BACKGROUND

Os odontoideum is a well identified anomaly of the craniovertebral junction. Since its initial description, there has been a continuous debate regarding the nature of its etiology: Whether congenital or traumatic. We sought to compare the gene expression profiles in patients with congenital os odontoideum, those with traumatic os odontoideum and controls.

METHODS

We have evaluated a pair of identical twins both with os odontoideum. We identified two additional patients with and four subjects without os odontoideum. We analyzed the gene expression profiles in these patients using a custom TaqMan microarray and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The relative gene expression profiles in the two identical twins, the two nontwin patients with os odontoideum and the controls were assessed.

RESULTS

A total of 213 genes with significantly different expression between the twin os odontoideum patients and the subjects without os odontoideum were detected. CACNG6, PHEX, CACNAD3, IL2, FAS, TUFT1, KIT, TGFBR2, and IGF2 were expressed at levels greater than 100-fold more in the twins. There were six genes with significantly different expression profiles in the twins as compared with the nontwin os odontoideum patients: CMK4, ATF1, PLCG1, TAB1, E2F3, and ATF4. There were no statistically significant differences in gene expression in the four patients with os odontoideum and the subjects without. Trends, however, were noted in MMP8, KIT, HIF1A, CREB3, PWHAZ, TGFBR1, NFKB2, FGFR1, IPO8, STAT1, COL1A1, and BMP3.

CONCLUSIONS

Os odontoideum has multiple etiologies, both traumatic and congenital and perhaps some represent a combination of the two. This work has identified a number of genes that show increased expression in a pair of twins with congenital os odontoideum and also demonstrates trends in gene expression profiles between a larger group of os odontoideum patients and non-os patients. A number of these genes are related to bone morphogenesis and maintenance.

Tanning ability is important, because it represents the ability of the skin to protect itself against ultraviolet (UV) radiation. Here, we sought to determine genetic regions associated with tanning ability. Skin pigmentation was measured at the outer forearm and buttock areas to represent facultative and constitutive skin color, respectively. In our study population consisting of isolated Mongolian subjects, with common histories of environmental UV exposure during their nomadic life, facultative skin color adjusted by constitutive skin color was used to indicate tanning ability. Through linkage analysis and family-based association tests of 345 Mongolian subjects, we identified 2 potential linkage regions regulating tanning ability on 5q35.3 and 12q13.2, having 6 and 7 significant single nucleotide polymorphisms (SNPs), respectively. Those significant SNPs were located in or adjacent to potential candidate genes related to tanning ability: GRM6, ATF1, WNT1, and SILV/Pmel17.

Pulmonary clear cell sarcoma is a rare malignant tumor that has rarely been reported and is challenging to diagnose, especially when differentiating from malignant melanoma. Currently, EWSR1-ATF1 is the key marker for distinguishing clear cell sarcoma from melanoma, but IHC has diagnostic limitations. We report a patient diagnosed with pulmonary clear cell sarcoma, in which an NGS was used to help with the pathological diagnosis. The exposure to the immune microenvironment in pulmonary clear cell sarcoma suggests that TIGIT-related drugs may be a new and effective treatment for this rare disease. Immune microenvironment-related markers, including PD-L1, CD8, TIM3, LAG3, and CD163, were negatively expressed in pulmonary clear cell sarcoma.

Keywords: EWS-ATF1; PD-L1; TIGIT; clear cell sarcoma; lung; next-generation sequencing.

In meiosis, multiple different DNA sequence motifs help to position homologous recombination at hotspots in the genome. How do the seemingly disparate <i>cis</i>-acting regulatory modules each promote locally the activity of the basal recombination machinery? We defined molecular mechanisms of action for five different hotspot-activating DNA motifs (<i>M26</i>, <i>CCAAT</i>, <i>Oligo-C</i>, <i>4095</i>, <i>4156</i>) located independently at the same site within the <i>ade6</i> locus of the fission yeast <i>Schizosaccharomyces pombe</i> Each motif promoted meiotic recombination (i.e., is active) within this context, and this activity required the respective binding proteins (transcription factors <em>Atf1</em>, Pcr1, Php2, Php3, Php5, Rst2). High-resolution analyses of chromatin structure by nucleosome scanning assays revealed that each motif triggers the displacement of nucleosomes surrounding the hotspot motif in meiosis. This chromatin remodeling required the respective sequence-specific binding proteins, was constitutive for two motifs, and was enhanced meiotically for three others. Hotspot activity of each motif strongly required the ATP-dependent chromatin remodeling enzyme Snf22 (Snf2/Swi2), with lesser dependence on Gcn5, Mst2 and Hrp3. These findings support a model in which most meiotic recombination hotspots are positioned by the binding of transcription factors to their respective DNA sites. The functional redundancy of multiple, sequence-specific protein-DNA complexes converges upon shared chromatin remodeling pathways that help provide the basal recombination machinery (Spo11/Rec12 complex) access to its DNA substrates within chromatin.

BACKGROUND

Meiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not fully understood. The fission yeast spo5+ gene encodes a meiosis-specific RNA-binding protein, which is required for the progression of meiosis II and spore formation. However, the target RNA molecules of Spo5 are yet to be identified. Characterization of meiosis-specific RNA-binding proteins will provide insight into how post-transcriptional regulation influence gene expression during sexual differentiation.

RESULTS

To assess the functional significance of RNA-recognition motifs (RRMs) of Spo5, we constructed a series of new spo5 truncated mutants and previously reported spo5 missense mutants. In addition, we isolated novel spo5 missense mutants. The phenotypic characteristics of these mutants indicated that the RRMs are essential for both the localization and function of the protein. Interestingly, Spo5 is exported from the nucleus to the cytoplasm via the Rae1-dependent mRNA export pathway, but is unlikely to be involved in global mRNA export. Furthermore, cytoplasmic localization of Spo5 is important for its function, which suggests the involvement of Spo5 in post-transcriptional regulation. We identified pcr1+ mRNA as one of the critical targets of Spo5. The pcr1+ gene encodes an activating transcription factor/cAMP response element binding (ATF/CREB) transcription factor family. Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.

CONCLUSIONS

Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway. RRMs are necessary for this process and also for the function of Spo5 after the nuclear export. Spo5 appears to influence the activity of pcr1+ mRNA, and the mechanism of how Spo5 stimulates the mRNA to promote the progression of meiosis II and spore formation remains an intriguing question for future research.

Clear cell carcinoma (CCC) is a rare low-grade malignant salivary gland carcinoma. EWSR1-ATF1 fusion has been characterized as a consistent finding in CCC, with breakpoints described between EWSR1 exon 11 and ATF1 exon 3. So far, over 100 cases of CCC harboring EWSR1 rearrangement arising from salivary gland of the oral cavity have been reported. Although EWSR1 involvement in these cases was confirmed by EWSR1 break-apart FISH indicating the translocation, sequence analysis for EWSR1-ATF1 fusion type has been reported only in three cases of CCC so far. Herein, we report a CCC case with novel EWSR1-ATF1 fusion (EWSR1 exon 15 and ATF1 exon 5) arising in minor salivary gland and review the role of the chimeric variants in some malignancies with EWSR1-ATF1 rearrangement. Current tumor was composed of the small nests of clear tumor cells and hyalized fibrous stroma. Immunohistochemically, the tumor was positive for AE1/AE3, CK5/6 and p63, negative for S100, Melan-A, SMA and CD10. After 8 months of follow-up, there are no evidence of recurrence.

Keywords: ATF1; Clear cell carcinoma; EWSR1; Hyalinizing clear cell carcinoma; Salivary gland tumor.

Penicillium oxalicum has received increasing attention as a potential cellulase-producer. In this study, a copper-controlled flippase recombination enzyme/recognition target (FLP/FRT)-mediated recombination system was constructed in P. oxalicum, to overcome limited availability of antibiotic resistance markers. Using this system, two crucial transcription repressor genes atf1 and cxrC for the production of cellulase and xylanase under solid-state fermentation (SSF) were simultaneously deleted, thereby leading to 2.4- to 29.1-fold higher cellulase and 78.9% to 130.8% higher xylanase production than the parental strain under SSF, respectively. Glucose and xylose released from hydrolysis of pretreated sugarcane bagasse achieved 10.6%-13.5% improvement by using the crude enzymes from the engineered strain Δatf1ΔcxrC::flp under SSF in comparison with that of the parental strain. Consequently, these results provide a feasible strategy for improved cellulase and xylanase production by filamentous fungi.

Keywords: Atf1; Cellulase; CxrC; FLP/FRT-mediated recombination system; Penicillium oxalicum.

Diminishing petroleum reserves and the rapid accumulation of greenhouse gases lead to increasing interest in microbial biofuels. In this study, a heterologous farnesyl acetate biosynthesis pathway was constructed in Escherichia coli for the first time. Firstly, the AtoB, ERG13, tHMG1, ERG12, ERG8, MVD1, Idi, IspA and PgpB were expressed to accumulate farnesol in the E. coli cells. Then the alcohol acetyltransferase (ATF1) was heterologous overexpressed for the subsequent esterification farnesol to farnesyl acetate. The engineered strain DG 106 accumulated 128 ± 10.5 mg/L of farnesyl acetate. Finally, the isopentenyl-diphosphate isomerase was further overexpressed, and the recombinant strain DG107 produced 201 ± 11.7 mg/L of farnesyl acetate. This study shows the novel method for the biosynthesis of the advanced biofuel farnesyl acetate directly from glucose and highlight the enormous designing strategies for metabolic engineering of bioproducts.

Clear cell sarcoma harbours recurrent translocation, resulting in EWSR1/ATF1 or less commonly EWSR1/CREB1 fusion. To date, six types of EWSR1/ATF1 fusion have been reported, of which three are in-frame and encode functional proteins. We present a reverse transcription - polymerase chain reaction analysis of a tumour near the hallux of the right foot. The sequencing of obtained fragments revealed the presence of a novel chimerical transcript-the in-frame fusion between EWSR1 exon 7 and ATF1 exon 6 that represents the fourth in-frame type of EWSR1/ATF1 fusion identified in clear cell sarcomas.

<AbstractText>Fibroblast growth factors (FGFs) are growth factors that have diverse biological activities including broad mitogenic and cell survival activities. They function through the activation of a specific tyrosine kinase receptor that transduces the signal by activating several intracellular signaling pathways.</AbstractText><AbstractText>To identify the different signaling pathways involved in the mechanism of action of FGF8 and FGF18 on ovine granulosa cells using mass spectrometry.</AbstractText><AbstractText>Ovine ovarian granulosa cells were harvested from adult sheep independently at the stage of the estrous cycle and were cultured at a density of 500,000 viable cells in 1 ml DMEM/F12 medium for five days. The cells were then treated on day 5 of culture with 10 ng/mL FGF8 and FGF18 for 30 minutes, and total cell protein was collected for mass spectrometry.</AbstractText><p><div><b>Results</b></div>Mass spectrometry showed that both FGF8 and FGF18 significantly induce simultaneous upregulation of several proteins, including <em>ATF1</em>, STAT3, MAPK1, MAPK3, MAPK14, PLCG1, PLCG2, PKCA, PIK3CA, RAF1, GAB1, and BAG2 ( <mml:math>></mml:math> 1.5-fold; p <mml:math><</mml:math> 0.01).</p><AbstractText><em>ATF1</em> and STAT3 are important transcription factors involved in cell growth, proliferation and survival, and consequently can hamper or rescue the normal ovine reproductive system function.</AbstractText>

BACKGROUND

Diluted preparations obtained from Apis mellifica are reported in the homeopathic literature to have anti-inflammatory activity. The present study was designed to explore the effects on global gene expression profiles of human cells by means of microarrays, using Apis mellifica mother tincture (TM) and its 3C, 5C, 7C dynamized dilutions; the technique employed allowed us to study the changes in gene expression at concentrations much lower than those associated with pharmacological responses.

METHODS

An RWPE-1 cell line (human immortalized prostate epithelial cells) was used to study the effects on global gene expression by transcriptomic analysis.

RESULTS

Apis mellifica TM and its 3C, 5C, 7C dynamized dilutions modulated hundreds of genes; using cluster analysis we observed groups of genes up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response. In particular, genes involved in cytokine expression, inflammatory processes, anti-oxidative responses and proteasome degradation were differentially, and sometimes divergently expressed by the TM or by Apis mellifica 3C, 5C and 7C dilutions. We confirmed these data by RT-PCR analyses on 5 selected candidate genes (IL1β, CD46, ATF1, UBE2Q2 and MT1X).

CONCLUSIONS

Apis mellifica TM modifies gene expression in human cells and has inhibitory effects on regulatory processes of inflammation; in addition, extremely diluted dynamized dilutions (3C, 5C and 7C) still exert significant effects on genes involved in inflammation and oxidative stress.

The resistant to glucose repression mutants of Schizosaccharomyces pombe (ird5, ird13, and ird14) have a high tolerance to oxidative stress induced by H2O2. In all ird mutants, the increased expression level of the fbp1 gene can be interpreted as a lack of glucose repression in these mutants. To investigate the mechanisms of the oxidative stress response in ird mutants, we analyzed the transcription of stress response-related genes, sod1, ctt1, atf1, pap1, and sty1, under stressed and non-stressed conditions. We then analyzed the phosphorylation state of the Sty1-MAP kinase in ird mutants. Our findings support the concept of an adaptive response to oxidative stress in these mutants. In addition, these results imply that either glucose signaling mechanisms leading to glucose repression and glucose utilization as an energy source are regulated apart from each other or, like Saccharomyces cerevisiae, S. pombe might have additional glucose detection systems.

Elongation factor 2 (EF-2) is a protein involved in peptide chain elongation in eukaryotes. We isolated the mouse EF-2 gene and characterized its promoter. We showed that the majority of enhancer elements were located within 500 bp of the flanking sequence and identified a factor binding site sequence (CGTCACGTGACGC) located between nucleotides -58 and -47 containing two CGTCA motifs separated by two nucleotides. The motif represents a half-site for binding of the cAMP response element (CRE) binding protein (CREB). Mutation analysis indicated that the presence of one CGTCA site alone conferred cAMP inducibility, but the presence of one or two CGTCA sites and spacing nucleotides elicited cAMP-independent, constitutive expression. UV cross-linking and DNA affinity chromatography revealed that three 40-, 43-, and 65-kD proteins bound to the CRE-like element. Of these, the 65-kD protein was unique to the CRE-like element. The 40-kD protein was ATF1 and the 43-kD protein with the molecular size of CREB was not CREB, on the basis of reactivity to their respective antibodies. Because ATF1 responds poorly to cAMP induction, it is likely the contributor to the constitutive expression rather than inductive expression of the CRE-like element, and, thus, the EF-2 gene.

The vasculoprotective properties of delphinidin are driven mainly by its action on endothelial cells. Moreover, delphinidin displays anti-angiogenic properties in both in vitro and in vivo angiogenesis models and thereby might prevent the development of tumors associated with excessive vascularization. This study was aimed to test the effect of delphinidin on melanoma-induced tumor growth with emphasis on its molecular mechanism on endothelial cells. Delphinidin treatment significantly decreased in vivo tumor growth induced by B16-F10 melanoma cell xenograft in mice. In vitro, delphinidin was not able to inhibit VEGFR2-mediated B16-F10 melanoma cell proliferation but it specifically reduced basal and VEGFR2-mediated endothelial cell proliferation. The anti-proliferative effect of delphinidin was reversed either by the MEK1/2 MAP kinase inhibitor, U-0126, or the PI3K inhibitor, LY-294002. VEGF-induced proliferation was reduced either by U-0126 or LY-294002. Under these conditions, delphinidin failed to decrease further endothelial cell proliferation. Delphinidin prevented VEGF-induced phosphorylation of ERK1/2 and p38 MAPK and decreased the expression of the transcription factors, CREB and ATF1. Finally, delphinidin was more potent in inhibiting in vitro cyclic nucleotide phosphodiesterases (PDEs), PDE1 and PDE2, compared to PDE3-PDE5. Altogether delphinidin reduced tumor growth of melanoma cell in vivo by acting specifically on endothelial cell proliferation. The mechanism implies an association between inhibition of VEGF-induced proliferation via VEGFR2 signalling, MAPK, PI3K and at transcription level on CREB/ATF1 factors, and the inhibition of PDE2. In conjunction with our previous studies, we demonstrate that delphinidin is a promising compound to prevent pathologies associated with generation of vascular network in tumorigenesis.

Superinduction of the catalase gene was observed in Schizosaccharomyces pombe cells treated with cycloheximide and hydrogen peroxide. The promoter analysis of the catalase gene revealed that element A (the region from -111 to -90, numbered with the transcription start site as +1), involved in the induction of the gene under oxidative stress, was required for superinduction by hydrogen peroxide and cycloheximide. Although Atf1 is a transcription factor responsible for the induction of the catalase gene by several stresses, a disruptant of atf1 exhibited superinduction. Moreover, in a deletion mutant that lacks element A but has an Atf1 binding site, the cells treated with hydrogen peroxide and cycloheximide expressed as much catalase mRNA as those treated with hydrogen peroxide alone. This suggests that cycloheximide does not stabilize the catalase mRNA but enhances the transcription via element A. Staurosporine, a strong inhibitor of protein phosphorylation, did not inhibit superinduction.

Fingolimod hydrochloride (FTY720) is the first-in-class immune modulator known as sphingosine 1-phosphate (S1P) receptor agonists. FTY720 has also been reported to exert a variety of physiological functions such as antitumor effect, angiogenesis inhibition, and Ca2+ mobilization. Here, we show that FTY720 treatment induced reactive oxygen species (ROS) accumulation, and investigated the effect of FTY720 on the stress-activated MAP kinase Spc1/Sty1, a functional homologue of p38 MAPK, using a Renilla luciferase reporter construct fused to the CRE, which gives an accurate measure of the transcriptional activity of Atf1 and thus serves as a faithful readout of the Spc1/Sty1 MAPK signaling in response to oxidative stresses. FTY720 stimulated the CRE responses in a concentration-dependent manner, which was markedly reduced by deletion of the components of the Spc1/Sty1 MAPK pathway. The blockade of ROS production by NAC (N-acetyl-L-cysteine) significantly reversed the FTY720-induced ROS accumulation, subsequent activation of the Spc1/Sty1 MAPK pathway, and inhibition of cell proliferation. Cells lacking the components of the Spc1/Sty1 MAPK exhibited higher sensitivity to FTY720 and higher ROS levels upon FTY720 treatment than in wild-type cells. Thus, our results demonstrate the usefulness of fission yeast for elucidating the FTY720-mediated signaling pathways involving ROS.

Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages. Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively. The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo. These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.

The Parkinson's disease protein DJ-1 is involved in various cellular functions including detoxification of dicarbonyl compounds, autophagy and oxidative stress response. DJ-1 homologs are widely found in both prokaryotes and eukaryotes, constituting a superfamily of proteins that appear to be involved in stress response. Schizosaccharomyces pombe contains six DJ-1 homologs, designated Hsp3101-Hsp3105 and Sdj1 (previously named SpDJ-1). Here we show that deletion of any one of these six genes somehow affects autophagy during prolonged stationary phase. Furthermore, deletions of each of these DJ-1 homologs result in reduced stationary phase survival. Deletion of sdj1 also increases the sensitivity of stationary-phase cells to oxidative stress induced by hydrogen peroxide (H2O2) whereas overexpression of sdj1 has the opposite effect. Consistent with their role in stationary phase, expression of hsp3101, hsp3102, hsp3105 and sdj1, and to a lesser extent hsp3103 and hsp3104, is increased in stationary phase. The induction of hsp3101, hsp3102, hsp3105 and sdj1 involves the Sty1-regulated transcription factor Atf1 but not the transcription factor Pap1. Our results firmly establish that S. pombe homologs of DJ-1 are stationary-phase associated proteins and are likely involved in autophagy and antioxidant defense in stationary phase of S. pombe cells.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter in human physiology and inflammatory disease, however, with limited knowledge of how signal transduction pathways are involved in immune cells. To examine the effects of sulfide on relevant intracellular signaling in human peripheral blood mononuclear cells (PBMCs), we stimulated healthy donor PBMCs with sodium hydrosulfide (NaHS, 1-1000μM) to mimic H2S stimulation, and analyzed phosphorylation of p38 mitogen activated protein kinase (MAPK) (pT180/pY182), NF-κB p65 (pS529), Akt (pS473) and CREB/ATF1 (pS133/pS63) with flow and mass cytometry. In contrast to transient effects in subsets of lymphocytes, classical monocytes demonstrated sustained phosphorylation of p38, Akt and CREB/ATF1. NaHS induced calcium dependent phosphorylation of p38, Akt and CREB, but not NF-κB, and the phosphorylation of Akt was partly dependent on p38, indicative of p38-Akt crosstalk. Attenuation of these effects by molecules targeting p38 and Hsp90 indicated Hsp90 as a possible target for H2S-induced activation of p38. These results provide a description of a NaHS-induced signal transduction pathway in human primary immune cells that may have relevance for the role of sulfides in inflammation.

During alcoholic fermentation, Saccharomyces cerevisiae synthesizes more than 400 different compounds with higher alcohols, acetate esters of higher alcohols and ethyl esters of medium-chain fatty acids being the most important products of its metabolism, determining the particular flavour profile of each wine. The concentration of the metabolites produced depends to a large extent on the strain used. The use of indigenous strains as starting cultures can lead to the production of wines with excellent organoleptic characteristics and distinct local character, superior in quality when compared to their commercial counterparts. However, the relationship of these wild-type genotypes, linked to specific terroirs, with the biosynthetic profiles of flavour metabolites is not completely clarified and understood. To this end, qRT-PCR was employed to examine, for the first time on the transcriptional level, the performance of an indigenous Saccharomyces cerevisiae strain (Z622) in its natural environment (Debina grape must). The expression of genes implicated in higher alcohols and esters formation was correlated with the concentrations of these compounds in the produced wine. Furthermore, by applying the same fermentation conditions, we examined the same parameters in a commercial strain (VL1) and compared its performance with the one of strain Z622.

Strain Z622, exhibited lower concentrations of 2-methylbutanol, 3-methylbutanol and 2-phenyl ethanol, than VL1 correlating with the elevated expression levels of transaminase and decarboxylase genes. Furthermore, the significantly high induction of ADH3 throughout fermentation of Z622 probably explains the larger population numbers reached by Z622 and reflects the better adaptation of the strain to its natural environment. Regarding acetate ester biosynthesis, Z622 produced higher concentrations of total acetate esters, compared with VL1, a fact that is in full agreement with the elevated expression levels of both ATF1 and ATF2 in strain Z622.

This study provides evidence on the transcriptional level that indigenous yeast Z622 is better adapted to its natural environment able to produce wines with desirable characteristics, i.e. lower concentrations of higher alcohol and higher ester, verifying its potential as a valuable starter for the local wine-industry.

The effect of adding amino acids on wine aroma is largely influenced by nutritional status of grape must. In this study, the effects of linoleic acid (LA) content on the aromatic function of branched-chain amino acids (BCAAs) addition were investigated in alcoholic fermentation of Cabernet Sauvignon wine. The results showed that initial LA content in must significantly influenced the effect of BCAAs addition on volatiles in final wine. Adding BCAAs (140 mg/L of l-leucine, 117 mg/L of l-isoleucine and 118 mg/L of l-valine) in must with low LA content (12 mg/L) promoted the production of most volatiles, including higher alcohols (isobutanol, 2-phenylethanol), fatty acids (hexanoic acid, octanoic acid, decanoic acid) and esters (ethyl acetate, isoamyl acetate, 2-phenethyl acetate and ethyl octanoate), which were well consistent with previous literatures. However, this function disappeared or even became inhibition with increasing LA content in must, especially in 120 mg/L LA must, the total contents of higher alcohol, acetate esters and ethyl esters were 33.9%, 18.1% and 54.2% lower than those in the control without BCAAs addition, respectively. The transcriptional data revealed that several major genes including GAP1, ADH1, ATF1, ACC1, FAS1 and OLE1 were marked repressed by high LA content. Our data indicated that LA can regulate the expressions of related functional genes to efficiently influence the formations of volatiles in BCAAs supplemented wines. Therefore, it is essential to consider initial content of unsaturated fatty acids (LA) in must when using the strategy that supplying amino acids (BCAAs) to modulate aromatic quality of wines.

Keratinocyte growth factor (KGF) plays a critical role for the normal development and morphogenesis of many different tissues and organs. Furthermore, its expression is induced during wound healing and in various chronic inflammatory diseases. To determine the molecular mechanisms which regulate KGF gene induction at the transcriptional level, we carried out in vitro studies using the human KGF promoter. We have identified a novel regulatory element, TGAGGTCAG, located between -39 and -46 bp (relative to the transcription start site) in the KGF basal promoter region, which binds to inducible transcription factors as determined by electrophoretic mobility shift assay. When cloned in front of a heterologous SV40 promoter this region conferred inducibility to forskolin, a stimulator of adenylate cyclase. In contrast, various mutated forms of this region were either partially or completely impaired in their ability to mediate induction to forskolin. The TGAGGTCAG sequence shared homology to both the cAMP responsive element (CRE) and CCAAT/enhancer binding protein (C/EBP) consensus binding sites. An oligonucleotide comprising a consensus CRE binding site partially competed for the nuclear protein binding to the TGAGGTCAG site. Gel mobility supershift assays indicated that two members of the activating transcription factor (ATF) family of CRE binding proteins, ATF1 and ATF2, were part of the nuclear protein complex bound to this regulatory region. Furthermore, purified recombinant ATF2 was able to directly recognize and bind the TGAGGTCAG sequence. In contrast, no evidence was obtained for C/EBP transcription factors being part of the complex. These results suggest that members of the ATF family are involved in mediating the transcriptional regulation of the KGF gene in response to extracellular stimuli via a novel CRE regulatory element.