Mutant P1 Research Articles

Unique expression and critical role of metallothionein 3 in the control of osteoclastogenesis and osteoporosis

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway

BackgroundToxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii.MethodsQuantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining.To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells.ResultsIn T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site.ConclusionsT. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.Graphical

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Simple SummaryGonadotropin-releasing hormone (GnRH) plays a pivotal role in fish reproduction. In the present study, transcription activator-like effector nuclease (TALEN) plasmids targeting the catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). After low fertility was observed, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants (individuals with human-induced sequence changes at the cfGnRH locus). Gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

In vivo analysis of the evolutionary conserved BTD-box domain of Sp1 and Btd during Drosophila development

The Sp family of transcription factors plays important functions during development and disease. An evolutionary conserved role for some Sp family members is the control of limb development. The family is characterized by the presence of three C2H2-type zinc fingers and an adjacent 10 aa region with an unknown function called the Buttonhead (BTD) box. The presence of this BTD-box in all Sp family members identified from arthropods to vertebrates, suggests that it plays an essential role during development. However, despite its conservation, the in vivo function of the BTD-box has never been studied. In this work, we have generated specific BTD-box deletion alleles for the Drosophila Sp family members Sp1 and buttonhead (btd) using gene editing tools and analyzed its role during development. Unexpectedly, btd and Sp1 mutant alleles that lack the BTD-box are viable and have almost normal appendages. However, in a sensitized background the requirement of this domain to fully regulate some of Sp1 and Btd target genes is revealed. Furthermore, we have also identified a novel Sp1 role promoting leg vs antenna identity through the repression of spineless (ss) expression in the leg, a function that also depends on the Sp1 BTD-box.

Insights Into the Role of Exposed Surface Charged Residues in the Alkali-Tolerance of GH11 Xylanase.

Thermostable and alkaline- or acid-stable xylanases are more advantageous in agricultural and industrial fields. In this study, a rational structure-based design was conducted based on a thermostable GH11 xylanase TlXynA from Thermomyces lanuginosus to improved pH-tolerance. Four mutant enzymes (P1, P2, P3, and P4) and five variants (N1, N2, N3, N4, and N5) were constructed by substituting surface charged residue combinations using site-directed mutagenesis. Compared to the native enzyme, two mutants P1 and P2 showed higher acid tolerance, especially at pH 3.0, presented 50 and 40% of their maximum activity, respectively. In addition, four mutants N1, N2, N3 and N4 had higher tolerance than the native enzyme to alkaline environments (pH 7.0–9.0). At pH 9.0, the residual activities of N1, N2, N3, and N4 were 86, 78, 77, and 66%, respectively. In summary, an improved pH-tolerance design principle is being reported.

New insights into cellular cholesterol acquisition: promoter analysis of human HMGCR and SQLE, two key control enzymes in cholesterol synthesis

BackgroundThe two control points of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) are known targets of the transcription factor sterol-regulatory element binding protein-2 (SREBP-2). Yet the location of the sterol-regulatory elements (SREs) and cofactor binding sites, nuclear factor-Y (NF-Y) and specificity protein 1 (Sp1), have not been satisfactorily mapped in the human SQLE promoter, or at all in the human HMGCR promoter. MethodsWe used luciferase reporter assays to screen the sterol-responsiveness of a library of predicted SRE, Sp1 and NF-Y site mutants and hence identify bone fide binding sites. We confirmed SREs via an electrophoretic mobility shift assay (EMSA) and ChIP-PCR. ResultsWe identified two SREs in close proximity in both the human HMGCR and SQLE promoters, as well as one NF-Y site in HMGCR and two in SQLE. In addition, we found that HMGCR expression is highly activated only when SREBP-2 levels are very high, in contrast to the low density lipoprotein receptor (LDLR), a result reflected in mouse models used in other studies. ConclusionsBoth HMGCR and SQLE promoters have two SREs that may act as a homing region to attract a single SREBP-2 homodimer, with HMGCR being activated only when there is absolute need for cholesterol synthesis. This ensures preferential uptake of exogenous cholesterol via LDLR, thereby conserving energy. General SignificanceWe provide the first comprehensive investigation of SREs and NF-Ys in the human HMGCR and SQLE promoters, increasing our fundamental understanding of the transcriptional regulation of cholesterol synthesis.

Enzyme-Triggered Defined Protein Nanoarrays: Efficient Light-Harvesting Systems to Mimic Chloroplasts.

The elegance and efficiency by which chloroplasts harvest solar energy and conduct energy transfer have been a source of inspiration for chemists to mimic such process. However, precise manipulation to obtain orderly arranged antenna chromophores in constructing artificial chloroplast mimics was a great challenge, especially from the structural similarity and bioaffinity standpoints. Here we reported a design strategy that combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts. Cricoid stable protein one (SP1) was utilized as a building block model. Under enzyme-triggered covalent protein assembly, mutant SP1 with tyrosine (Tyr) residues at the designated sites can couple together to form nanostructures. Through controlling the Tyr sites on the protein surface, we can manipulate the assembly orientation to respectively generate 1D nanotubes and 2D nanosheets. The excellent stability endowed the self-assembled protein architectures with promising applications. We further integrated quantum dots (QDs) possessing optical and electronic properties with the 2D nanosheets to fabricate chloroplast mimics. By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions, we successfully developed an artificial light-harvesting system. The assembled protein nanosheets structurally resembled the natural thylakoids, and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls. Therefore, the coassembled system was meaningful to explore the photosynthetic process in vitro, as it was designed to mimic the natural chloroplast.

Molecular characterization of porcine circovirus-like virus P1 in eastern China

A large-scale epidemiological study of porcine circovirus-like virus P1 was carried out from 2007 to 2015 in China, which revealed a high level of P1 infection. Thirty-nine sequences obtained from pigs in eastern China showed remarkable genetic diversity, with the P1 predominant sequences and the mutant P1 with ORF1 or ORF2 containing an additional amino-acid extension at the C-terminus. This information may be useful for understanding the evolution of P1 circovirus-like virus.

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5′-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the −134 to −19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.

O-linked N-acetylglucosaminylation of Sp1 interferes with Sp1 activation of glycolytic genes

Glycolysis, the primary pathway metabolizing glucose for energy production, is connected to the hexosamine biosynthetic pathway (HBP) which produces UDP-N-acetylglucosamine (UDP-GlcNAc), a GlcNAc donor for O-linked GlcNAc modification (O-GlcNAc), as well as for traditional elongated glycosylation. Thus, glycolysis and O-GlcNAc are intimately associated. The present study reports the transcriptional activation of glycolytic genes by the transcription factor Sp1 and the O-GlcNAc-mediated suppression of Sp1-dependent activation of glycolytic genes. O-GlcNAc-deficient mutant Sp1 stimulated the transcription of nine glycolytic genes and cellular production of pyruvate, the final product of glycolysis, to a greater extent than wild-type Sp1. Consistently, this mutant Sp1 increased the protein levels of the two key glycolytic enzymes, phosphofructokinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), to a greater extent than wild-type Sp1. Finally, the mutant Sp1 occupied GC-rich elements on PFK and GAPDH promoters more efficiently than wild-type Sp1. These results suggest that O-GlcNAcylation of Sp1 suppresses Sp1-mediated activation of glycolytic gene transcription.

Major Histocompatibility Complex Class I Core Promoter Elements Are Not Essential for Transcription in vivo

The role of core promoter elements in regulating transcription initiation is largely unknown for genes subject to complex regulation. Major histocompatibility complex class I genes are ubiquitously expressed and governed by tissue-specific and hormonal signals. Transcription initiates at multiple sites within the core promoter, which contains elements homologous to the canonical elements CCAAT, TATAA, Sp1 binding site (Sp1BS), and Initiator (Inr). To determine their functions, expression of class I transgenes with individually mutated elements was assessed. Surprisingly, all mutant promoters supported transcription. However, each mutated core promoter element had a distinct effect on expression: CAAT box mutations modulated constitutive expression in nonlymphoid tissues, whereas TATAA-like element mutations dysregulated transcription in lymphoid tissues. Inr mutations aberrantly elevated expression. Sp1BS element mutations resulted in variegated transgene expression. RNA polymerase II binding and histone H3K4me3 patterns correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the wild-type, TATAA-like, and CAAT mutant promoters were activated by gamma interferon, the Sp1 and Inr mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each plays a distinct role in promoter activity, chromatin structure, tissue-specific expression, and extracellular signaling.

Mutational Analysis of the P1 Phosphorylation Domain in Escherichia coli CheA, the Signaling Kinase for Chemotaxis

The histidine autokinase CheA functions as the central processing unit in the Escherichia coli chemotaxis signaling machinery. CheA receives autophosphorylation control inputs from chemoreceptors and in turn regulates the flux of signaling phosphates to the CheY and CheB response regulator proteins. Phospho-CheY changes the direction of flagellar rotation; phospho-CheB covalently modifies receptor molecules during sensory adaptation. The CheA phosphorylation site, His-48, lies in the N-terminal P1 domain, which must engage the CheA ATP-binding domain, P4, to initiate an autophosphorylation reaction cycle. The docking determinants for the P1-P4 interaction have not been experimentally identified. We devised mutant screens to isolate P1 domains with impaired autophosphorylation or phosphotransfer activities. One set of P1 mutants identified amino acid replacements at surface-exposed residues distal to His-48. These lesions reduced the rate of P1 transphosphorylation by P4. However, once phosphorylated, the mutant P1 domains transferred phosphate to CheY at the wild-type rate. Thus, these P1 mutants appear to define interaction determinants for P1-P4 docking during the CheA autophosphorylation reaction.

Strategy for Naturelike Designer Transcription Factors with Reduced Toxicity

For clinical applications, the biological functions of DNA-binding proteins require that they interact with their target binding site with high affinity and specificity. Advances in randomized production and target-oriented selection of engineered artificial DNA-binding domains incited a rapidly expanding field of designer transcription factors (TFs). Engineered transcription factors are used in zinc-finger nuclease (ZFN) technology that allows targeted genome editing. Zinc-finger-binding domains fabricated by modular assembly display an unexpectedly high failure rate having either a lack of activity as ZFNs in human cells or activity at "off-target” binding sites on the human genome causing cell death. To address these shortcomings, we created new binding domains using a targeted modification strategy. We produced two SP1 mutants by exchanging amino acid residues in the alpha-helical region of the transcription factor SP1. We identified their best target binding sites and searched the NCBI HuRef genome for matches of the nine-base-pair consensus binding site of SP1 and the best binding sites of its mutants. Our research concludes that we can alter the binding preference of existing zinc-finger domains without altering its biological functionalities.

Core promoter elements are not essential for transcription in mammals

Core promoter elements are thought to be the docking site for transcription factors binding, and thereby essential for transcription initiation. The role of core promoter elements has been studied for decades in reporter systems. the central model claim that each cis sequence has an epigenetic role in navigating transcription initiation. We use here the MHC class I gene as model to study transcription in a transgenic mouse. MHC class I genes are ubiquitously expressed and subject to both tissue-specific and hormonal regulation. The core promoter contains four conventional elements: CCAAT, a TATAA-like element, an Sp1 binding site and a canonical Inr. The in vivo function of these elements was determined by mutating each individually within the context of the native gene. Surprisingly, none of the elements was essential for transcription since no single mutation eliminated transcription. Indeed, mutation of any one of the elements resulted in increased promoter activity, indicating that these elements function as transcriptional modulators. Further, each of the elements was found to have a distinct function, contributing uniquely to tissue-specificity, hormonal responses or both. The core promoter elements do not affect start site selection, demonstrating that they do not invoke a cryptic promoter. However, they do modulate relative start site usage. The patterns of chromatin modification reflect the expression status of the different promoters. In tissues where the different promoters constructs support active transcription, histone H3K4 trimethylation is high and H3K9 trimethylation is low. Conversely, H3K9 trimethylation is high and H3K4 trimethylation low across the gene in tissues where the promoter constructs are less active. The wild type promoter is activated by interferon, while the Inr and Sp1 mutants repress transcription in response to interferon treatment. Finally, the CAAT element was found to have dual function, both as a transcriptional regulator and as a barrier element. The barrier function correlated with the binding of C/EBP and CTCF to the wild type CAAT element and variegated expression across generations when the CAAT element was mutated. Remarkably, these results demonstrate that none of the elements homologous to canonical core promoter elements are necessary for promoter activity. However, they do contribute to the fine tuning of the tissue specific patterns of expression, extracellular signaling, overall promoter activity and chromatin modifications.

Manganese regulates caspase-3 gene promoter activity by inducing Sp1 phosphorylation in PC12 cells

Chronic manganese exposure causes selective toxicity to the dopaminergic system, resulting in a parkisonian-like neurological condition known as manganism. Manganese causes a typical apoptosis, which includes activation of the caspase cascade and DNA fragmentation in PC12 cells. Caspase-3 is a major contributor to the execution of neuronal apoptosis. In a previous study, we demonstrated that caspase-3 cleavage and expression of pro-caspase-3 mRNA and protein increased in PC12 cells treated with manganese, but this response was not observed with other apoptosis inducers. To understand the molecular mechanisms that regulate expression of caspase-3 in manganese-treated PC12 cells, we characterized the 5′-flanking region of the rat caspase-3 gene and identified both a core promoter and a manganese-responsive region that contains three putative Sp1 binding sites. Furthermore, manganese treatment induced robust Sp1 phosphorylation and increased its DNA binding activity. Overexpression of mutant Sp1 lacking phosphorylation sites attenuated Sp1's ability to stimulate manganese-induced caspase-3 promoter activity. In conclusion, our results indicate that Sp1 phosphorylation is required for manganese-induced transactivation of caspase-3.

SP-1 regulation of MMP-9 expression requires Ser586 in the PEST domain.

Rac1, a small GTPase, regulates macrophage MMP (matrix metalloproteinase)-9 in an ERK (extracellular-signal-regulated kinase)- and SP (specificity protein)-1-dependent manner. SP-1 contains a PEST (Pro-Glu-Ser-Thr) domain that may modulate protein stability. We hypothesize that Thr578, Ser586 and/or Ser587 in the PEST domain are required for SP-1 stability and MMP-9 expression secondary to activation of ERK, a serine/threonine kinase. We determined the effects of Rac1 and ERK on MMP-9 expression driven by SP-1WT (wild-type) and the SP-1 mutants T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP9 promoter activity in cells expressing SP-1WT, SP-1T578A and SP-1S587A, but not SP-1S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP9 transcription in cells expressing SP-1WT, but not SP-1S586A. As Rac1 activation and ERK inactivation increased degradation of SP-1WT and not SP-1S586A, the results of the present study suggest that SP-1 stability mediated at Ser586 regulates MMP9 transcription. Ex vivo, alveolar macrophages obtained from patients with asbestosis had less MMP-9 expression that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that Ser586 in the PEST domain of SP-1 is important for MMP9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.

Organizational Requirements of the SaeR Binding Sites for a Functional P1 Promoter of the sae Operon in Staphylococcus aureus

In Staphylococcus aureus, the SaeRS two-component system controls the expression of multiple virulence factors. Of the two promoters in the sae operon, P1 is autoinduced and has two binding sites for the response regulator SaeR. In this study, we examined the organizational requirements of the SaeR binding sites in P1 for transcription activation. Mutational studies showed that both binding sites are essential for binding to phosphorylated SaeR (P-SaeR) and transcription activation. When the 21-bp distance between the centers of the two SaeR binding sites was altered to 26 bp, 31 bp, 36 bp, or 41 bp, only the 31-bp mutant retained approximately 40% of the original promoter activity. When the -1-bp spacing (i.e.,1-bp overlap) between the primary SaeR binding site and the -35 promoter region was altered, all mutant P1 promoters failed to initiate transcription; however, when the first nucleotide of the -35 region was changed from A to T, the mutants with 0-bp or 22-bp spacing showed detectable promoter activity. Although P-SaeR was essential for the binding of RNA polymerase to P1, it was not essential for the binding of the enzyme to the alpha-hemolysin promoter. When the nonoptimal spacing between promoter elements in P1 or the coagulase promoter was altered to the optimal spacing of 17 bp, both promoters failed to initiate transcription. These results suggest that SaeR binding sites are under rather strict organizational restrictions and provide clues for understanding the molecular mechanism of sae-mediated transcription activation.

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H 2) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H 2 yield (HY) of 0.65 mol H 2/mol acetate- and 2.50 mol H 2/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H 2/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H 2 production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H 2 production. When pH was controlled to 7.0, strain KD131 wt evolved more H 2 by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H 2 production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH 4) 2SO 4. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H 2 production in expending reducing power.

'Tachisuzuka', a new rice cultivar with high straw yield and high sugar content for whole-crop silage use

‘Tachisuzuka’ is a new rice (Oryza sativa L.) cultivar for whole-crop silage (WCS) use. Agronomic characters were recorded from 2007 to 2009. The culm length of Tachisuzuka was 11 cm longer than that of Kusanohoshi. The short panicle of Tachisuzuka was morphologically similar to that of the short panicle 1 (sp1) mutant. Tachisuzuka exhibited high lodging resistance in the yellow ripening stage, the optimal period for harvesting, and a month after yellow ripening. The straw yield of Tachisuzuka was remarkably higher than that of Kusanohoshi but its grain yield was lower. A portion of grains are undigested and excreted in feces by cattle fed with rice WCS, and this loss of nutrients should not be disregarded. The low grain/straw ratio of Tachisuzuka indicates that the loss of nutrients in cattle fed with Tachisuzuka WCS should be lower than with Kusanohoshi WCS. Low sugar content of rice plants is considered a cause of silage deterioration. The sugar content of Tachisuzuka was markedly higher than that of Kusanohoshi.