Analysis of gene expression levels in individual bacterial cells without image segmentation

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.     

Proteomic analysis of the brain tissues from a transgenic mouse model of amyloid β oligomers

Amyloid β (Aβ) oligomers are presumed to be one of the causes of Alzheimer's disease (AD). Previously, we identified the E693Δ mutation in amyloid precursor protein (APP) in patients with AD who displayed almost no signals of amyloid plaques in amyloid imaging. We generated APP-transgenic mice expressing the E693Δ mutation and found that they possessed abundant Aβ oligomers from 8months of age but no amyloid plaques even at 24months of age, indicating that these mice are a good model to study pathological effects of Aβ oligomers. To elucidate whether Aβ oligomers affect proteome levels in the brain, we examined the proteins and phosphoproteins for which levels were altered in 12-month-old APP(E693Δ)-transgenic mice compared with age-matched non-transgenic littermates. By two-dimensional gel electrophoresis (2DE) followed by staining with SYPRO Ruby and Pro-Q Diamond and subsequent mass spectrometry techniques, we identified 17 proteins and 3 phosphoproteins to be significantly changed in the hippocampus and cerebral cortex of APP(E693Δ)-transgenic mice. Coactosin like-protein, SH3 domain-bind glutamic acid-rich-like protein 3 and astrocytic phosphoprotein PEA-15 isoform 2 were decreased to levels less than 0.6 times those of non-transgenic littermates, whereas dynamin, profilin-2, vacuolar adenosine triphosphatase and creatine kinase B were increased to levels more than 1.5 times those of non-transgenic littermates. Furthermore, 2DE Western Blotting validated the changed levels of dynamin, dihydropyrimidinase-related protein 2 (Dpysl2), and coactosin in APP(E693Δ)-transgenic mice. Glyoxalase and isocitrate dehydrogenase were increased to levels more than 1.5 times those of non-transgenic littermates. The identified proteins could be classified into several groups that are involved in regulation of different cellular functions, such as cytoskeletal and their interacting proteins, energy metabolism, synaptic component, and vesicle transport and recycling. These findings indicate that Aβ oligomers altered the levels of some proteins and phosphoproteins in the hippocampus and cerebral cortex, which could illuminate novel therapeutic avenues for the treatment of AD.     

Citreorosein inhibits degranulation and leukotriene C4 generation through suppression of Syk pathway in mast cells

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati (P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C(4) (LTC(4)) generation in mast cells. Cit suppresses both degranulation and the generation of LTC(4) in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca(2+), phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A(2) (PLA(2)) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC(4) generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation.     

Fumigant toxicity of plant essential oils against Camptomyia corticalis (Diptera: Cecidomyiidae)

The toxicity of 98 plant essential oils against third instars of cecidomyiid gall midge Camptomyia corticalis (Loew) (Diptera: Cecidomyiidae) was examined using a vapor-phase mortality bioassay. Results were compared with that of a conventional insecticide dichlorvos. Based on 24-h LC50 values, all essential oils were less toxic than dichlorvos (LC50, 0.027 mg/cm3). The LC50 of caraway (Carum carvi L.) seed, armoise (Artemisia vulgaris L.), clary sage (Salvia sclarea L.), oregano (Origanum vulgare L.), lemongrass [Cymbopogon citratus (DC.) Stapf], niaouli (Melaleuca viridiflora Gaertner), spearmint (Mentha spicata L.), cassia especial (Cinnamomum cassia Nees ex Blume), Dalmatian sage (Salvia offcinalis L.), red thyme (Thymus vulgaris L.), bay [Pimenta racemosa (P. Mill.) J.W. Moore], garlic (Allium sativum L.), and pennyroyal (Mentha pulegium L.) oils is between 0.55 and 0.60 mg/cm3. The LC50 of cassia (C. cassia, pure and redistilled), white thyme (T. vulgaris), star anise (Illicium verum Hook.f.), peppermint (Mentha X piperita L.), wintergreen (Gaultheria procumbens L.), cinnamon (Cinnamomum zeylanicum Blume) bark, sweet marjoram (Origanum majorana L.), Roman chamomile [Chamaemelum nobile (L.) All.], eucalyptus (Eucalyptus globulus Labill.), rosemary (Rosmarinus officinalis L.),Virginian cedarwood (Juniperus virginiana L.), pimento berry [Pimenta dioica (L.) Merr.], summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia Mill.), and coriander (Coriandrum sativum L.) oils is between 0.61 and 0.99 mg/cm3. All other essential oils tested exhibited low toxicity to the cecidomyiid larvae (LC50, >0.99 mg/cm3). Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the active essential oils as potential larvicides for the control of C. corticalis populations as fumigants with contact action.     

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK‐mediated signaling

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast‐like cells were induced in the co‐cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti‐osteoprotegerin (OPG) antibody was added to the co‐cultures. mRNA levels of receptor activator of nuclear factor‐kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D₃. Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force‐induced OPG up‐regulation. Inhibition of extracellular signal‐regulated kinase (ERK) signaling by a pharmacological inhibitor (10 µM PD98059) or by siERK transfection suppressed the force‐induced OPG up‐regulation along with the augmentation of osteoclast‐like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation. J. Cell. Biochem. 106: 1010–1019, 2009. © 2009 Wiley‐Liss, Inc.     

Interferon‐γ suppresses Na+–H+ exchanger in cultured human endolymphatic sac epithelial cells

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na⁺–H⁺ exchanger (NHE) is a major determinant of intracellular pH (pH_i), and facilitates Na⁺ and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN‐γ on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, ‐2, and ‐3 isoforms was determined by real‐time RT‐PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH‐sensitive fluorescent dye, 2′,7′‐bis(carbonylethyl)‐5(6)‐carboxyfluorescein (BCECF), and a NHE‐inhibitor, 3‐methylsulfonyl‐4‐piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, ‐2, and ‐3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and ‐2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na⁺‐dependent pH_i recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN‐γ (50 nM for 24 h) suppressed mRNA expression of NHE1 and ‐2. IFN‐γ also suppressed functional activity of both NHE1 and ‐2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN‐γ suppresses the expression and functional activity of NHE1 and ‐2. J. Cell. Biochem. 107: 965–972, 2009. © 2009 Wiley‐Liss, Inc.     

p35 interacts with α‐tubulin and organelle proteins: Nuclear translocation of p35 in dying cells

We identified heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A1, the translocase of the transporter outer membrane 40 (TOM40), and α‐tubulin as new interaction partners of anti‐apoptotic protein p35 using MS‐based functional proteomics with GST‐p35 fusion protein as a bait, and using a pull‐down assay with p35‐6His followed by Western blot analysis. p35 was localized in the cytoplasm and in distinct organelles such as the nucleus and mitochondria. p35 was more abundant in the cytoplasm than it was in the nucleus. It co‐localized with α‐tubulin in the cytoplasm in the absence of a death stimulus. However, while cells were undergoing death induced by actinomycin D, cytoplasmic p35 was translocated into the nucleus; this process was inhibited by deletions of the N‐ and C‐terminal domains containing leucine‐rich motifs. Gene delivery of p35 using recombinant adenoviruses inhibited cytoplasmic compartmentalization of hnRNP C1/C2 and hnRNP A1 in dying cells. This study demonstrated translocation of p35 into the nuclei, as well as protection of the hnRNPs from redistribution in cells undergoing death. We propose an active role for p35 in maintaining the integrity of nuclear proteins during cell death.     

Novel GH10 Xylanase,with a Fibronectin Type 3 Domain,from Cellulosimicrobium sp. Strain HY-13, a Bacterium in the Gut of Eisenia fetida

The gene encoding a novel modular xylanase from Cellulosimicrobium sp. strain HY-13 was identified and expressed in Escherichia coli, and its truncated gene product was characterized. The enzyme consisted of three distinct functional domains, an N-terminal catalytic GH10 domain, a fibronectin type 3 domain, and C-terminal carbohydrate-binding module 2.Most known microbial xylanases, which decompose primarily β-1,4-xylosic polysaccharides in an endo fashion, are currently affiliated with the two glycoside hydrolase (GH) families 10 and 11. Compared to GH11 xylanases, GH10 xylanases generally have a molecular mass of >30 kDa and an acidic pI. In addition, GH10 xylanases are frequently found in nature as modular enzymes that consist of a catalytic GH10 domain with one or more substrate-binding domains, such as a cellulose-binding domain, carbohydrate-binding module (CBM), or xylan-binding domain (1, 5, 12). However, no modular xylanase with a fibronectin type 3 (Fn3) domain has been characterized to date, even though Fn3 modules are often found in bacterial carbohydrolases such as cellulases, amylases, pullulanases, polygalacturonidases, and chitinases. It is now believed that the Fn3 domains in bacterial carbohydrolases participate in promotion of the hydrolysis of carbohydrate substrates by modifying their surfaces (10, 17).Gut microorganisms from invertebrates have recently attracted a great deal of attention as sources of novel fibrolytic enzymes with unique molecular structures and distinct substrate specificities (2-4, 7, 15). However, no study has been conducted to evaluate xylanolytic enzymes from the gut microorganisms of earthworms that may participate in the digestion of cellulosic or hemicellulosic foods taken up by the hosts. Here, we report a novel GH10 xylanase (XylK1) with an Fn3 domain from Cellulosimicrobium sp. strain HY-13 KCTC 11302BP (11), which was isolated from the digestive tract of the earthworm Eisenia fetida.Amplification of a partial sequence of the Cellulosimicrobium sp. strain HY-13 xylanase gene from the genomic DNA was conducted using the degenerate primers designed on the basis of conserved regions (WDVVNE and ITELDI) in the GH10 xylanases. The upstream primer (KF) was 5′-TGGGACGTCSTCAACGAG-3′, and the downstream primer (KR) was 5′-GATGTCGAGCTCSGTGAT-3′, which produced a 342-bp DNA fragment. Cloning of the full xylK1 gene was performed by repeated genomic walking and nested-PCR methods using a DNA Walking SpeedUp premix kit (Seegene). To overproduce mature XylK1, its encoding gene was cloned into the NdeI/HindIII sites of a pET-28a(+) vector (Novagen). Likewise, a partial sequence containing the GH10 domain (Ala34 to Leu345) of XylK1 was amplified using the primers tKF (5′-CATATGGCCACCGAGCCGCTCG-3′) and tKR (5′-AAGCTTTCAGGACCTCGGCGATCGC-3′) and subsequently also cloned into the same expression vector. When overexpressed in recombinant Escherichia coli BL21 cells harboring pET-28a(+)/xylK1, most recombinant proteins (rXylK1) were produced as inactive inclusion bodies. Therefore, after solubilization of the isolated inclusion bodies, on-column refolding and purification of rXylK1 was conducted using a HisTrap HP (GE Healthcare, Sweden) (5-ml) column attached to a fast-performance liquid chromatography (LC) system (Amersham Pharmacia Biotech, Sweden) according to the manufacturer''s instructions. The active rXylK1 proteins were then purified to electrophoretic homogeneity by gel permeation chromatography using a HiLoad 26/60 Superdex 200 prep-grade (Amersham Biosciences, Sweden) column, as previously described (11). Like rXylK1, the recombinant proteins (rXylK1ΔFn3) without both an Fn3 domain and CBM 2 were also purified by the method described above because they were produced as insoluble inclusion bodies. The relative molecular mass of the denatured rXylK1 was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel, and the protein concentrations were assayed by using the Bradford reagent (Bio-Rad). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis was conducted using an Ultraflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Germany) at the Korea Basic Science Institute (Daejeon, South Korea). The binding capacity of recombinant enzymes with/without the Fn3 domain to carbohydrate polymers was determined as described elsewhere (4). Xylanase activity was routinely assayed by measuring the amount of reducing sugars released from birch wood xylan by using the 3,5-dinitrosalicylic acid reagent. The standard assay mixture (0.5 ml) consisted of birch wood xylan (1.0%) or p-nitrophenyl (PNP)-sugar derivatives (5 mM) with suitably diluted enzyme solution (0.05 ml) in 50 mM sodium phosphate buffer (pH 6.0), and the catalytic reaction was performed at 55°C for 10 min. One international unit of xylanase activity for xylans or PNP-sugar derivatives was defined as the amount of enzyme required to produce 1 μmol of reducing sugar or PNP, respectively, per min under standard assay conditions. Enzymatic hydrolysis of birch wood xylan (10 mg) (Sigma Co.), xylooligosaccharides (1 mg each) (Megazyme International Ireland, Ireland), and cellooligosaccharides (1 mg each) (Seikagaku Biobusiness Co., Japan) was conducted using purified rXylK1 (2 μg) in 0.1 ml of 50 mM sodium phosphate buffer (pH 6.0) for 3 or 6 h at 37°C, during which time the enzyme remained fairly stable. The reaction mixture was then heated to 100°C for 5 min to stop the enzyme reaction. The hydrolysis products were identified by LC-MS, as previously described (11).The isolated XylK1 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ859907","term_id":"265509370","term_text":"FJ859907"}}FJ859907) contained a 1,671-bp open reading frame that encodes a protein of 556 amino acids with a deduced molecular mass of 58,296 Da and a calculated pI of 4.59. It was predicted that the signal sequence cleavage site of premature XylK1 was between Ala33 and Ala34, which may generate a mature XylK1 of 523 amino acids with a deduced molecular mass of 54,843 Da and a calculated pI of 4.49 (Fig. ​(Fig.1).1). The results of a protein BLAST survey revealed that XylK1 was a unique modular xylanase composed of an N-terminal catalytic GH10 (Leu38-to-Asp330) domain, an Fn3 (Pro359-to-Gly430) domain, and a C-terminal CBM 2 (Cys454 to Cys553), which was very comparable to the domain architectures of other related GH10 enzymes (Fig. ​(Fig.2).2). To the best of our knowledge, no xylanase with domain architecture identical to that of XylK1 with an Fn3 domain has been reported to date, although an uncharacterized modular xylanase (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ABQ06877","term_id":"146156023","term_text":"ABQ06877"}}ABQ06877) consisting of an N-terminal Fn3 domain and a C-terminal GH10 domain from Flavobacterium johnsoniae UW101 was previously identified through a genome survey. As shown in Fig. ​Fig.1,1, the catalytic GH10 domain of XylK1 showed the highest sequence identity (67%) with that of the Cellulomonas fimi xylanase ({"type":"entrez-protein","attrs":{"text":"AAA56792","term_id":"144429","term_text":"AAA56792"}}AAA56792) among other GH10 enzymes available in the NCBI database. However, its CBM 2 was 64% identical to that of Cellulomonas fimi GH6 cellulase ({"type":"entrez-protein","attrs":{"text":"AAC36898","term_id":"456029","term_text":"AAC36898"}}AAC36898). The highest sequence identity (70%) of the Fn3 domain in XylK1 was obtained when it was compared to that of the Acidothermus cellulolyticus 11B GH48 enzyme ({"type":"entrez-protein","attrs":{"text":"ABK52390","term_id":"117648288","term_text":"ABK52390"}}ABK52390), which degrades cellulose. The two conserved residues of Glu161 (acid/base catalyst) and Glu266 (catalytic nucleophile) were predicted in the active site of premature XylK1.Open in a separate windowFIG. 1.Alignment of the deduced amino acid sequence of GH10 xylanase from Cellulosimicrobium sp. strain HY-13 with those of other GH10 xylanases. Shown are sequences (GenBank accession numbers) of Cellulosimicrobium sp. strain HY-13 (Csp) xylanase ({"type":"entrez-nucleotide","attrs":{"text":"FJ859907","term_id":"265509370","term_text":"FJ859907"}}FJ859907), Cellulomonas fimi (Cfi) xylanase ({"type":"entrez-protein","attrs":{"text":"AAA56792","term_id":"144429","term_text":"AAA56792"}}AAA56792), Streptomyces coelicolor (Sco) A3 xylanase ({"type":"entrez-protein","attrs":{"text":"CAB61191","term_id":"6434744","term_text":"CAB61191"}}CAB61191), Streptomyces ambofaciens (Sam) xylanase ({"type":"entrez-protein","attrs":{"text":"CAJ88420","term_id":"117164871","term_text":"CAJ88420"}}CAJ88420), Acidothermus cellulolyticus 11B (Ace) xylanase ({"type":"entrez-protein","attrs":{"text":"ABK51955","term_id":"117647853","term_text":"ABK51955"}}ABK51955), and Thermobifida alba (Tal) xylanase ({"type":"entrez-protein","attrs":{"text":"CAB02654","term_id":"1621277","term_text":"CAB02654"}}CAB02654). The identical and similar amino acids are shown by black and gray boxes, respectively. The predicted signal peptide is indicated by a black bar. The internal peptide sequences used in the design of degenerate oligonucleotides for PCR are marked by arrows. Highly conserved amino acid residues that play an essential role in the catalytic reaction are indicated by asterisks. GH10, Fn3, and CBM 2 domains are outlined by solid, dashed, and dotted lines, respectively.Open in a separate windowFIG. 2.Domain architectures of Cellulosimicrobium sp. strain HY-13 xylanase and the following related bacterial GH10 xylanases (GenBank accession numbers): Cellulosimicrobium sp. strain HY-13 xylanase ({"type":"entrez-nucleotide","attrs":{"text":"FJ859907","term_id":"265509370","term_text":"FJ859907"}}FJ859907) (A), Streptomyces thermocarboxydus HY-15 xylanase ({"type":"entrez-nucleotide","attrs":{"text":"EU880430","term_id":"215261531","term_text":"EU880430"}}EU880430) (B), Cellulomonas fimi xylanase ({"type":"entrez-protein","attrs":{"text":"AAZ76373","term_id":"73427793","term_text":"AAZ76373"}}AAZ76373) (C), Streptomyces thermoviolaceus xylanase ({"type":"entrez-protein","attrs":{"text":"BAD02382","term_id":"38524461","term_text":"BAD02382"}}BAD02382) (D), Pseudomonas fluorescens xylanase ({"type":"entrez-protein","attrs":{"text":"P23030","term_id":"294862476","term_text":"P23030"}}P23030) (E), and Cellvibrio japonicus xylanase (YP 001982932) (F).The molecular mass of the purified enzyme was estimated to be approximately 42.0 kDa, which was smaller than the deduced molecular mass (57,138 Da) of intact His-tagged rXylK1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). In addition, MALDI-TOF MS analysis revealed that the purified His-tagged rXylK1 with a calculated molecular mass of 45,169 Da was a smaller protein than the intact rXylK1. These results indicate that rXylK1 was formed by proteolytic cleavage at the C terminus region because the enzyme was able to tightly bind to a His tag column. Based on the calculated molecular mass (45,169 Da) of the truncated rXylK1, it is assumed that the intact rXylK1 was processed at the Val439-Thr440 site in a hinge region between the Fn3 domain and the C-terminal CBM 2 of the premature XylK1. The deduced molecular mass (45,179 Da) of rXylK1 with the Val439 residue at the C-terminal extremity was very close to the molecular mass (45,169 Da) of the enzyme calculated by MALDI-TOF MS analysis. A similar C-terminal processing of some modular xylanases with a cellulose-binding domain by proteases has also been observed when they are expressed in E. coli (8, 14). It is likely that the C-terminal truncation does not induce a significant alteration of the binding affinity of rXylK1 with an Fn3 domain to carbohydrate polymers since the truncated enzyme could still bind to both Avicel and insoluble oat spelt xylan (Table ​(Table1).1). In this case, only weak catalytic activity of the rXylK1 (<3% of its original activity [0.5 IU]) was recovered from an enzyme solution after binding. It was of great interest that no rXylK1ΔFn3 was bound to Avicel, although the enzyme could still not only bind to insoluble oat spelt xylan but also catalyze the hydrolysis of xylosic polymers. The specific activity (27 IU/mg) of rXylK1ΔFn3 for birch wood xylan was evaluated to be approximately 19% of that (143 IU/mg) of rXylK1 with the Fn3 domain for the same substrate. Taken together, the binding ability of the C-terminal CBM 2-lacking rXylK1 to Avicel and insoluble oat spelt xylan clearly suggests that the Fn3 domain plays an important role in enzyme-substrate binding because rXylK1ΔFn3 was not bound to Avicel. A significant decrease in the catalytic activity of rXylK1ΔFn3 induced by deletion of the Fn3 domain also suggests that the Fn3 domain may take part in the promotion of the catalytic hydrolysis of xylosic substrates by modifying their surfaces, as shown in other GHs (10, 17). The maximum catalytic activity of rXylK1 toward birch wood xylan was observed at pH 6.0 and 55°C, and it maintained over 80% of its highest activity at a relatively broad pH range of 5.0 to 9.0 during the reaction period of 15 min. These high activities of rXylK1 in alkaline pH ranges suggest that it is a peculiar enzyme that can be distinguished from xylanases of other invertebrate-symbiotic microorganisms, which showed weak hydrolytic activities toward xylan at the same alkaline pHs (4, 11, 15). At 55°C, the half-life of rXylK1 was approximately 10 min, which indicates that it is a typical mesophilic enzyme. Compared to many other xylanases that were completely inhibited by Hg²⁺ (11, 13), rXylK1 was partially inhibited (by 40% relative to its original activity) by 1 mM Hg²⁺. In addition, the enzyme was relatively suppressed by <25%, relative to its original activity, in the presence of some divalent cations at a concentration of 1 mM, in the order of Ca²⁺ > Cu²⁺ > Ba²⁺. No significant alterations of rXylK1 activity by Mn²⁺ and Co²⁺ were interesting to note because the xylanases from Streptomyces sp. strain S9 [12] and Aeromonas caviae ME-1 [14] have been negatively affected by the compound. In this study, the catalytic activity of rXylK1 increased by approximately 1.4-fold when the reaction was conducted in the presence of 1 mM Fe²⁺. The promotion of rXylK1 activity by Fe²⁺ was comparable to previous observations of xylanase inhibition by the metal ion (6, 15). The rXylK1 was relatively unaffected by sulfhydryl reagents (5 mM) such as sodium azide, iodoacetamide, and N-ethylmaleimide, while the enzyme lost 68% of its original activity when preincubated with 5 mM EDTA for 10 min. The complete inhibition of rXylK1 by 5 mM N-bromosuccinimide was in good agreement with the fact that three Trp residues in the highly conserved region of the GH10 enzymes are critically involved in enzyme-substrate interaction, as shown for Streptomyces lividans (16) and Geobacillus stearothermophilus T-6 (18) GH10 xylanases. It was predicted that the three residues Trp118, Trp306, and Trp314 in premature XylK1 might be responsible for catalysis and substrate binding of the enzyme. It is also noteworthy that the catalytic activity of His-tagged rXylK1 increased significantly, by approximately 1.8-fold, when the reaction was conducted in the presence of Tween 80 or Triton X-100 at a concentration of 0.5%. It is assumed that the nonionic-detergent-induced activation of His-tagged rXylK1 might be due to the direct interaction of the recombinant enzyme with the Tween 80 or Triton X-100 molecule, which may lead to an alteration of the enzyme-substrate interaction. Indeed, the stimulation of His-tagged rXylK1 activity was insignificant when the enzyme reaction was conducted in the presence of the detergents without preincubation with the same compounds for 10 min. As with rXylK1, it has been reported that the catalytic activity of a His-tagged esterase from Bacillus megaterium 20-1 expressed in E. coli is greatly stimulated by various nonionic detergents (9).

TABLE 1.

Binding of rXylK1 and rXylK1ΔFn3 to hydrophobic polysaccharides

Characterization of three Arabidopsis homologs of human RING membrane anchor E3 ubiquitin ligase

Ubiquitination affects diverse physiological processes in eukaryotic cells. AtRMA1 was previously identified as an Arabidopsis homolog of human RING membrane-anchor E3 ubiquitin (Ub) ligase. Here, we identified two additional AtRMA homologs, AtRMA2 and AtRMA3. The predicted AtRMA proteins contain a RING motif and a trans-membrane domain in their N-terminal and extreme C-terminal regions, respectively. Bacterially expressed AtRMAs exhibited E3 ligase activity in vitro, which was abrogated by mutation of the conserved cysteine residue in their RING domains. In vivo targeting experiments using an Arabidopsis protoplast-transfection system showed that all three AtRMAs are localized to the ER. Although RT-PCR analysis indicated that AtRMA mRNAs were expressed constitutively in all tissues examined, their promoter activities were differentially detected in a tissue-specific fashion in AtRMA-promoter::GUS transgenic Arabidopsis plants. The AtRMA1 and AtRMA3 genes are predominantly expressed in major tissues, such as cotyledons, leaves, shoot–root junction, roots, and anthers, while AtRMA2 expression is restricted to the root tips and leaf hydathodes. We suggest that a ubiquitnation pathway involving these AtRMA E3 Ub ligases may play a role in the growth and development of Arabidopsis.