Very recently a new designation of "Malignant Neuroectodermal Gastrointestinal Tumor" has been proposed for an aggressive form of neuroectodermal tumor with features similar to that of Clear Cell Sarcoma of Soft Tissue, however without a melanocytic differentiation. Also known as "clear cell sarcoma-like tumors of the gastrointestinal tract", these tumors show some features strongly suggesting an origin from a gastrointestinal neuroectodermal precursor cell unable to differentiate along the melanocytic lineage. They occur mainly in young and middle-aged adults, and have a poor prognosis with a high rate of liver and lymphnode metastases. Histologically they are composed of epithelioid or oval-to spindle cells with a sheet-like or nested pattern of growth, strongly positive for neural markers (S-100, SOX10, and vimentin) and negative for the melanocytic ones. EWSR1 gene rearrangements including EWSR1-ATF1 or EWSR1-CREB1 GENE fusions are typically assessed in these tumors. Here we report a case of malignant neuroectodermal gastrointestinal tumor which immunophenotypically unusually expressed FLI-1, occurring in a 29-year-old man with a previous medical history of Ewing sarcoma. We finally suggest that this case might be a further evidence of a link between these two entities.

The highly conserved fission yeast Pmk1 MAPK pathway plays a key role in cell integrity by regulating Atf1, which belongs to the ATF/cAMP-responsive element-binding (CREB) protein family. We identified and characterized ecm33(+), which encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. We demonstrated that the gene expression of Ecm33 is regulated by two transcription factors Atf1 and a MADS-box-type transcription factor Mbx1. We identified a putative ATF/CREB-binding site and an RLM1-binding site in the ecm33(+) promoter region and monitored the transcriptional activity of Atf1 or Mbx1 in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of the CRE and six tandem repeats of the Rlm1-binding sequence, respectively. These reporter genes reflect the activation of the Pmk1 pathway by various stimuli, thereby enabling the real-time monitoring of the Pmk1 cell integrity pathway. Notably, the Deltaecm33 cells displayed hyperactivation of the Pmk1 signaling together with hypersensitivity to Ca(2+) and an abnormal morphology, which were almost abolished by simultaneous deletion of the components of the Rho2/Pck2/Pmk1 pathway. Our results suggest that Ecm33 is involved in the negative feedback regulation of Pmk1 cell integrity signaling and is linked to cellular Ca(2+) signaling.

Transcription factors of the cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family were implicated in the expression of T cell-specific genes and in the expression of oncogenic retroviruses associated with leukemia in T and B lymphocytes. To study the regulation of CREB/ATF transcription factors during lymphocyte activation, studies were pursued in primary cultures of resting murine splenic T and B lymphocytes stimulated via the Ag receptor. Using consensus/CRE and proliferating cell nuclear Ag (PCNA)/CRE as probes in the DNA binding assay, we showed that a marked induction of CRE binding is associated with activation of splenic T lymphocytes with anti-CD3 Ab. CRE binding was markedly induced after 48 h; it gradually declined at 72 h, but remained elevated above control levels after 120 h. Most significant, activation by anti-CD3 was associated with a marked induction of cAMP levels that preceded the onset of DNA synthesis and the induction of IL-2 secretion and reached a peak after 48 h (9.5- to 11-fold), concomitant with the peak in CRE binding. Rapamycin, a potent immunosuppressant, inhibited the induction of cAMP levels by anti-CD3 concomitant with inhibition of CRE binding activity and arrest of DNA synthesis. A marked induction in CRE binding after 48 h was also found in splenic B lymphocytes stimulated by LPS and anti-Ig and was correlated with a 3- to 4-fold increase in the intracellular levels of cAMP. Two inducible CRE complexes were found to bind to consensus/CRE and PCNA/CRE; the major complex contained primarily CREB homodimers and was constitutively expressed in resting lymphocytes. Conversely, stimulation of lymphocytes was associated with formation of a new, slow migrating CRE complex that demonstrated high inducibility in both consensus/CRE and PCNA/CRE. We show that this de novo inducible CRE complex contains CREB and ATF2, but not ATF1. Taken collectively, these results suggest that recruitment of CREB and ATF2 to the promoter of genes is tightly regulated during activation of T and B lymphocytes and implicate a cross-talk of cAMP and non-cAMP pathways in the regulation of transcriptional processes at late stages of activation in T and B lymphocytes stimulated via the Ag receptor.

We have developed a simple method to purify sequence-specific DNA-binding proteins directly from crude cell extracts by using DNA affinity latex beads. The method enabled us to purify not only DNA-binding proteins, but also their associated proteins. Using beads bearing the ATF/E4TF3 site from the adenovirus E4 gene promoter, a protein kinase activity was copurified with the ATF/E4TF3 family. We found that the kinase interacted with ATF1 in vitro efficiently. The kinase did not bind directly to DNA. The kinase mainly phosphorylated ATF1 on serine 36, which was one of target amino acids for casein kinase (CK) II. Biological features of the kinase were the same as those of CKII and an anti-CKII serum reacted with the kinase, indicating that the kinase was CKII. Moreover, it was clearly shown that one of CKII subunits, the CKII alpha protein bound to glutathione-S-transferase (GST) fusion ATF1 but not GST in vitro. It has been reported that a specific CKII inhibitor, 5,6-dichloro-1-beta-D-ribo-furanosylbenzimidazole (DRB) inhibits transcription by RNA polymerase II [Zandomeni et al., (1986) J. Biol. Chem. 261, 3414-3419]. Taken together, these results suggest that ATF/E4TF3 may recruit the CKII activity to a transcription initiation machinery and stimulate transcription.

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.

Soft tissue tumours represent a heterogeneous group of mesenchymal lesions and their classification continues to evolve as a result of incorporating advances in cytogenetic and molecular techniques. In the last decade, traditional diagnostic approaches were supplemented with a significant number of reliable molecular diagnostic tools, detecting tumour type specific genetic alterations. Additionally, the successful application of some of these techniques to formalin fixed, paraffin embedded tissue enabled a broader range of clinical material to be subjected to molecular analysis. However, despite all these remarkable advances, the realisation that some of the genetic abnormalities are not fully histotype specific and that certain gene aberrations can be shared among different sarcoma types, otherwise completely unrelated clinically or immunophenotypically, has introduced some drawbacks in surgical pathology practice. One such common example is the presence of EWSR1 gene rearrangements by fluorescence in situ hybridisation (FISH), a test now preferred over the elaborate RT-PCR testing, in a variety of benign and highly malignant soft tissue tumours, in addition to a subset of carcinomas. Furthermore, the presence of identical gene fusions in completely different sarcoma types (i.e., EWSR1-ATF1, EWSR1-CREB1) or in non-mesenchymal malignancies (epithelial or haematological) has raised skepticism as to their diagnostic utility, and their lack of specificity has been compared to the limitations of other ancillary techniques, in particular immunohistochemistry. This review catalogues the main groups of genes that behave in a promiscuous manner within recurrent fusion events in soft tissue tumours. Although we acknowledge that the present molecular classification of soft tissue tumours is much more complex than two decades ago, when EWSR1 gene rearrangements had been described as the hallmark of Ewing sarcoma, we make the strong argument that with very few exceptions, the prevalence of fusion transcripts in most sarcomas is such that they come to define these entities and can be used as highly specific molecular diagnostic markers in the right clinical and pathological context.

The Ets-related DNA-binding protein human GA-binding protein (hGABP) alpha interacts with the four ankyrin-type repeats of hGABPbeta to form an hGABP tetrameric complex that stimulates transcription through the adenovirus early 4 (E4) promoter. Using co-transfection assays, this study demonstrated that the hGABP complex mediated efficient activation of transcription from E4 promoter synergistically with activating transcription factor (ATF) 1 or cAMP response element-binding protein (CREB), but not ATF2/CRE-BP1. This synergy also partially occurred when hGABPalpha was used alone in place of the combination of hGABPalpha and hGABPbeta. hGABP activated an artificial promoter containing only ATF/CREB-binding sites under coexistence of ATF1 or CREB. Consistent with these results, physical interactions of hGABPalpha with ATF1 or CREB were observed in vitro. Functional domain analyses of the physical interactions revealed that the amino-terminal region of hGABPalpha bound to the DNA-binding domain of ATF1, which resulted in the formation of ternary complexes composed of ATF1, hGABPalpha, and hGABPbeta. In contrast to hGABPalpha, hGABPbeta did not significantly interact with ATF1 and CREB. Taken together, these results indicate that hGABP functionally interacts with selective members of the ATF/CREB family, and also suggest that synergy results from multiple interactions which mediate stabilization of large complexes within the regulatory elements of the promoter region, including DNA-binding and non-DNA-binding factors.

Vitamin D deficiency is common in the Caucasian population and is associated with increased incidence and unfavorable outcome of many diseases, including various types of cancer, infectious, cardiovascular, and autoimmune diseases. Individual factors that predispose for a person's vitamin D status, such as skin type, have been identified, but limited data exist on genetic determinants of serum 25-hydroxyvitamin D (25[OH]D) concentration. We have tested the hypothesis that variants of genes (single nucleotide polymorphisms [SNPs]) involved in skin pigmentation are predictive of serum 25(OH)D levels. Serum 25(OH)D and SNPs (n = 960) related to genes with relevance for skin pigmentation (tyrosinase [TYR], TYR-related protein 1 [TYRP1], dopachrome tautomerase [DCT], oculocutaneous albinism II [OCA2], two pore segment channel 2 [TPCN2], solute carrier family 24 A4 [SLC24A4], solute carrier family 45 A2 [SLC45A2], agouti signalling peptide [ASIP], cyclic AMP-dependent transcription factor [ATF1], microphthalmia-associated transcription factor [MITF], proopiomelanocortin [POMC], cAMP-dependent protein kinase catalytic subunit beta [PRKACB], cAMP-dependent protein kinase catalytic subunit gamma [PRKACG], cAMP-dependent protein kinase type I-alpha regulatory subunit [PRKAR1A], cAMP-dependent protein kinase type II-alpha regulatory subunit [PRKAR2A], cAMP-dependent protein kinase type II-beta regulatory subunit [PRKAR2B], tubulin beta-3 chain/melanocortin receptor 1 [TUBB3/MC1R], Cadherin-1 [CDH1], catenin beta 1 [CTNNB1], Endothelin 1 [EDN1], endothelin 3 [EDN3], endothelin receptor type B [EDNRB], fibroblast growth factor 2 [FGF2], KIT, KIT ligand [KITLG], nerve growth factor [NGF], interferon regulatory factor 4 [IRF4], exocyst complex component 2 [EXOC2], and tumor protein 53 [TP53]) were analyzed in a cohort of participants of the Ludwigshafen Risk and Cardiovascular Health Study (n = 2970). A total of 46 SNPs were associated (P <.05) with lower or higher serum 25(OH)D levels as compared with the total cohort (median, 15.5 ng/mL). Although 1 SNP in the EXOC2 gene reached the aimed significance level after correction for multiple comparisons (false discovery rate) and was associated with a Δ25(OH)D value more than 5.00 ng/mL, 11 SNPs located in the TYR (n = 4), PRKACG (n = 1), EDN1 (n = 3), TYRP1 (n = 1), and microphthalmia-associated transcription factor (n = 2) genes reached the aimed significance level after false discovery rate correction but were not associated with Δ25(OH)D value more than 5.00 ng/mL. We conclude that variants of genes involved in skin pigmentation are predictive of serum 25(OH)D levels in the Caucasian population. Our data indicate that out of the variants in 29 different genes analyzed, variants of 11 genes, including EXOC2, TYR, and TYRP1, have the highest impact on vitamin D status. Our results have a fundamental importance to understand the role of sunlight, skin pigmentation, and vitamin D for the human evolution.

In fission yeast, the Sty1/Spc1/Phh1 mitogen-activated protein kinase (MAPK) pathway is known to be involved in multiple-stress responses. It is currently thought that the Sty1 MAPK cascade is mediated by histidine kinases and phosphorelay proteins in response to oxidative stress signals. However, studies of the exact transduction mechanism of multiple-stress responses are lacking. Thus, in response to various stimuli, we monitored the Sty1 MAPK pathway through the downstream transcription factor Atf1 in living cells using a highly sensitive luciferase reporter gene. Surprisingly, in cadmium and low glucose (LG) medium, Atf1 activation was observed even in the absence of all of the four fission yeast MAPK kinase kinases (MAPKKKs); whereas in osmotic stress, Atf1 activation was abolished. Thus, the osmotic stress likely mediates the MAPK activation via MAPKKKs, whereas a cadmium or LG condition activates the MAPK in a MAPKKK-independent manner. On the other hand, knockout of tyrosine phosphatase gene pyp1(+) abolished the Atf1 response to cadmium and LG, but not to osmotic stress, suggesting that Pyp1 is a sensor for cadmium and LG.

OBJECTIVE

Ankaferd® Blood Stopper (ABS) is an herbal extract that has historically been used as a hemostatic agent in traditional Turkish medicine. ABS is comprised of a standardized herbal mixture of T. vulgaris, G. glabra, V. vinifera, A. officinarum, and U. dioica. ABS's basic mechanism of action is the formation of an encapsulated protein web, which represents the focal point for vital erythrocyte masses. The hemostatic effects of ABS have been observed in vitro and in vivo. ABS was registered as a hemostatic agent for external hemorrhages and dental bleeding following phase I randomized, double-blind crossover placebo-controlled clinical research, and safety and efficacy reports. In terms of the potential use of ABS, transcription factors may be novel factors that play a role in the hemostatic and other pleiotropic effects of ABS.

METHODS

Hence, the present study aimed to investigate the effects of ABS on endothelium, and possible transcription factor changes in HUVEC (human umbilical vein endothelial cells) and the erythrocyte membrane profile. ABS (5 μL and 50 μL) was administered to HUVEC (in 75 cm2; ~75% fullness) for 5 min and 15 min.

RESULTS

ABS caused significant increases in the level of activation of the following transcription factors; AP2, AR, CRE/ATF1, CREB, E2F1-5, E2F6, EGR, GATA, HNF-1, ISRE, Myc-Max, NF-1, NFkB, p53, PPAR, SMAD 2/3, SP1, TRE/AP1, and YY1. Following erythrocyte membrane isolation, protein complexes were undissolved, but denatured. The protein complex formed was resistant to heat and detergent. Trypsin and sonication were used in order to break this complex; the complex dissolved and erythrocyte membrane proteins were released in SDS-PAGE.

CONCLUSIONS

ABS established a very fast and solid protein web, and increased the level of transcription factor activation. Therefore the cellular effects of ABS could be related to different intracellular biological pathways.

In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24-41 and 508-525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.

Recent generation of genetically modified Creb1 mutant mice has revealed an important role for CREB (cAMP responsive element binding protein) and the related proteins CREM (cAMP responsive element modulator) and ATF1 (activating transcription factor 1) in cell survival, in agreement with previous studies using overexpression of dominant-negative CREB (dnCREB). CREB and ATF1 are abundantly expressed in T cells and are rapidly activated by phosphorylation when T cells are stimulated through the T cell antigen receptor. We show that T cell-specific loss of CREB in mice, in combination with the loss of ATF1, results in reduced thymic cellularity and delayed thymic recovery following sublethal irradiation but no changes in T cell development or activation. These data show that loss of CREB function has specific effects on thymic T lymphocyte proliferation and homeostasis in vivo.

Mesothelioma is a rare but very aggressive tumor derived from mesothelial cells. A number of often complex but nonrandom cytogenetic abnormalities have been found in these tumors, resulting in loss of chromosome bands 14q32 and 22q12 in more than 35% of the cases. In this study, we used RNA sequencing to search for fusion transcripts in a mesothelioma carrying a t(14;22)(q32;q12) as the sole chromosomal aberration and found an EWSR1-YY1 and its reciprocal YY1-EWSR1 fusion transcript. Screening 15 additional cases of mesothelioma from which we had RNA but no cytogenetic information, we identified one more tumor carrying an EWSR1-YY1 fusion gene but not the reciprocal YY1-EWSR1 transcript. RT-polymerase chain reaction and sequencing showed that in both cases exon 8 of EWSR1 (nucleotide 1,139, accession number NM_013986 version 3, former exon 7 in sequence with accession number X66899) was fused to exon 2 of YY1 (nucleotide 1,160, accession number NM_003403 version 3). The EWSR1 breakpoint in exon 8 in the EWSR1-YY1 chimeric transcript is similar to what is found in other fusions involving EWSR1 such as EWSR1-FLI1, EWSR1-DDIT3, and EWSR1-ATF1. The EWSR1-YY1-encoded protein is an abnormal transcription factor with the transactivation domain of EWSR1 and the DNA-binding domain of YY1. This is the first study to detect a specific fusion gene in mesothelioma (the reason how frequent the EWSR1-YY1 fusion is remains uncertain) and also the first time that direct involvement of YY1 in oncogenesis has been demonstrated.

Neuroblastoma cells having stem cell-like qualities are widely employed models for the study of neural stem/progenitor cell proliferation and differentiation. We find that human BE(2)C neuroblastoma cells possess a signaling cascade initiated by Ca(2+) influx via voltage-dependent calcium channels and the N-methyl-D-aspartate (NMDA) receptor and culminating in nuclear calmodulin-dependent protein kinase IV (CaMKIV)-mediated phosphorylation and activation of the transcription factors Ca(2+)/cyclic AMP-response element-binding protein (CREB) and ATF1 (activating transcription factor-1). This pathway functions to maintain BE(2)C cells in an undifferentiated, proliferative state. Parallel to this Ca(2+)-dependent pathway is a hormone-responsive program by which retinoic acid (RA) initiates the differentiation of BE(2)C cells toward a neuronal lineage. This is evidenced by RA-dependent induction of the cell cycle inhibitor p21/Cip1 (Cdk-interacting protein 1) and cell cycle arrest, induction of the neuroblastic marker doublecortin and of the neuron-specific intermediate filament protein, peripherin, and by RA-stimulated extension of neuritic processes. During neuronal differentiation there is a complex antagonistic interplay between these two major signaling pathways. RA down-regulates expression of CaMKIV and one of its upstream activators, CaMKK1 (calmodulin-dependent protein kinase kinase 1). This is accompanied by RA-induced suppression of activating phosphorylation of CREB with a time course paralleling that of CaMKIV down-regulation. RA-induced repression of the Ca(2+)/calmodulin-dependent protein kinase kinase/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation, as shown by the effect of RNA interference of CaMKIV to markedly accelerate RA-dependent up-regulation of p21/Cip1 and doublecortin expression and RA-promoted neurite outgrowth. RA-induced repression of the CaMKIV signaling pathway may represent an early event in retinoid-dependent neuronal differentiation.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2¸ EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae- and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.

The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi-copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2.5-fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse-field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus-type gene, which has a relatively low level of homology with the S. cerevisiae-type gene, is also usually detected. Interestingly, an S. bayanus-type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2-lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg-ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively.

BACKGROUND

A key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase). This pathway is also prevalent in fruit, producing short and medium chain volatile esters during ripening. In this work, a series of six AATases from Saccharomyces and non-Saccharomyces yeasts as well as tomato fruit were evaluated with respect to their activity, intracellular localization, and expression in Saccharomyces cerevisiae and Escherichia coli cell hosts. The series of AATases includes Atf1 and Atf2 from S. cerevisiae, as well as AATases from S. pastorianus, Kluyveromyces lactis, Pichia anomala, and Solanum lycopersicum (tomato).

RESULTS

When expressed in S. cerevisiae, Atf1, Atf2, and an AATase from S. pastorianus localized to lipid droplets, while AATases from non-Saccharomyces yeasts and tomato fruit did not localize to intracellular membranes and were localized to the cytoplasm. All AATases studied here formed intracellular aggregates when expressed in E. coli, and western blot analysis revealed that expression levels in E. coli were upwards of 100-fold higher than in S. cerevisiae. Fermentation and whole cell lysate activity assays of the two most active AATases, Atf1 from S. cerevisiae and an AATase from tomato fruit, demonstrated that the aggregates were enzymatically active, but with highly reduced specific activity in comparison to activity in S. cerevisiae. Activity was partially recovered at lower expression levels, coinciding with smaller intracellular aggregates. In vivo and in vitro activity assays from heterologously expressed Atf1 from S. cerevisiae, which localizes to lipid droplets under homologous expression, demonstrates that its activity is not membrane dependent.

CONCLUSIONS

The results of these studies provide important information on the biochemistry of AATases under homologous and heterologous expression with two common microbial hosts for biochemical processes, S. cerevisiae and E. coli. All studied AATases formed aggregates with low enzymatic activity when expressed in E. coli and any membrane localization observed in S. cerevisiae was lost in E. coli. In addition, AATases that were found to localize to lipid droplet membranes in S. cerevisiae were found to not be membrane dependent with respect to activity.

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers' yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.

Clear cell sarcoma of soft tissue (CCS-ST) is a rare malignant neoplasm characterized by a tumor-defining translocation [t(12;22) (q13;q12)], resulting in the EWS-ATF1 gene fusion. An extremely limited number of visceral CCS-ST cases have been reported in the literature. Here the authors report a visceral CCS-ST in a Hispanic adolescent male with a large infiltrative mass involving the small bowel. The tumor was evaluated by light microscopy, immunocytochemistry, electron microscopy, cytogenetics, and molecular genetics. The tumor cells were strongly positive for S-100 protein, but negative for HMB-45. Rare premelanosomes were identified only after an extensive search with electron microscopy. Cytogenetics showed a characteristic t(12;22)(q13;q12) for CCS-ST with isochromosome 18q and trisomy 22. An EWS exon 8 sense primer and an antisense ATF1 primer were employed for detection of the CCS-ST tumor-defining EWS-ATF1 translocation, using reverse transcriptase-polymerase chain reaction techniques (RT-PCR), and the fusion gene breakpoint underwent DNA sequencing. This tumor is exceptional, because it is the first visceral CCS-ST that has been confirmed by RT-PCR and DNA sequencing. This case also illustrates the necessity of a multimodal approach to tumor diagnosis, and the utility of cytogenetics and molecular pathology in confirming the diagnosis of CCS-ST and eliminating conventional metastatic or primary visceral malignant melanoma as a consideration.

Chimeric proteins resulting from characteristic chromosomal translocations are believed to play a key role in the development of neoplasia. The consistent chromosomal translocation t(12;22) found in Clear Cell sarcoma (CCS) fuses the genes for Ewing's sarcoma protein (EWS) and activating transcription factor 1 (ATF1). Contribution of the chimeric EWS/ATF1 protein to maintenance of the tumor phenotype was investigated using intracellular expression of an inhibitory anti-ATF1 single chain antibody fragment (scFv4). Transfection of scFv4 into a cell line (SU-CCS-1) derived from CCS resulted in a 90% reduction in cyclic AMP response element-driven reporter activity. The delivery of scFv4 into SU-CCS-1 cells by a Moloney sarcoma retroviral vector (SRalpha-Fv4) significantly reduced viability and induced apoptosis as measured by terminal deoxynucleotidetransferase-mediated dUTP-biotin nick end labeling and flow cytometry. Conversely, scFv4 had no effect on viability of HeLa cells. The level of EWS/ATF1 expression was found to be significantly higher in primary tumor tissue than in SU-CCS-1 cells or in 293T cells following introduction of an EWS/ATF1 expression vector. These studies demonstrate a direct role for the EWS/ATF1 fusion protein in maintaining tumor cell viability of Clear Cell sarcoma and indicate that intracellular antibodies may be used to achieve a phenotypic knockout of tumor-related proteins as a method to explore their function.

BACKGROUND

Many types of sarcomas are characterized by specific chromosomal translocations that result in the production of novel chimeric genes. Detection of these fusion genes could be a sensitive molecular diagnostic assay. However, to the authors' knowledge there have been few systemic comparisons between the current histopathologic diagnosis and the presence or absence of particular fusion genes in patients with adult soft tissue sarcomas (STSs).

METHODS

Total RNA was extracted from 75 cases of STS and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of a variety of fusion transcripts. The results of the molecular assay were compared with standard histopathologic diagnoses.

RESULTS

Of the 18 tumors diagnosed as synovial sarcoma, 17 (94%) expressed SYT-SSX chimeric transcripts. All nine myxoid liposarcomas were positive for FUS-CHOP fusion transcripts. Of the four cases of Ewing sarcoma, two had an EWS-FLI1 fusion transcript and one had an EWS-ERG fusion transcript. A clear cell sarcoma had a EWS-ATF1 fusion transcript. None of 19 cases of malignant fibrous histiocytoma nor 3 leiomyosarcomas contained a fusion transcript. Three cases with an initial diagnosis other than synovial sarcoma expressed a SYT-SSX fusion transcript. A review of the slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. There was a trend for biphasic synovial sarcoma to contain the SYT-SSX1 fusion.

CONCLUSIONS

The authors believe RT-PCR assay for the detection of a specific fusion gene provides a useful tool for confirmation of the diagnosis of adult STS in diagnostically difficult cases and in retrospective studies.

BACKGROUND

Network Component Analysis (NCA) has been used to deduce the activities of transcription factors (TFs) from gene expression data and the TF-gene binding relationship. However, the TF-gene interaction varies in different environmental conditions and tissues, but such information is rarely available and cannot be predicted simply by motif analysis. Thus, it is beneficial to identify key TF-gene interactions under the experimental condition based on transcriptome data. Such information would be useful in identifying key regulatory pathways and gene markers of TFs in further studies.

RESULTS

We developed an algorithm to trim network connectivity such that the important regulatory interactions between the TFs and the genes were retained and the regulatory signals were deduced. Theoretical studies demonstrated that the regulatory signals were accurately reconstructed even in the case where only three independent transcriptome datasets were available. At least 80% of the main target genes were correctly predicted in the extreme condition of high noise level and small number of datasets. Our algorithm was tested with transcriptome data taken from mice under rapamycin treatment. The initial network topology from the literature contains 70 TFs, 778 genes, and 1423 edges between the TFs and genes. Our method retained 1074 edges (i.e. 75% of the original edge number) and identified 17 TFs as being significantly perturbed under the experimental condition. Twelve of these TFs are involved in MAPK signaling or myeloid leukemia pathways defined in the KEGG database, or are known to physically interact with each other. Additionally, four of these TFs, which are Hif1a, Cebpb, Nfkb1, and Atf1, are known targets of rapamycin. Furthermore, the trimmed network was able to predict Eno1 as an important target of Hif1a; this key interaction could not be detected without trimming the regulatory network.

CONCLUSIONS

The advantage of our new algorithm, relative to the original NCA, is that our algorithm can identify the important TF-gene interactions. Identifying the important TF-gene interactions is crucial for understanding the roles of pleiotropic global regulators, such as p53. Also, our algorithm has been developed to overcome NCA's inability to analyze large networks where multiple TFs regulate a single gene. Thus, our algorithm extends the applicability of NCA to the realm of mammalian regulatory network analysis.

Regulation and the role of the sod1+ gene encoding CuZnSOD were investigated in fission yeast Schizosaccharomyces pombe. The amount of sod1+ mRNA decreased in the stationary phase, consistent with the decrease in enzyme activity. The transcript increased by treatment with oxidants such as H(2)O(2) and menadione (MD). Induction by H(2)O(2) was rapid and transient, being dependent on Wis1-Spc1-Atf1 pathway of signal transduction, whereas induction by MD was slow and sustained longer, being independent of Wis1 pathway. Wis1 and Spc1 also turned out to down-regulate sod1+ gene at the stationary phase. Tetrad analysis following sod1+ gene disruption revealed that the sod1Delta cells were not viable, even on rich media. Repression of the sod1+ gene expression by thiamine through nmt1 promoter resulted in the arrest of cell cycle progression following S phase, possibly between G(2) and cytokinesis. The current and previous observations that the viability of Schizosaccharomyces pombe cells, unlike Saccharomyces cerevisiae, critically depends on the action of oxidative defense enzymes in the cytosol, such as CuZnSOD and glutathione reductase, suggest that S. pombe can serve as a good model system to study the effect of oxidative stress on cell proliferation.