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The COVID-19 pandemic posed challenges to the tourism sector globally. We investigated changes in visitor demographics, satisfaction level, and its determinants pre- and peri-COVID-19. Data were collected using questionnaire surveys in 2019 and 2021 within Banff National Park (BNP). The data analyses were based on a sample size of 1183 respondents by conducting factor analysis, correlation analysis and stepwise regression analysis. Results highlight that there were fewer international visitors and more local and domestic visitors during the pandemic. Park attributes were evaluated at a higher satisfaction level peri-COVID-19. The quality of the Park facilities and services were the most important satisfaction determinants pre- and peri-COVID-19, and all the Park COVID-19 measures and actions received positive experience from visitors. This research fills this knowledge gap by developing a better understanding in the change of visitor demographics and satisfaction level in BNP under the context of the pandemic. It also provides implication for both scholars and practitioners to understand the impacts of the pandemic on Park visitation. The study can provide insights for utilizing the pandemic as a transformative strength and for mitigating its negative impact on tourism industry., (© The Author(s) 2023.)

Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.

Glucocorticoid (GC) hormones induce apoptosis in T-cell and pre-B-cell acute lymphoblastic leukemia (ALL) cells. Steroid-mediated apoptosis requires a threshold level of the glucocorticoid receptor (GR) protein, and increasing the intracellular GR levels in ALL cells would augment their hormone sensitivity. A protein transduction domain (PTD) approach was used to accomplish this. We produced an HIV Tat PTD domain fusion protein (Tat-GR(554-777)) that potentially competes for the degradation of GR protein by the ubiquitin-proteasome system and should thus increase its intracellular levels by "stabilizing" the GR. We also designed a fusion peptide for the c-Myb DNA binding domain, Tat-c-Myb DBD, since the biological function of this peptide as a dominant negative inhibitor of the c-Myb protein was already known. Purified, bacterially expressed Tat-c-Myb DBD and Tat-GR(554-777) exhibited highly efficient transduction into cultured ALL cell lines including 697 (pre-B-ALL) and CEM-C7 (T-ALL) cells. As expected, the transduced Tat-c-Myb DBD peptide inhibited steroid-mediated stimulation of a GR promoter-luciferase reporter gene. Significantly, transduced Tat-GR(554-777) effectively increased intracellular GR levels in the GC-resistant T-ALL cell line, CEM-C1, and in the pre-B-ALL 697 cell line. Furthermore, transduction of Tat-GR(554-777) rendered GC-resistant CEM-C1 cells sensitive to steroid killing and further sensitized 697 cells to steroid. The use of Tat-fusion peptide transduction may eventually lead to innovative therapeutic modalities to improve the clinical response of patients suffering from T-cell and pre-B-cell acute lymphoblastic leukemia by increasing steroid responsiveness and perhaps converting steroid-resistant leukemia to a hormone-responsive phenotype.

Glucocorticoid (GC) hormones are widely used in the treatment of acute lymphoblastic leukemia (ALL). Whereas a high level of GC receptor (GR) protein is associated with the sensitivity of ALL cells to steroid-mediated apoptosis, the auto-up-regulation of human (h)GR mRNA and protein is also found in hormone-sensitive ALL cell lines. We have characterized the hGR gene-proximal promoters for DNA sequences and transcription factors required for hormone responsiveness in T lymphoblasts. Sequences at -4559/-4525 and -2956/-2916, relative to the translation start site, function as strong composite GC response units (GRUs). Both GRUs include adjacent protein recognition sequences for the c-Myb transcription factor and the GR as a DNA cassette. An Ets-binding sequence overlaps the GR-binding site in the -4559/-4525 GRU, whereas an Ets-binding site present in the -2956/-2916 GRU does not overlap the GR/c-Myb-binding cassette. The Ets protein family member, PU.1, blocks hormonal activation of the -4559/-4525 GR/c-Myb-binding cassette but does not interfere with the responsiveness of the -2956/-2916 GRU. Thus, the hGR 1A GRU (described previously), the -4559/-4525 GRU, and the -2956/-2916 GRU have a similar structure and can mediate cell type-specific hormonal auto-up-regulation of hGR promoter activity in steroid-sensitive ALL cells. However, subtle differences in the GRU architecture result in differential sensitivity of the promoters to Ets family members such as PU.1. The architecture of the GRU and the spectrum of specific transcription factors present in different types of ALL might allow the development of a tailored therapy to enhance steroid sensitivity in ALL patients.

Steroid auto-regulation of the human glucocorticoid receptor (hGR) 1A promoter in lymphoblast cells resides largely in two DNA elements (footprints 11 and 12). We show here that c-Myb and c-Ets family members (Ets-1/2, PU.1, and Spi-B) control hGR 1A promoter regulation in T- and B-lymphoblast cells. Two T-lymphoblast lines, CEM-C7 and Jurkat, contain high levels of c-Myb and low levels of PU.1, whereas the opposite is true in IM-9 B-lymphoblasts. In Jurkat cells, overexpression of c-Ets-1, c-Ets-2, or PU.1 effectively represses dexamethasone-mediated up-regulation of an hGR 1A promoter-luciferase reporter gene, as do dominant negative c-Myb (c-Myb DNA-binding domain) or Ets proteins (Ets-2 DNA-binding domain). Overexpression of c-Myb in IM-9 cells confers hormone-dependent up-regulation to the hGR 1A promoter reporter gene. Chromatin immunoprecipitation assays show that hormone treatment causes the recruitment of hGR and c-Myb to the hGR 1A promoter in CEM-C7 cells, whereas hGR and PU.1 are recruited to this promoter in IM-9 cells. These observations suggest that the specific transcription factor that binds to footprint 12, when hGR binds to the adjacent footprint 11, determines the direction of hGR 1A promoter auto-regulation. This leads to a "molecular switch" model for auto-regulation of the hGR 1A promoter.

Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. When fused to Flag and enhanced green fluorescent protein (EGFP) tags at the carboxy terminus, all transcript variants can be correctly translated in vitro and in vivo. The Flag-tagged hGRalpha protein variants can functionally bind to a glucocorticoid response element (GRE) and can mediate hormonal stimulation of a pGRE-luciferase reporter gene. Compared to the "classical", native hGRalpha, these four variants exhibit a cell type-specific activation of a reporter gene, and this is influenced by the hGRalpha 3' untranslated region in the hGR transcript. When equal amounts of the cDNAs for these GRalpha variant proteins are transfected into cells, they can exhibit lower or higher transcriptional activation compared to the classical GR. Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.

The newly described 1A promoter of the human glucocorticoid receptor (hGR) gene contains an interferon (IFN) regulatory factor element (IRF-E), a binding motif for the family of proteins termed IFN regulatory factors (IRFs) that are regulated by IFNs. To examine the in vivo role of IFNs in hGR gene regulation, human T cell lines (CEM-C7 and Jurkat) were treated with IFN gamma. IFN gamma rapidly induces the expression of IRF-1 proteins in a dose- and time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A promoter IRF-E after IFN gamma treatment, whereas IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1 and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however, only IRF-1 activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in T cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Additional studies revealed that the glucocorticoid steroid hormone, dexamethasone (DEX), completely blocks IFN induction of IRF-1 mRNA levels. This could explain the lack of any greater apoptotic response to a combination of DEX plus IFN compared with the response to DEX alone. In addition, treatment with IFN gamma alone does not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter) in T lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1-driven transcription between the hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such as methylation.

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.

Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene in humans cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with progressive neuro-degeneration, cancer predisposition, immunodeficiency, and hypersensitivity to ionizing radiation. The ATM gene is highly conserved across a wide range of species. In an attempt to establish a zebrafish (Danio rerio) model of ataxia-telangiectasia, we cloned the coding sequence of the catalytic domain of the zebrafish homologue of ATM and found it to contain an open reading frame encoding 907 amino acids at the carboxyl terminus of the zebrafish ATM (zATM). The catalytic domain of zATM shares 67% and 66% homology with human ATM (hATM) and mouse ATM (mATM), respectively. The full-length mRNA encoding zATM is found to be approximately 11 kb by Northern hybridization, and the expression of zATM is observed in different adult and embryonic tissues. Overexpression of a kinase-inactive zATM domain in human cells has a dominant-negative effect against hATM function. Expression of the altered zATM in ZF4 cells leads to an A-T-like phenotype in response to ionizing radiation. These results taken together indicate that zATM is the homologue of hATM. Furthermore, using the kinase-inactive form of zATM should allow manipulation of zATM function in fish cells.

At least three promoters (1A, 1B, and 1C) control the expression of mRNA transcripts for the human glucocorticoid receptor (hGR) protein. An hGR 1A promoter/exon sequence (-218/+269) contains at least 12 deoxyribonuclease (DNase) I footprints that contain bound protein. Whereas four of these footprints (FP6, FP7, FP8, and FP11) contain bound hGR in protein-DNA complexes that are formed, only two (FP7 and FP11) appear to be important for the up-regulation of hGR 1A promoter/exon activity in T-lymphoblasts. Furthermore, the activity of these DNA elements depends upon the promoter context, leading to a redundant and complex regulation of expression of the hGR 1A promoter/exon. FP7 appears to be required for hormonal responsiveness in the absence of upstream sequences (+41/+191), whereas the hormonal responsiveness of FP11 requires a functional, adjacent FP12 DNA sequence. FP12 contains overlapping binding sites for the protooncogene transcription factors c-Myb and c-Ets. It seems likely that binding of either c-Myb or c-Ets to FP12 is necessary for the direct or indirect binding of the hGR to FP11 (a nonconsensus glucocorticoid response element), and the resultant steroid-responsiveness of the hGR 1A promoter/exon sequence. We propose that the identity of the accessory transcription factor bound to FP12 (c-Myb or c-Ets) may determine the nature of regulation (positive or negative) of hGR gene expression by hormone, and that this may be important for hormone-induced apoptosis in T cell acute lymphoblastic leukemia.

A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from the mold Aspergillus giganteus MDH 18894 has been developed. alpha-Sarcin and AFP were purified simultaneously by carboxymethylcellulose 52 cation-exchange chromatography and Blue Sepharose CL-6B affinity chromatography. By this method, 4.2 mg of alpha-sarcin and 6.8 mg of AFP were obtained from 2 liters of medium. Compared with other purification methods such as gel-filtration chromatography, this procedure was simple and specific. The purified alpha-sarcin and AFP were homogeneous characterized on SDS-polyacrylamide gel. The enzymatic activity of several ribosome-inactivating proteins such as alpha-sarcin, trichosanthin, and cinnamomin was significantly inhibited by the dye Cibacron blue F3GA. In 50 microliter of reaction mixture, 10 microM of the dye could inhibit 50% activity of cinnamomin (7 x 10(-9) M), whereas 50% inhibition of the enzymatic activity of trichosanthin (7 x 10(-9) M) and alpha-sarcin (1 x 10(-7) M) required 100 and 50 microM of the dye, respectively., (Copyright 1998 Academic Press.)