BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.Polysaccharides are essential biopolymers performing diverse biological functions, ranging from energy storage to osmoregulation and cell wall formation. Extracellular polysaccharides, including cellulose, chitin, and alginate, are synthesized inside the cell from nucleotide-activated sugars and are transported across the cell membrane during their synthesis. This remarkable task is performed by membrane-integrated glycosyltransferases (GTs) that couple polymer elongation with translocation (1, 2).Cellulose is a linear polymer of glucose molecules linked via β-1,4 glycosidic linkages (3, 4) and is primarily formed by vascular plants, but also by some algae, protists, and bacteria (4–6). Cellulose is synthesized by cellulose synthase (CesA), a family 2 member of GTs (7) that processively polymerizes UDP-activated glucose via an evolutionarily conserved mechanism (2). CesAs contain eight predicted transmembrane (TM) segments and at least one extended intracellular domain adopting a GT-A fold (2, 8). The intracellular GT-A domain is responsible for donor and acceptor sugar binding, as well as for catalyzing the GT reaction, and the membrane-embedded part forms a TM pore in close juxtaposition with the catalytic site, thereby allowing translocation of the nascent polysaccharide (2).Although most eukaryotic CesAs are believed to form supramolecular complexes that organize the secreted glucans into cable-like structures, i.e., the cellulose microfibrils (9), many Gram-negative bacteria synthesize cellulose as a biofilm component (10, 11). Biofilm formation is stimulated by the bacterial messenger cyclic-di-GMP (c-di-GMP) (12), which affects a diverse group of enzymes via interaction with either covalently or noncovalently attached c-di-GMP-binding domains, such as PilZ (13–15).Bacterial cellulose synthase (Bcs) is a multicomponent protein complex encoded in an operon containing at least three genes, bcsA, B, and C (16, 17). BcsA is the catalytic subunit that synthesizes cellulose and forms the TM pore across the inner membrane and BcsB is a large periplasmic protein that is anchored to the inner membrane via a single C-terminal TM helix. BcsB may guide the polymer across the periplasm toward the outer membrane via two carbohydrate-binding domains (CBDs) (2). BcsA and BcsB are fused into a single polypeptide chain in some species (18). BcsC is predicted to form a β-barrel in the outer membrane, preceded by a large periplasmic domain containing tetratricopeptide repeats likely involved in complex assembly (16). Most cellulose synthase operons also code for a periplasmic cellulase, BcsZ, whose biological function is unknown, yet it appears to enhance cellulose production in vivo (19, 20). Although most biofilm-forming bacteria likely produce amorphous cellulose that is embedded in a 3D matrix of polysaccharides, proteinaceous fibers, and nucleic acids (21), some bacteria produce cellulose microfibrils resembling those synthesized by eukaryotic cells (22). In such bacteria, CesA complexes are linearly arranged along the cell axis, and the CesA operons encode at least one additional subunit, BcsD, that might facilitate the linear organization of the synthases (18).Despite the numerous studies available on a large number of pro- and eukaryotic model systems, revealing the mechanism of cellulose synthesis and translocation has been hampered by difficulties in reconstituting functional CesAs in a purified system, either from eukaryotic or prokaryotic enzymes (23–26). To date, cellulose biosynthetic activities have only been recovered from detergent extracts of native membranes (24–26).To overcome these challenges, we reconstituted an active cellulose synthetic system in vitro from a purified Rhodobacter sphaeroides BcsA-B complex (27). The purified complex efficiently synthesizes amorphous, high-molecular-weight (HMW) cellulose on incubation with UDP-glucose (UDP-Glc) and c-di-GMP, both in a detergent-solubilized state and after reconstitution into proteoliposomes (PLs). We show that cellulose elongation occurs directly from UDP-Glc without lipid-linked intermediates, reveal that c-di-GMP activates the synthase, and demonstrate the strict substrate specificity of BcsA for UDP-Glc. Furthermore, we demonstrate that BcsB is crucial for the catalytic activity of BcsA and localize the region required for cellulose synthesis within BcsB’s C-terminal, membrane-associated domain that packs against the TM region of BcsA.     

Aerobic physical training has little effect on cardiovascular autonomic control in aging rats subjected to early menopause

We investigated and compared the effects of physiological menopause (PM) and early menopause (EM) and the adaptations promoted by physical training on the cardiovascular autonomic control of aged rats. Female Wistar rats (N = 72) were assigned to 3 groups: control (22 weeks old rats, undergoing sham surgery in the 10th week of life), PM (82 weeks old rats, undergoing sham surgery in the 10th week of life) and EM (82 weeks old rats, undergoing ovariectomy in the 10th week of life). In each group, half of the rats were subjected to swimming training over a period of 10 weeks. Sedentary PM and EM groups had higher basal mean arterial pressure (MAP) and heart rate (HR) and lower intrinsic HR compared to the sedentary control group. Physical training reduced MAP in PM group. All trained groups had lower basal HR; however, only control and PM-trained groups showed decreased intrinsic HR. The assessment of cardiac autonomic balance showed that PM and EM sedentary groups exhibited sympathetic predominance compared to control group. After physical training, only EM group presented sympathetic predominance. HR variability (pulse interval) was similar among all sedentary groups. However, control and PM-trained groups showed lower power in low frequency band (LF; 0.2–0.75 Hz) and higher power in high frequency band (HF; 0.75–3.0 Hz). The analysis of systolic arterial pressure variability revealed that PM and EM sedentary groups had higher LF power. However, PM group showed lower LF power following physical training. Finally, PM and EM groups had a reduction in spontaneous baroreflex sensitivity, that was attenuated by physical training. The overall results suggest that PM or EM promotes similar negative effects on MAP, HR and cardiovascular autonomic control. However, unlike the PM group, physical training was not able to mitigate all negative effects of EM on cardiovascular autonomic control.     

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.Most tissue and cell types produce and release exosomes, a distinct population of microvesicles ranging from about 30 to 150 nm in size. Exosomes are formed in the endocytic compartment of multivesicular bodies (1) and are secreted in various body fluids under normal and pathological conditions (2, 3). Extensive studies have now implicated exosomes in many biological processes such as tissue injury and immune responses by transfer of antigens, antigen presentation (2, 4), and the shuttling of proteins, mRNAs, and microRNAs (miRNA) between cells (5). As such, it has been postulated that exosomes play a crucial role in cell communication and in the transfer of genetic information between cells (5).The role of exosomes and other secretory vesicles in the transfer of pathogen-derived antigens and virulence factors is emerging (6, 7). Whether release of vesicles from infected cells contributes to immune control and clearance of infection by the host is still not clear. For example, the HIV Gag protein recruits the outward vesicle-budding machinery of exosomes to form free virions (8). Recently, it has been shown that exosomes released from HIV-infected cells contain negative regulatory factor, which induces apoptosis of uninfected cells (9). Epstein-Barr virus-infected B cells also secrete exosomes that contain virally encoded miRNA (10). This study further demonstrates the delivery of naturally occurring functional genetic elements to neighboring cells via exosomes, indicating that viral particles or molecules associated with viral infection can be transmitted to adjacent uninfected cells via exosomes and become functional. More recently, the hepatitis A virus has shown to be able to escape humoral immunity by cloaking itself in cellular membranes on release from host cells. These virus-containing microvesicles, resembling exosomes, were shown to protect virions from antibody-mediated neutralization (11).Hepatitis C virus (HCV) infection, a leading liver disease, has been shown to have multiple routes of transmission. Apart from classical transmission by free viral particles, an antibody-resistant cell-to-cell transmission route also has been described (12). Indeed, HCV is known to evade humoral immune responses, as indicated by a lack of resistance to HCV reinfection in i.v. drug users (13), HCV reinfection during liver transplantation (14), and an ongoing difficulty of developing effective vaccines. The role of exosomes in HCV infection is still largely unknown. One earlier paper reported the presence of viral RNA in exosomes isolated from plasma of HCV-infected patients (15) but did not show exosome-mediated transmission of infection. More recent studies suggest that HCV virus assembly and release in hepatocytes may be linked to the exosome secretory pathway (16) and that hepatocyte-derived exosomes can transfer viral RNA to plasmacytoid dendritic cells, triggering their activation and IFN-α production (17). However, the role of exosomes in the cell-to-cell transmission route of HCV between hepatocytes has not been demonstrated. The aim of our study was to investigate transmission of HCV infection by hepatocyte-derived exosomes in the presence of neutralizing antibodies (nAbs) in vitro that could explain the ineffectiveness of prophylactic nAbs and agents targeting the entry of HCV into a cell. We further observe that this route of infection can generate productive viral infection.     

Colonic preparation before colonoscopy in constipated and non-constipated patients:A randomized study

AIM: To compare the efficacy of different doses of sodium phosphate(NaP) and polyethylenglicol(PEG) alone or with bisacodyl for colonic cleansing in constipated and non-constipated patients.METHODS: Three hundred and forty-nine patients,older than 18 years old,with low risk for renal damage and who were scheduled for outpatient colonoscopy were randomized to receive one of the following preparations(prep): 90 mL of NaP(prep 1);45 mL of NaP + 20 mg of bisacodyl(prep 2);4 L of PEG(prep 3) or 2 L of PEG + 20 mg of bisacodyl(prep 4).Randomization was stratified by constipation.Patients,endoscopists,endoscopists’ assistants and data analysts were blinded.A blinding challenge was performed to endoscopist in order to reassure blinding.The primary outcome was the efficacy of colonic cleansing using a previous reported scale.Secondary outcomes were tolerability,compliance,side effects,endoscopist perception about the necessity to repeat the study due to an inadequate colonic preparation and patient overall perceptions.RESULTS: Information about the primary outcome was obtained from 324 patients(93%).There were no significant differences regarding the preparation quality among different groups in the overall analysis.Compliance was higher in the NaP preparations being even higher in half-dose with bisacodyl: 94%(prep 1),100%(prep 2),81%(prep 3) and 87%(prep 4)(2 vs 1,3 and 4,P < 0.01;1 vs 3,4,P < 0.05).The combination of bisacodyl with NaP was associated with insomnia(P = 0.04).In non-constipated patients the preparation quality was also similar between different groups,but endoscopist appraisal about the need to repeat the study was more frequent in the half-dose PEG plus bisacodyl than in whole dose NaP preparation: 11%(prep 4) vs 2%(prep 1)(P < 0.05).Compliance in this group was also higher with the NaP preparations: 95%(prep 1),100%(prep2) vs 80%(prep 3)(P < 0.05).Bisacodyl was associated with abdominal pain: 13%(prep 1),31%(prep 2),21%(prep 3) and 29%(prep 4),(2,4 vs 1,2,P < 0.05).In constipated pati     

Effect of Different Insoles on Postural Balance: A Systematic Review

[Purpose] The aim of the present study was to perform a systematic review of the literature on the effect of different insoles on postural balance. [Subjects and Methods] A systematic review was conducted of four databases. The papers retrieved were evaluated based on the following inclusion criteria: 1) design: controlled clinical trial; 2) intervention: insole; 3) outcome: change in static postural balance; and 4) year of publication: 2005 to 2012. [Results] Twelve controlled trials were found comparing the effects of different insoles on postural balance. The papers had methodological quality scores of 3 or 4 on the PEDro scale. [Conclusion] Insoles have benefits that favor better postural balance and control.Key words: Postural balance, Proprioception, Foot     

Aromatase expression in the eutopic endometrium of myomatous uteri: the influence of the menstrual cycle and oral contraceptive use.

AIMS: To detect aromatase expression in the endometrium of myomatous uteri and to correlate it with the location of the myoma, phase of the menstrual cycle, the presence of menorrhagia and oral contraceptive use. METHOD: Aromatase p450 expression was measured using immunohistochemical methods in the endometrium of 116 patients. Sixty-one patients had menorrhagia associated with intramural/submucous myomas and nine had subserous myomas and no excessive bleeding. Forty-six patients had no uterine pathology and served as controls. Nineteen out of 61 patients with menorrhagia were oral contraceptive users at the time of the examination. Endometrial samples were obtained by hysteroscopy in all cases. RESULTS: Aromatase p450 expression was detected more frequently in the eutopic endometrium of patients with submucous or intramural myomas than in those in the subserous group, and was significantly greater during the proliferative phase than during the luteal phase or following the use of oral contraceptives. In normal uteri, aromatase expression was detected in the endometrium in less than 10% of users. CONCLUSIONS: Aromatase expression in the endometrium was affected by the location of the myoma, the presence of symptoms, and the phase of the menstrual cycle. Oral contraceptives, on the other hand, inhibited aromatase expression in the eutopic endometrium of patients with submucous/intramural myomas.     

A competitive interaction model predicts the effect of WAY-100,635 on the time course of R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin-induced hypothermia.

The objective of this investigation was to characterize quantitatively the pharmacodynamic interaction between N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide (WAY-100,635) and R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT) in vivo. The 8-OH-DPAT-induced change in body temperature was used as a pharmacodynamic endpoint. Four groups of rats each received 1 mg/kg 8-OH-DPAT in 5 min during computer-controlled infusions of physiological saline or WAY-100,635, targeted at steady-state concentrations of 20, 85, and 170 ng/ml. Body temperature was monitored continuously with a telemetric system, and frequent blood samples were obtained to determine the pharmacokinetics of both drugs. Large differences in pharmacokinetics were observed between WAY-100,635 and R-8-OH-DPAT, reflected in values of the terminal elimination half-life of 33 and 143 min, respectively. Infusion of WAY-100,635 had no influence on the pharmacokinetics of R-8-OH-DPAT. With regard to the pharmacodynamics, clear antagonism of the R-8-OH-DPAT-induced hypothermia was observed. The complex pharmacological effect versus time profiles of R-8-OH-DPAT were analyzed on the basis of an indirect physiological response model with set point control coupled to a competitive interaction model for an agonist and antagonist acting at a common receptor. This model converged, yielding precise estimates of the pharmacodynamic parameters of both WAY-100,635 and R-8-OH-DPAT, which were independent of the infusion rate of WAY-100,635. The estimated in vivo binding constant of WAY-100,635 was 0.98 ng/ml (2.3 nM), which is very similar to the reported value from in vitro receptor binding assays. The findings of this investigation show that, in contrast to earlier reports in the literature, WAY 100,635 behaves as a pure competitive antagonist at the 5-hydroxytryptamine(1A) receptor in vivo.     

Protein kinase C inhibitors, H7 and calphostin C, inhibit induction of DNA synthesis by cytosolic extracts of exponentially growing neuroblastoma cells in isolated nuclei

Cytoplasmic extracts from proliferating Neuro-2a cells contain a protein factor, ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. Cytoplasmic extracts derived from quiescent-made Neuro-2a cells contain none or very little ADR activity, but this activity can be generated after a brief exposure of cytosolic extracts to a membrane-enriched fraction derived from exponentially growing Neuro-2a cells. ADR activity appears at the beginning of the S phase of the cell cycle. Moreover it appears to be a protease, because aprotinin inhibits ADR activity. ADR activity can be also inhibited by the protein kinase C inhibitors, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7) and calphostin C.