of intracellular signalling pathways. H292 cells were treated with S. griseus chitinase with different concentrations and times. The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction. Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to S. griseus chitinase. Cells exposed to S. griseus chitinase showed higher level of IL-8 protein production and mRNA expression. Cells stimulated by S. griseus chitinase resulted in the activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-kB) pathways. Inhibitors of Ca(2+)-dependent PKC (Ro-31-8220, calphostin C and Go6976) significantly abolished chitinase-induced expression of IL-8. However, Ca(2+)-independent PKC inhibitor (rottlerin) did not inhibit IL-8 expression. Through ERK inhibitor (U0126) and NF-kB inhibitor (caffeine acid phenethyl ester) treatment, it was proven that ERK and NF-kB regulated chitinase-induced IL-8 expression. We concluded that S. griseus chitinase-induced IL-8 expression was regulated by the activation of Ca(2+/-)-dependent PKC, ERK and NF-kB in human airway epithelial cells.

Increased serum B cell-activating factor level in children with atopic dermatitis.

BACKGROUND: B cell-activating factor (BAFF) is a tumour necrosis factor superfamily member best known for its role in the survival and maturation of B cells. BAFF activity is seen in naïve and effector/memory T cells. AIM: To investigate the level and role of BAFF in serum of patients with atopic dermatitis (AD). METHODS: Levels of serum BAFF, a proliferation-inducing ligand (APRIL) and total serum IgE level, and total eosinophil count were measured in 245 children. RESULTS: Patients were characterized as having atopic eczema (AE) (n = 90) or non-AE (n = 77); the remainder were healthy control subjects (n = 78). Serum BAFF level in children with AE (1625.04 +/- 708.32 pg/mL) was significantly higher than in non-AE children (1194.69 +/- 448.44 pg/mL, P < 0.0001) or healthy controls (1062.89 +/- 444.74 pg/mL, P < 0.0001). Serum APRIL level was not different between the three groups. Serum BAFF level significantly correlated with total serum IgE level (gamma = 0.42, P < 0.0001) and total eosinophil count. It was also positively correlated with serum BAFF and egg-specific IgE level (gamma = 0.252, P = 0.045) in AE. CONCLUSIONS: Serum BAFF level is high in AE and might be a useful marker for AE.

Interleukin-18 is a critical factor for vascular endothelial growth factor-enhanced migration in human gastric cancer cell lines.

Cell migration and angiogenesis are key steps in tumor metastasis. However, the mechanism of migration regulated by vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis, is not completely understood. This study examined the relationship between VEGF and migration, along with the mechanism involved in the VEGF-regulated migration of human gastric cancer cells. The level of cell migration was increased by recombinant human (rh)VEGF-165 in the VEGF receptor-2-expressing SNU-601 cells. Interleukin (IL)-18 is associated with the malignant progression of tumors. Accordingly, this study examined the effect of IL-18 on the migration of cancer cells in order to identify the factors involved in VEGF-enhanced migration. Inhibiting IL-18 markedly reduced the level of VEGF-enhanced migration, and IL-18 increased cell migration directly through filamentous-actin polymerization and tensin downregulation. It was confirmed that rhVEGF-165 increased IL-18 production significantly. An antioxidant and an extracellular signal-regulated kinase (ERK)1/2-specific inhibitor blocked rhVEGF-165-enhanced IL-18 production. Accordingly, rhVEGF-165 increased the generation of region of interest (ROI) and activated the ERK1/2 pathway. These results suggest that rhVEGF-165 enhances IL-18 production via the generation of ROI and ERK1/2 phosphorylation, which results in the increased migration of gastric cancer cells.

Combined effect of tumour necrosis factor-alpha and interleukin-13 polymorphisms on bronchial hyperresponsiveness in Korean children with asthma.

BACKGROUND: TNF-alpha and IL-13, two pivotal pro-inflammatory cytokines, are increased in asthmatic airways and may be linked to asthma susceptibility and/or bronchial hyperresponsiveness (BHR). OBJECTIVE: We investigated the association between the TNF-alpha-308G/A polymorphism and asthma susceptibility or asthma-related phenotypes in Korean children with asthma, and tested for a combined effect with IL-13 polymorphisms. METHODS: Asthmatic children (n=719) and non-atopic healthy control children (n=243) were evaluated for asthma phenotypes including total serum IgE and BHR to methacholine. Genotypes were determined by PCR-restriction fragment length polymorphism analysis. RESULTS: The allele frequency of TNF-alpha-308A in asthmatics (14.1%) was higher than that in control children [8.7%, odds ratio (OR) 1.72, 95% confidence interval (CI) 1.05-2.82]. Significantly lower PC(20) values were found in asthmatic children carrying one or two copies of the TNF-alpha risk allele (-308A) vs. those homozygous for the common allele (P=0.026). Combined analysis revealed that atopic asthmatic children co-inherited the risk alleles of TNF-alpha-308G/A and IL-13 +2044G/A more frequently than control children (aOR 1.91, 95% CI 1.00-3.65), and asthmatic children co-inheriting both risk alleles had significantly lower PC(20) values vs. asthmatic children homozygous for the common alleles (P=0.024). CONCLUSION: The TNF-alpha promoter polymorphism (-308G/A) may be associated with asthma susceptibility and BHR in Korean children with asthma. In addition, there appears to be a synergistic effect between the TNF-alpha promoter polymorphism and an IL-13 coding region polymorphism in terms of asthma susceptibility and BHR in this population.

Reevaluation of the earlobe types in Koreans.

The shape of the earlobes has a variety of genetic significance. This study analyzed the frequencies of the earlobe shapes in the Korean population. Data were collected on randomly selected 500 males and 500 females in Daegu Metropolitan City, with all participant ages being in their twenties. Obtuse angled earlobes accounted for 41.2% of the earlobes observed, while acute angled earlobes prevalence was calculated at 38.8% and right angled earlobe was 20.0% of the total (sexes combined). In men, the acute angled earlobe was the most frequent type (43.0%), while the obtuse angled earlobe was the most frequent type in females (45.2%). These differences were statistically significant (p = 0.015). Overall, attached type earlobe (61.2%) was more frequent than free type earlobe. The attached type earlobe was more common in both sex groups (57.0% in male and 65.4% in female), and the proportion was significantly higher for females (p = 0.006). In conclusion, the findings in this study suggest that the attached earlobe type is the most common among Koreans, and the proportion of earlobe types among males and females is significantly different. Further studies are needed to understand the genetic background of earlobe types among Koreans.

Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency.

Mice lacking the Gadd45a gene are susceptible to ionizing radiation-induced tumors. Increased levels of Gadd45a transcript and protein are seen after treatment of cells with ionizing radiation as well as many other agents and treatments that damage DNA. Because cells deficient in Gadd45a were shown to have a partial defect in the global genomic repair component of the nucleotide excision repair pathway of UV-induced photoproducts, dimethylbenzanthracene (DMBA) carcinogenesis was investigated because this agent produces bulky adducts in DNA that are also repaired by nucleotide excision repair. Wild-type mice and mice deficient for Gadd45a were injected with a single i.p. dose of DMBA at 10-14 days of age. The latency for spontaneous deaths was slightly decreased for Gadd45a-null mice compared with wild-type mice. At 17 months, all surviving animals were killed, and similar percentages of each genotype were found to have tumors. However, nearly twice as many Gadd45a-null than wild-type mice had multiple tumors, and three times as many had multiple malignant tumors. The predominant tumor types in wild-type mice were lymphoma and tumors of the intestines and liver. In Gadd45a-null mice, there was a dramatic increase in female ovarian tumors, male hepatocellular tumors, and in vascular tumors in both sexes. In wild-type mice, this dose of DMBA induced a >5-fold increase in Gadd45a transcript in the spleen and ovary, whereas the increase in liver was >20-fold. Nucleotide excision repair, which repairs both UV- and DMBA-induced DNA lesions, was substantially reduced in Gadd45a-null lymphoblasts. Mutation frequency after DMBA treatment was threefold higher in Gadd45a-null liver compared with wild-type liver. Therefore, lack of basal and DMBA-induced Gadd45a may result in enhanced tumorigenesis because of decreased DNA repair and increased mutation frequency. Genomic instability, decreased cell cycle checkpoints, and partial loss of normal growth control in cells from Gadd45a-null mice may also contribute to this process.

Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes.

C-terminal truncations of the NFKB2 p100 gene product have been observed in a number of cases of human cutaneous T cell lymphomas, as well as human B-cell lymphomas and myelomas. The contribution of these alterations to lymphomagenesis is not understood; however, truncation at amino acid 666 to generate 80 - 85 kD proteins in the HUT78 cell line is associated with addition of a short (serine-alanine-serine) fusion at the 3' end of p80HT, as well as with increased expression of NFKB2 mRNA. We therefore examined the effects of p80HT on the regulation of NFKB2 expression, as well as the properties of a series of other tumor-associated, and site directed mutations of NFKB2. While p80HT had not itself acquired novel transcriptional activation properties with respect to the NFKB2 P1 or P2 promoters or the IL-6 kappaB promoter, p80HT had lost the potent inhibitory (IkappaB-like) activity associated with the wild-type, p100 gene product. Loss of the inhibitory property depended on the SAS residues in the fusion protein, direct truncation at aa666 was fully inhibitory, as was a substitution of three alanines for the SAS residues. The presence of as few as two C-terminal ankyrin motifs was sufficient for inhibition of NF-kappaB-mediated transcriptional activation. Assays of a series of additional lymphoma-associated NF-kappaB-2 truncation suggested that the C-terminal truncation associated with these proteins was also associated with a loss of the IkappaB-like activities of p100 NF-kappaB-2, for at least some NF-kappaB target promoters. Thus, the loss of IkappaB-like activity of lymphoma-associated NFKB2 mutations may play an important role in the genesis of a subset of human lymphomas.

Functional analysis of the interaction of a tissue-specific factor with an upstream enhancer element of the rat prolactin gene.

A nuclear factor which binds to an upstream element of the rat PRL gene has been identified and the functional properties of the factor-DNA interaction have been assessed by mutagenesis of the factor binding sites. Gel mobility shift assays have been used to identify a factor which binds to a fragment from the -1712 to -1494 region of the rat PRL gene. The DNA binding factor is present in nuclear extracts from PRL-producing GH3 cells, but not in nuclear extracts from several other cell lines. Although previous studies have shown that the estrogen receptor binds to this region of DNA, chromatography on heparin-agarose demonstrated that the factor detected by mobility shift assay is probably not the estrogen receptor. Nuclease protection experiments demonstrate that the factor binds to a discrete region at positions -1666 to -1652. The protected region includes half of a palindrome, TCATTAT ... ATAATGA. Mutagenesis by T to G transversions of either both halves of this symmetrical sequence, or only the upstream portion shown to interact with the factor substantially reduced factor binding as assessed by gel mobility shift assay. Transfer of fusion genes containing this region of DNA into GH3 cells demonstrated that the -1712 to -1494 region has a basal enhancer activity which is reduced severalfold by T to G mutagenesis of the complete dyad symmetry at positions -1665 to -1644. The results suggest that the -1712 to -1494 region of the rat PRL gene contains two relatively independent elements. One element, located at positions -1582 to -1569, interacts with the estrogen receptor and mediates estrogenic stimulation of transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep.

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.

Characterization of a non-tissue-specific, 3',5'-cyclic adenosine monophosphate-responsive element in the proximal region of the rat prolactin gene.

The nature of a DNA element located in the -100 to -85 region of the rat PRL gene has been characterized. Previous studies demonstrated that this region may contribute to basal and hormonally regulated expression of the PRL gene. As this region contains a sequence with similarity to a consensus cAMP-responsive element (CRE), a possible role for the cAMP response element binding protein (CREB) has been explored. A point mutation which made the PRL CRE-like sequence less like a consensus CRE had little effect on basal or cAMP-stimulated expression of a PRL-luciferase reporter gene. DNase footprint studies demonstrated that the proximal region of the PRL gene does not contain a high affinity CREB binding site. Mobility shift experiments demonstrated that the major GH3 nuclear protein which interacts with the -100 to -85 region of the PRL gene in vitro is not CREB. Transfection of a dominant inhibitor of CREB action had little or no effect on expression of an indicator gene containing the PRL proximal region. Thus, the PRL proximal region does not contain a high affinity CREB binding site, and it is unlikely that CREB plays a major role in expression of the PRL gene. The functional capabilities of the -100 to -85 region of the PRL gene were then tested in a transfection assay. Synthetic multimers of this region were found to be sufficient to permit a transcriptional response to cAMP or TRH in GH3 cells and cAMP in Rat-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene.

Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.

Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin alpha-subunit gene.

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes.

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)

The affinity of allergen specific IgE and the competition between IgE and IgG for the allergen in Amb a V sensitive individuals.

We have calibrated a solid state RAST assay with affinity purified allergen-specific IgE. We then utilized the calibrated assay to measure the average affinity of individual IgE-containing sera in terms of the average association constant < K > for purified allergen Amb a V. The binding data yielded linear reciprocal plots indicating that the range of affinities of the responding clones was narrow. The range of the average association constant for the IgE-Amb a V complex was 0.9-26 x 10(10) M-1. The average affinity of the corresponding IgG response in the same individual, estimated by inhibition studies of IgE binding, was 10(7) M-1 in one case and lower than 10(6) M-1 in all the other cases.