Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: Insights into polysaccharide binding mechanism of CBM21 family

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield β‐d ‐glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N‐terminal starch binding domain (SBD) and a C‐terminal catalytic domain connected by an O‐glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only α‐(1,6)‐linked isomaltotriose (RoSBD‐isoG3) and isomaltotetraose (RoSBD‐isoG4) have been determined at 1.2 and 1.3 Å, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD‐isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch. Proteins 2014; 82:1079–1085. © 2013 Wiley Periodicals, Inc.     

Is Early Oral Feeding after Gastric Cancer Surgery Feasible? A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Aim

To assess the feasibility and safety of early oral feeding (EOF) after gastrectomy for gastric cancer through a systematic review and meta-analysis based on randomized controlled trials.

Methods

A literature search in PubMed, Embase, Web of Science and Cochrane library databases was performed for eligible studies published between January 1995 and March 2014. Systematic review was carried out to identify randomized controlled trials comparing EOF and traditional postoperative oral feeding after gastric cancer surgery. Meta-analyses were performed by either a fixed effects model or a random effects model according to the heterogeneity using RevMan 5.2 software.

Results

Six studies remained for final analysis. Included studies were published between 2005 and 2013 reporting on a total of 454 patients. No significant differences were observed for postoperative complication (RR = 0.95; 95%CI, 0.70 to 1.29; P = 0.75), the tolerability of oral feeding (RR = 0.98; 95%CI, 0.91 to 1.06; P = 0.61), readmission rate (RR = 1; 95%CI, 0.30 to 3.31; P = 1.00) and incidence of anastomotic leakage (RR = 0.31; 95%CI, 0.01 to 7.30; P = 0.47) between two groups. EOF after gastrectomy for gastric cancer was associated with significant shorter duration of the hospital stay (WMD = −2.36; 95%CI, −3.37 to −1.34; P<0.0001) and time to first flatus (WMD = −19.94; 95%CI, −32.03 to −7.84; P = 0.001). There were no significant differences in postoperative complication, tolerability of oral feeding, readmission rates, duration of hospital stay and time to first flatus among subgroups stratified by the time to start EOF or by partial and total gastrectomy or by laparoscopic and open surgery.

Conclusions

The result of this meta-analysis showed that EOF after gastric cancer surgery seems feasible and safe, even started at the day of surgery irrespective of the extent of the gastric resection and the type of surgery. However, more prospective, well-designed multicenter RCTs with more clinical outcomes are needed for further validation.     

Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca²⁺ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca²⁺ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca²⁺ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.     

High glucose promotes gap junctional communication in cultured neonatal cardiac fibroblasts via AMPK activation

Cardiac fibroblasts are known to be essential for adaptive responses in the pathogenesis of cardiovascular diseases, and increased intercellular communication of myocardial cells and cardiac fibroblasts acts as a crucial factor in maintaining the functional integrity of the heart. AMP-activated kinase (AMPK) is a key stress signaling kinase, which plays an important role in promoting cell survival and improving cell function. However, the underlying link between AMPK and gap junctional communication (GJIC) is still poorly understood. In this study, a connection between AMPK and GJIC in high glucose-mediated neonatal cardiac fibroblasts was assessed using fibroblast migration, measurement of dye transfer and connexin43 (Cx43) expression. 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and Compound C (CC) were used to regulate AMPK activity. The levels of cell migration and Cx43 protein expression in neonatal cardiac fibroblasts increased during high glucose treatment, accompanied by developed dye transfer. In addition, high glucose induced abundant phosphorylation of AMPK. Suppression of AMPK phosphorylation using CC reduced dye transfer, cell migration and Cx43 protein expression in neonatal cardiac fibroblasts, whereas the activation of AMPK using AICAR mimicked the high glucose-mediated cell migration, Cx43 protein expression and dye transfer enhancement. AMPK appears to participate in regulating GJIC in high-glucose-treated neonatal cardiac fibroblasts, including cell migration, dye transfer, Cx43 expression and distribution.     

CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.     

Fine-mapping of an Arabidopsis cell death mutation locus

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between lGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.