Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose I_α and III_I were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose I_α and III_I are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose I_α and III_I. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose I_α and III_I. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose I_α and III_I and similar fractions of productive binding on cellulose I_α and III_I. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose I_α and III_I with similar translational rates. With structural models of cellulose I_α and III_I, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.     

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.     

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal‐regulated kinase 1/2

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an ‘acute’ phase (0–15 min) and ‘tonic’ phase (16–120 min), which is accompanied by significant phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post‐formalin injection. To uncover a possible relationship between the slow‐onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of ‘tonic’ phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre‐treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin‐induced ‘tonic’ phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the ‘tonic’ phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP‐NK1 receptor–ERK1/2 system.

     

Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca^2 + concentration. The oligomerization of STIM1, which triggers extracellular Ca^2 + influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca^2 + concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca^2 + concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca^2 + concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca^2 + influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca^2 + loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca^2 +-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.     

Infection with Porphyromonas gingivalis Exacerbates Endothelial Injury in Obese Mice

Background

A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity.

Methods

In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression.

Results

Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate.

Conclusions

Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.     

The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa, representing the four basic genomes of the genus, by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on the chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Hybridization of the pSc119.2 sequence to the chromosomes and Southern blots of digested DNA detected signals in Hordeum bulbosum, Hordeum chilense, Hordeum marinum and Hordeum murinum 4x, but not in Hordeum murinum 2x and Hordeum vulgare ssp. spontaneum. A maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. The species carrying the same I-genome differed in the presence (Hordeum bulbosum) or absence (Hordeum spontaneum) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of Hordeum murinum, but its absence in the diploid cytotype, suggests that the tetraploid is not likely to be a simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences give information about both the interrelationships of the species and the evolution of the repetitive sequences. Received: 17 March 1999 / Accepted: 16 June 1999     

Micropattern-immobilization of heparin to regulate cell growth with fibroblast growth factor

Heparin was immobilized on a polystyrene plate in a specificpattern by photolithography. Heparin was coupled with azidoaniline. Thederivatized heparin was cast on the polystyrene plate from aqueoussolution. After drying, the plate was photo-irradiated in the presence of aphotomask. The micropatterning was confirmed by staining with a dye,ethydium bromide. Since heparin has negative charges, the cationic dyewas adsorbed on the regions where heparin was immobilized. In thepresence fibroblast growth factor (FGF), the growth of mouse fibroblastSTO cells was enhanced only on the heparin-immobilized regions. Thisresult indicated that micropattern-immobilized heparin activated FGF forcell growth activity.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.