Involvement of GDH3-encoded NADP+-dependent glutamate dehydrogenase in yeast cell resistance to stress-induced apoptosis in stationary phase cells.

Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP(+)-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.

Mutational biosynthesis of tacrolimus analogues by fkbO deletion mutant of Streptomyces sp. KCTC 11604BP.

Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.

TCA cycle-independent acetate metabolism via the glyoxylate cycle in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the accepted theory is that due to TCA cycle dysfunction, the Δcit1 mutant lacking the mitochondrial enzyme citrate synthase (Cit1) cannot grow on acetate, regardless of the presence of the peroxisomal isoenzyme (Cit2). In this study, we re-evaluated the roles of Cit1 and Cit2 in acetate utilization and examined the pathway of acetate metabolism by analysing mutants defective in TCA or glyoxylate cycle enzymes. Although Δcit1 cells showed significantly reduced growth on rich acetate medium (YPA), they exhibited growth similar to Δcit2 and the wild-type cells on minimal acetate medium (YNBA). Impaired acetate utilization by Δcit1Δcit2 cells on YNBA was restored by ectopic expression of either Cit2 or its cytoplasmically localized variants. Deletion of any of the genes for the enzymes solely involved in the TCA cycle (IDH1, KGD1 and LSC1), except for SDH1, caused little defect in acetate utilization on YNBA but resulted in significant growth impairment on YPA. In contrast, cells lacking any of the genes involved in the glyoxylate cycle (ACO1, FUM1, MLS1, ICL1 and MDH2) did not grow on either YNBA or YPA. Deletion of SFC1 encoding the succinate-fumarate carrier also caused similar growth defects on YNBA. Our results suggest that in S. cerevisiae the glyoxylate cycle functions as a competent metabolic pathway for acetate utilization on YNBA, while both the TCA and glyoxylate cycles are essential for growth on YPA.

Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways.

The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.

Yeast cells lacking the CIT1-encoded mitochondrial citrate synthase are hypersusceptible to heat- or aging-induced apoptosis.

In Saccharomyces cerevisiae, the initial reaction of the tricarboxylic acid cycle is catalyzed by the mitochondrial citrate synthase Cit1. The function of Cit1 has previously been studied mainly in terms of acetate utilization and metabolon construction. Here, we report the relationship between the function of Cit1 and apoptosis. Yeast cells with cit1 deletion showed a temperature-sensitive growth phenotype, and they displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e., reactive oxygen species (ROS) accumulation and nuclear fragmentation, DNA breakage, and phosphatidylserine translocation, when exposed to heat stress. On long-term cultivation, cit1 null strains showed increased potentials for both aging-induced apoptosis and adaptive regrowth. Activation of the metacaspase Yca1 was detected during heat- or aging-induced apoptosis in cit1 null strains, and accordingly, deletion of YCA1 suppressed the apoptotic phenotype caused by cit1 null mutation. Cells with cit1 deletion showed higher tendency toward glutathione (GSH) depletion and subsequent ROS accumulation than the wild type, which was rescued by exogenous GSH, glutamate, or glutathione disulfide (GSSG). These results led us to conclude that GSH deficiency in cit1 null cells is caused by an insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power required for reduction of GSSG to GSH.

The putative C2H2 transcription factor RocA is a novel regulator of development and secondary metabolism in Aspergillus nidulans.

Multiple transcriptional regulators play important roles in the coordination of developmental processes, including asexual and sexual development, and secondary metabolism in the filamentous fungus Aspergillus nidulans. In the present study, we characterized a novel putative C2H2-type transcription factor (TF), RocA, in relation to development and secondary metabolism. Deletion of rocA increased conidiation and caused defective sexual development. In contrast, the overexpression of rocA exerted opposite effects on both phenotypes. Additionally, nullifying rocA resulted in enhanced brlA expression and reduced nsdC expression, whereas its overexpression exerted the opposite effects. These results suggest that RocA functions as a negative regulator of asexual development by repressing the expression of brlA encoding a key asexual development activator, but as a positive regulator of sexual development by enhancing the expression of nsdC encoding a pivotal sexual development activator. Deletion of rocA increased the production of sterigmatocystin (ST), as well as the expression of its biosynthetic genes, aflR and stcU. Additionally, the expression of the biosynthetic genes for penicillin (PN), ipnA and acvA, and for terrequinone (TQ), tdiB and tdiE, was increased by rocA deletion. Thus, it appears that RocA functions as a negative transcriptional modulator of the secondary metabolic genes involved in ST, PN, and TQ biosynthesis. Taken together, we propose that RocA is a novel transcriptional regulator that may act either positively or negatively at multiple target genes necessary for asexual and sexual development and secondary metabolism.

High Galacto-Oligosaccharide Production and a Structural Model for Transgalactosylation of ß-Galactosidase II from Bacillus circulans.

The transgalactosylase activity of ß-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. ß-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of ß-galactosidase II from B. circulans (ß-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of ß-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of ß-Gal-II with other ß-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on ß-Gal-II might provide molecular insights into the transgalactosylation process of the ß-galactosidases in GOS production.

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet.

A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85 degrees C, respectively. AJ kept 85% of the initial activity after heating at 100 degrees C for 20 min on the zymogram gel. The Michaelis constant (K (m)) and K (cat) values of AJ towards alpha-casein were 0.38 mM and 19.73 s(-1), respectively. AJ cleaved the A alpha-chain of fibrinogen but did not affect the B beta- and gamma-chains, indicating that it is an alpha-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100 degrees C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.

The NADP+-dependent glutamate dehydrogenase Gdh1 is subjected to glucose starvation-induced reversible aggregation that affects stress resistance in yeast.

The yeast Saccharomyces cerevisiae has two isoforms of NADP+-dependent glutamate dehydrogenase (Gdh1 and Gdh3) that catalyze the synthesis of glutamate from α-ketoglutarate and NH4+. In the present study, we confirmed that Gdh3, but not Gdh1, mainly contributes to the oxidative stress resistance of stationary-phase cells and found evidence suggesting that the insignificance of Gdh1 to stress resistance is possibly resulted from conditional and reversible aggregation of Gdh1 into punctuate foci initiated in parallel with post-diauxic growth. Altered localization to the mitochondria or peroxisomes prevented Gdh1, which was originally localized in the cytoplasm, from stationary phase-specific aggregation, suggesting that some cytosolic factors are involved in the process of Gdh1 aggregation. Glucose starvation triggered the transition of the soluble form of Gdh1 into the insoluble aggregate form, which could be redissolved by replenishing glucose, without any requirement for protein synthesis. Mutational analysis showed that the N-terminal proximal region of Gdh1 (NTP1, aa 21-26, TLFEQH) is essential for glucose starvation-induced aggregation. We also found that the substitution of NTP1 with the corresponding region of Gdh3 (NTP3) significantly increased the contribution of the mutant Gdh1 to the stress resistance of stationary-phase cells. Thus, this suggests that NTP1 is responsible for the negligible role of Gdh1 in maintaining the oxidative stress resistance of stationary-phase cells and the stationary phase-specific stresssensitive phenotype of the mutants lacking Gdh3.

Genome-wide drug-induced haploinsufficient screening of fission yeast for identification of hydrazinocurcumin targets.

Hydrazinocurcumin (HC), a synthetic derivative of curcumin, has been reported to inhibit angiogenesis via unknown mechanisms. Understanding the molecular mechanisms of the drug's action is important for the development of improved compounds with better pharmacological properties. A genomewide drug-induced haploinsufficiency screening of fission yeast gene deletion mutants has been applied to identify drug targets of HC. As a first step, the 50% inhibition concentration (IC50) of HC was determined to be 2.2 microM. The initial screening of 4,158 mutants in 384-well plates using robotics was performed at concentrations of 2, 3, and 4 microM. A second screening was performed to detect sensitivity to HC on the plates. The first screening revealed 178 candidates, and the second screening resulted in 13 candidates, following the elimination of 165 false positives. Final filtering of the condition-dependent haploinsufficient genes gave eight target genes. Analysis of the specific targets of HC has shown that they are related to septum formation and the general transcription processes, which may be related to histone acetyl transferase. The target mutants showed 65% growth inhibition in response to HC compared with wild-type controls, as shown by liquid culture assay.

Genome-wide identification of haploinsufficiency in fission yeast.

Abnormal phenotypes resulting from haploinsufficiency (HI) are due to the loss of one allele. Recent studies in budding yeast have shown that HI originates from insufficient protein levels or from a stoichiometric imbalance between subunits of protein complexes. In humans, however, HI often involves transcription factors. Therefore, the species differences in HI and the molecular mechanisms of species-specific HI remain under investigation. In this study, HI in fission yeast was systematically surveyed. HI in fission yeast affected genes related to signaling and to basic cellular processes, as observed in budding yeast. These results suggest that there are species differences in HI and that the HI that occurs in fission yeast is intermediate to and HI in budding yeast and humans.

Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome.

Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.

Differential Control of Asexual Development and Sterigmatocystin Biosynthesis by a Novel Regulator in Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans primarily reproduces by forming asexual spores called conidia and produces the mycotoxin sterigmatocystin (ST), the penultimate precursor of aflatoxins. It has been known that asexual development and ST production are tightly co-regulated by various regulatory inputs. Here, we report that the novel regulator AslA with a C2H2 domain oppositely regulates development and ST biosynthesis. Nullifying aslA resulted in defective conidiation and reduced expression of brlA encoding a key activator of asexual development, which indicates that AslA functions as an upstream activator of brlA expression. aslA deletion additionally caused enhanced ST production and expression of aflR encoding a transcriptional activator for ST biosynthetic genes, suggesting that AslA functions as an upstream negative regulator of aflR. Cellular and molecular studies showed that AslA has a trans-activation domain and is localized in the nuclei of vegetative and developing cells but not in spores, indicating that AslA is likely a transcription factor. Introduction of the aslA homologs from distantly-related aspergilli complemented the defects caused by aslA null mutation in A. nidulans, implying a functional conservancy of AslA. We propose that AslA is a novel regulator that may act at the split control point of the developmental and metabolic pathways.

Mutational and functional analysis of the cryptic N-terminal targeting signal for both mitochondria and peroxisomes in yeast peroxisomal citrate synthase Cit2p.

We previously found that the peroxisomal citrate synthase of Saccharomyces cerevisiae, Cit2p, contains a cryptic targeting signal for both peroxisomes (PTS) and mitochondria (MTS) within its 20-amino acid N-terminal segment [Lee et al. (2000) J. Biochem. 128, 1059-1072]. In the present study, the fine structure of the cryptic signal was scrutinized using green fluorescent protein fusions led by variants of the N-terminal segment. The minimum ranges of the cryptic signals for mitochondrial and peroxisomal targeting were shown to consist of the first 15- and 10-amino acid N-terminal segments, respectively. Substitution of the 3rd Val, 6th Leu, 7th Asn, or 8th Ser with Ala abolished the cryptic MTS function, however, no single substitution causing an obvious defect in PTS function was found. Neither the 15-amino acid N-terminal segment nor the C-terminal SKL sequence (PTS1) was necessary for Cit2p to restore the glutamate auxotrophy caused by the double Deltacit1 Deltacit2 mutation. The Cit2p variant lacking PTS1 [Cit2(DeltaSKL)p] partially restored the growth of both the Deltacit1 Deltacit2 and Deltacit1 mutants on acetate, while that carrying intact PTS1 or lacking the N-terminal segment [Cit2p, Cit2((DeltaNDeltaSKL))p, and Cit2((DeltaN))p] did not. It is thus suggested that the potential of the N-terminal segment as an ambidextrous targeting signal can be unmasked by deletion of PTS1.

IFN-gamma regulates the expression of B7-H1 in dermal fibroblast cells.

BACKGROUND: Programmed cell death ligand 1 (B7-H1) was recently cloned in antigen presenting cells (APCs) and represents a third member of the B7 family. Thus, B7-H1 may be a novel target for clinical intervention in human inflammatory disease. OBJECTIVE: The aim of this study is to investigate the signal transduction mechanism and transcriptional regulation of B7-H1 expression in human dermal fibroblasts. METHODS: We performed reverse transcription PCR (RT-PCR) for the detection of mRNA expression, luciferase reporter assays with B7-H1 promoter constructs, and Western blot analysis. RESULTS: From RT-PCR analysis, IFN-gamma can induce the expression of B7-H1 mRNA in dermal fibroblast. This expression is similar to the results of luciferase reporter assay with B7-H1 promoter. Western blot analysis and EMSA revealed that NF-kappaB transcription factors mediate the induction of B7-H1 expression via the transient phosphorylation of ERK1/2 and PI3K when cells are stimulated by IFN-gamma. Also, Specific destruction of the NF-kappaB binding site abolished the induction of the promoter activity by IFN-gamma. CONCLUSION: Our data not only provides the first evidence to demonstrate that dermal fibroblast express the B7-H1 mRNA in the process of skin inflammation, but also suggests the involvement of NF-kappaB and MAPK and PI3K, that may play some important roles in inflammation process in human skin diseases.

Differential expression of the chsE gene encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions.

Expression of chsE encoding one of the five chitin synthases of Aspergillus nidulans was analyzed. Expression of chsE was moderate in conidiophores, but somewhat weaker in vegetative mycelia. During sexual development, chsE was expressed strongly in young cleistothecia and hülle cells, but little in mature sexual structures. Deletion of chsE caused a significant decrease in the chitin content of the cell wall during early sexual development. Expression of chsE was increased by substituting glucose with lactose or by addition of 0.6M KCl or NaCl, but affected little by substituting glucose with sodium acetate. Consequently, chsE was shown to have a mode of expression distinct from those of the other chitin synthase genes, chsA, chsB and chsC.

A method for direct application of human plasmin on a dithiothreitol-containing agarose stacking gel system.

A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1 %) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells.

BACKGROUND AND OBJECTIVES: Endothelial and monocytic cells appear to play a key role in the initiation and progression of atherosclerosis and restenosis via the upregulation of inflammatory cytokines and the formation of oxidized low-density lipoprotein (ox-LDL). However, the role of smooth muscle cells (SMCs) has been underestimated and is not well understood. It was investigated for the first time that native LDL stimulates human SMCs to secrete IL-8. The aim of this study was to investigate the signaling pathway involved in the upregulation of IL-8, induced by LDL in human aortic SMCs. METHODS AND RESULTS: LDL-induced IL-8 expression (mRNA and protein) is specific to SMCs and likely to be regulated at the transcription level in dose- and time-dependent manners, as judged by experiments with actinomycin D and ELISA. Although both p38 and ERK 1/2 MAPKs were activated by LDL, only p38 MAPK is responsible for the LDL effects, as evidenced by a complete blockade of IL-8 upregulation by SB203580. Pretreatment with catalase significantly decreased the extent of IL-8 upregulation, indicating that H2O2 is necessary for the LDL response. Activation of activator protein (AP)-1, but not nuclear factor (NF)-kappaB, by p38 or H2O2 appears to be necessary along with the concomitant upregulation of c-fos and c-JUN, as judged by electrophoretic mobility shift and luciferase reporter assays. CONCLUSIONS: These data demonstrated that LDL stimulates SMCs to induce IL-8 production in dose- and time-dependent manners at the transcription level and that the LDL signaling in hAoSMCs is conveyed via the generation of H2O2, the phosphorylation of p38 MAPK, the activation of AP-1, and the participation of NF-kappaB.

Negative regulation of the vacuole-mediated resistance to K(+) stress by a novel C2H2 zinc finger transcription factor encoded by aslA in Aspergillus nidulans.

In fungi and plants, vacuoles function as a storage and sequestration vessel for a wide variety of ions and are responsible for cytosolic ion homeostasis and responses to ionic shock. In the filamentous fungus Aspergillus nidulans, however, little is known about the molecular genetic mechanisms of vacuolar biogenesis and function. In the present study, we analyzed the function of the aslA gene (AN5583) encoding a novel C2H2-type zinc finger transcription factor (TF) in relation to K(+) stress resistance, vacuolar morphology, and vacuolar transporters. The mutant lacking aslA showed increased mycelial growth and decreased branching at high K(+) concentrations. Deletion of aslA also caused elevated K(+) stress-inducible expression of the genes, nhxA (AN2288), vnxA (AN6986), and vcxA (AN0471), encoding putative endosomal and vacuolar cation/H(+) exchangers, as well as cpyA and vpsA genes encoding the proteins involved in vacuolar biogenesis. Interestingly, vacuolar fragmentation induced by K(+) stress was alleviated by aslA deletion, resulting in persistence of unfragmented vacuoles. In the presence of bafilomycin, an inhibitor of vacuolar H(+)-ATPase, the mutant phenotype was suppressed in terms of growth rates and vacuolar morphology. These results together suggest that the C2H2-type zinc finger TF AslA attenuates the K(+) stress-inducible expression of the genes encoding the ion pumps involved in vacuolar sequestration of K(+) ions powered by vacuolar H(+)-ATPase, as well as the proteins that function in vacuolar biogenesis.