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Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley β-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins), the antagonists of serine proteases, were unknown in the bacterial kingdom until recently. Kang et al. in this issue of Molecular Microbiology report the cloning and functional analysis of the three serpin genes from the thermophilic anaerobic bacterium Clostridium thermocellum. Two of the serpins contain a dockerin module for location in the extracellular hydrolytic multienzyme complex, the cellulosome. The susceptibility of cellulosome to proteolytic degradation and the presence of a serine protease in the same complex provoke speculation that protease inhibitor/protease pairs could play hitherto unrecognized roles in protein stability and regulation in bacteria. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the Clostridium thermocellum gene licB, coding for a thermoactive β1,3-1,4-glucanase, has been determined. The gene is located downstream, but in opposite orientation to the β-glucosidase gene bglA. A coding region of 1002 bp is flanked by canonical promoter and transcription terminator sequences. The primary translation product of the licB gene has a predicted molecular mass of 37 896 Da. The protein sequence can be divided into several discrete segments: an N-terminal signal peptide, a catalytic region, a segment rich in Pro and Thr residues and a C-terminal reiterated domain. The catalytic region shows close similarity to lichenases of bacilli (52-58% identity) and Fibrobacter succinogenes (35% identity), but is unrelated to barley β-1,3-1,4-glucanases. it consists of two domains, which in the case of the F. succinogenes lichenase are arranged in reversed order to that of C. thermocellum and Bacillus lichenases. The C-terminal reiterated domain of C. thermocellum lichenase is homologous to the duplicated non-catalytic domain of endo-β-l,4glucanases and xylanase Z from the same organism. This domain is considered a characteristic feature of clostridial cellulases organized as multienzyme complex (cellulosome). The β-l,3-1,4-glucanase encoded by the licB gene might therefore be an additional enzyme component of the C. thermocellum cellulosome. [ABSTRACT FROM AUTHOR]

Subcutaneous mast cells (MCs) are vulnerable to mechanical stimulation from external environment. Thus, MCs immune function could be modulated by their mechanosensitivity. This property has been identified as the trigger mechanism of needling acupuncture, a traditional oriental therapy. Previously we have demonstrated the release of adenosine triphosphate (ATP), a stress‐responsive signalling molecule, from mechanical‐perturbed MCs. The current work explores its underlying mechanisms. We noticed that propagation of intracellular free Ca2+ occurred among HMC‐1 cells in response to 50% hypotonic shock. Additionally, amplifying cascade of ATP‐induced ATP release was observed in RBL‐2H3 cells stimulated by medium displacement, which could be mimicked by exogenous ATP (exoATP). Secondary ATP liberation induced by low level (50 nmol/L) of exoATP was reduced by inhibiting ecto‐ATPase‐dependent ADP production with ARL67156, or blocking P2 receptors with suramin or PPADS, or with specific P2Y13 receptor antagonist MRS2211, or siRNA. Secondary ATP release induced by higher dose (200 μmol/L) of exoATP, sufficient to stimulate P2X7 receptor, was attenuated by suramin, PPADS or specific P2X7 receptor antagonist BBG, or siRNA. Finally, RT‐PCR confirmed mRNA expression of P2Y13 and P2X7 in RBL‐2H3 cells. Additionally, such secondary ATP release was attenuated by DPCPX, specific antagonist of adenosine A1 receptor, but not by MRS2179, specific inhibitor of P2Y1 receptor. In summary, mechanosensitive ATP release from MCs is facilitated by paracrine/autocrine stimulation of P2Y13 and P2X7 receptors. This multi‐receptor combination could mediate transmission of information from a local site to distal areas, enabling communication with multiple surrounding cells to coordinate and synchronize their function. [ABSTRACT FROM AUTHOR]

We compared individual-participant and jackknife-based methods for scoring the onset latencies of event-related potential (ERP) components using a diffusion process as a model for an ERP. We studied “ramp-like” components in which the true ERP increases or decreases monotonically, except for noise. If the growth rates of such components vary across participants, the jackknife-based measure can easily have only 10%–20% as much error variance as the traditional method, and this advantage is magnified with more participants. We also studied ∩-shaped or “bump-like” components. Jackknifing generally yielded smaller error variances with these components too, especially when the component's peak amplitude varied across participants, but less so if the component's peak latency varied. These results help illuminate the reasons for the superiority of jackknife-based onset latency measures over traditional measures in recent simulations. [ABSTRACT FROM AUTHOR]

Glutamate transporters play an essential role in regulating temporal and spatial glutamate concentration in the synaptic cleft. The excitatory amino acid carrier 1 (EAAC1) is a neuronal-specific transporter. Opioid receptors are found to modulate the glutamatergic system in brain, but the regulatory mechanism remains to be investigated. Here we report that the δ-opioid receptor (DOR) co-expressed with EAAC1 in Xenopus oocytes, but not the µ-opioid receptor, down-regulates EAAC1 function, and that [d-Pen2,5]-enkephalin stimulation of DOR can counteract the down-regulation of the EAAC1-mediated uptake. Results from co-immunoprecipitation and immunofluorescence microscopy in both oocytes and rat hippocampal neurons indicate co-localization and suggest direct interaction between DOR and EAAC1. The results suggest that DOR can reduce EAAC1 function by direct protein–protein interaction and that activation of DOR releases the inhibitory interaction. We suggest that this mechanism may be important for modulation of the glutamatergic system following opioid stimulation. [ABSTRACT FROM AUTHOR]

Abstract: Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity. [Copyright &y& Elsevier]

Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na+- and Cl–-coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM). This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N-glycosylation mutations and dMM reduced theVmax value, while not increasing theKm value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle. This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding. [ABSTRACT FROM AUTHOR]

An α-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60°C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p-nitrophenyl-α-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg[SUP-1] respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using [SUP1]H-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable β-glucosidase. The secondary structure of RamA consists of 27% α-helices and 50% β-sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family. [ABSTRACT FROM AUTHOR]