The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

AtSAP5, one of approximately 14 members of the Stress Associated Protein gene family in Arabidopsis, was identified by its expression in response to salinity, osmotic, drought and cold stress. AtSAP5 shows strong homology to OSISAP1, an A20/AN1-type zinc finger protein implicated in stress tolerance in rice. To evaluate the function of AtSAP5 in the regulation of abiotic stress responses, transgenic Arabidopsis plants that over-express AtSAP5 (35S::AtSAP5) were characterized, along with wild-type and T-DNA knock-down plants. Plants that over-express AtSAP5 showed increased tolerance to environmental challenges including salt stress, osmotic stress and water deficit. Comparison of gene expression patterns between 35S::AtSAP5 transgenic plants and wild-type plants under normal conditions and water deficit stress indicated that over-expression of AtSAP5 correlates with up-regulation of drought stress responsive gene expression. Analysis of transgenic plants that express GFP-AtSAP5 showed that it is localized primarily in nuclei of root cells and recombinant AtSAP5 has E3 ubiquitin ligase activity in vitro. These results indicate that AtSAP5 has E3 ligase activity and acts as a positive regulator of stress responses in Arabidopsis.

The purpose of this study was to examine what consumer characteristics are related to willingness to purchase and selection of children's organic cotton clothing (OCC). A survey was conducted on mothers of pre‐schoolers to collect data on mothers' environmental characteristics, involvement with OCC and children's clothing, children's clothing purchasing behaviour and willingness to purchase OCC. A buying scenario experiment was used to examine whether price and other product characteristics influenced the participants' selection of OCC. It was found that a mother's environmental concerns, environmental purchasing behaviour and recycling behaviour significantly affected their involvement in OCC, which further significantly determined mother's willingness to purchase OCC. However, mothers were not willing to pay a premium for purchasing children's OCC. A majority (59.5%) of the mothers who selected OCC in the buying scenario indicated fabric softness was the main reason for their selection. Preparing environmental education materials for consumers and producing OCC with high quality and good performance would help improve the acceptance of OCC in the market.

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

SUMMARYThe LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the a-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while a-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.

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Purpose – The purpose of this paper is to explore whether brand community characteristics (perceived personalization and familiarity among members) affect brand community engagement through customer-to-customer (C2C) interaction. Design/methodology/approach – A survey questionnaire was distributed to members of online brand communities to test the research hypotheses. Findings – The findings showed that the relationships among the brand community constructs are significant. C2C interaction mediates the relations between the characteristic variables and brand community engagement. Furthermore, the findings revealed that brand community trust moderates the effects of perceived personalization on the quality of C2C interaction and on brand community engagement. It also moderates the relations between perceived familiarity among community members and each of brand community engagement and the quality of C2C interaction. Practical implications – Marketers should utilize a brand community’s C2C interaction for its marketing strategies. Moreover, managing brand communities by focussing on perceived personalized service and the familiarity of members can also ultimately increase community engagement by enhancing the quality of C2C communication. Originality/value – This study argues that firms need to manage online brand communities intuitively in order to increase members’ community engagement. To do so, they need to allocate spaces in which C2C communication can actively occur within brand communities, for example, in a discussion forum.

ClpA is the ATPase component of the ATP-dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites. A ClpA variant (referred to as ClpAT) carrying threonine in place of the 169th methionine has recently been shown to be highly soluble but indistinguishable from the wild-type, 84-kDa ClpA in its ability to hydrolyze ATP and to support the casein-degrading activity of ClpP. Therefore, site-directed mutagenesis was performed to generate mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys220 or Lys501 with Thr). ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolysis. Without ATP, ClpAT and both of its mutant forms behaved as trimeric molecules as analyzed by gel filtration on a Sephacryl S-300 column. With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K220T remained trimeric. With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer. These results suggest that the first ATP-binding site of ClpA is responsible for hexamer formation, while the second is essential for ATP hydrolysis. When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran together with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and to support the ClpP-mediated proteolysis. Similar results were obtained in the presence of 2 mM ATP and also with the mixture with ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was significantly higher than the sum of that of each protein, particularly in the presence of 2 mM ATP, although its ability to support the proteolysis by ClpP remained unchanged. These results suggest that a rapid exchange of the subunits, possibly as a trimeric unit, occurs between the ClpAT proteins in the presence of ATP and leads to the formation of mixed hexameric molecules.