Search

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator).

Streptococcus pneumoniae (S. pneumoniae) is the most important pathogenic bacteria occurred in wide range of habitat, which cause of several diseases, including pneumonia, meningitis, otitis media, bacteremia and other common infections. Every year, approximately 1.6 million people are seriously affected and ultimately die as a result of pneumococcal infections worldwide, affecting people’s lives and health. Relevant studies in recent years have demonstrated the important role of microRNAs (miRNAs) in a variety of diseases. Further research on the mechanism of microRNA and the relationship between microRNA and diseases has shown that microRNA may become a new biological marker for disease diagnosis and may also provide a new approach to disease treatment. The function of microRNA in pneumococcal infections has also been gradually reported, including pneumococcal nasopharyngeal colonization, pneumonia, sepsis, bacteremia, heart invasion, meningitis, acute otitis media, osteoarthritis and other diseases. This article will overview the mechanism and research progress of microRNA in infections caused by S. pneumoniae.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2, poses a significant threat to public health worldwide, and diabetes is considered a risk factor for the rapid progression and poor prognosis of COVID-19. Limited immune function is a clinical feature of COVID-19 patients, and diabetes patients have defects in innate and adaptive immune functions, which may be an important reason for the rapid progression and poor prognosis of COVID-19 in patients with diabetes. We review the possible multiple effects of immune impairment in diabetic patients on the immune responses to COVID-19 to provide guidance for the diagnosis and treatment of diabetic patients with COVID-19.

Acute pancreatitis (AP) is an inflammatory disease caused by the abnormal activation of pancreatic enzymes in the pancreas, with a considerably high morbidity and mortality. However, the etiological factor and pathogenesis of AP are still unclear. This study was aimed to explore the role and mechanism of interferon regulatory factor 9 (IRF9) in the occurrence of AP and to provide experimental and theoretical foundation for AP diagnosis and treatment. AP model in vitro was established by caerulein-induced group. Small interfering RNA (siRNA) was designed and constructed to silence IRF9 gene. After siRNA transfected and caerulein treated successfully, the expression levels of IRF9, SIRT1, and acetylated p53 (Ac-p53) were determined by qRT-PCR and Western blot. The apoptosis, proliferation, and migration of AR42J cells were checked by flow cytometry, MTT, and transwell assay. Dual-luciferase reporter assay was implemented to validate the regulatory effect of IRF9 on SIRT1. Here, our study showed that the expression of IRF9 and Ac-p53 was increased, SIRT1 was decreased, and cell apoptosis, proliferation, and migration of AR42J cells were increased after caerulein induced. IRF9 gene silencing upregulated SIRT1, downregulated Ac-p53, and inhibited cell apoptosis, proliferation, and migration. Dual-Luciferase reporter assay showed that IRF9 could negatively regulate SIRT1. The potential mechanism was that IRF9 could modulate cell apoptosis, proliferation, migration, and bind the promoter of SIRT1 to repress SIRT1-p53. It hinted that IRF9 showed a novel function in AP by modulating cell apoptosis, proliferation, migration, and suppressing SIRT1-p53. IRF9 might be a good potential treatment target for AP.

Interferon regulatory factor 9 (IRF9) acts as a negative regulator of sirtuin-1 (SIRT1) to participate in many diseases. However, the role of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury is unclear. To explore the function of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury and provide theoretical guidance for disease diagnosis and treatment. Model rats were established by intraperitoneal injection of 20% l-arginine. Apoptosis of kidney tissue was determined by TUNEL staining. Expressions of IRF9, SIRT1, p53, and acetylated p53 were detected by qRT-PCR and Western blot. Dual-Luciferase Reporter Assay was carried out to validate the regulation of IRF9 on SIRT1. Pancreatic and renal injury was more serious, and apoptosis of kidney epithelial cells increased in acute pancreatitis (AP) and hyperlipidemia acute pancreatitis (HLAP) group. IRF9, p53, and acetylated p53 were up-regulated, and SIRT1 was down-regulated in AP and HLAP group (p

Objectives: To analyze the clinical characteristics of severe acute pancreatitis (SAP) patients retrospectively and explore the effective factors in death from severe acute pancreatitis (SAP). Methods: The required data were collected from 234 SAP patients admitted to our department from January 2013 to December 2020 and then analyzed retrospectively. According to the prognosis, all patients were admitted within 72 hours of onset and were assigned to the death and survival groups. The participants’ clinical and demographic information, laboratory indices when patients were brought to the intensive care unit (ICU), and organ failure were analyzed using univariate and logistic multivariate regression. The logistic regression (LR) model was developed and evaluated by the receiver operating characteristic (ROC) curve. Results: In this study, the total mortality rate was 11.96% (95% CI, 8.1 - 16.8%). The univariate analysis revealed a significant relationship between SAP-related death with age, ICU admission within 24 hours of onset, APACHE II score, serum amylase, serum albumin, PaO2, acute respiratory distress syndrome (ARDS), renal insufficiency, and other diseases (P < 0.05). The multivariate logistic regression analysis further demonstrated that ICU admission within 24 hours of onset, serum albumin, ARDS, and renal insufficiency were independent early prognostic factors of SAP (P < 0.05). LR model: Y = -0.108 - 1.852 × ICU admission within 24 hours of onset -0.102 × serum albumin + 1.790 × ARDS + 1.150 × renal insufficiency. The area under the curve (AUC) and 95% CI of the LR model were 0.864 (0.811 - 0.917) with the optimal threshold of 2.246. The sensitivity and specificity were 0.709 and 0.929, respectively. Conclusions: The SAP patients or acute pancreatitis (AP) patients at risk of developing SAP should be transferred to ICU at the earliest convenience. Moreover, hypoalbuminemia, ARDS, and renal insufficiency indicate poor prognosis.

Background: Acute pancreatitis (AP) is an acute abdomen caused by abnormal activation of trypsin. AMPK-SIRT1 pathway has been reported to be related to various diseases, but the function in AP remains unclear. This study is designed to investigate the mechanism and effect of AMPK-SIRT1 pathway in AP.Methods: An experimental AP model of AR42J cells was stimulated with caerulein after pretreated with compound C or metformin. The mRNA and protein expressions of genes were analyzed by qRT-PCR and western blot. Cell apoptosis, proliferation and migration were measured using flow cytometry, MTT and transwell assay. Results: After pretreated with metformin, expressions of p-AMPKα, SIRT1 were elevated, ace-p53, ace-NF-κB were attenuated, cell apoptosis, proliferation, and migration were decreased. After pretreated with compound C, the reverse effects occurred. p-AMPKα and SIRT1 expressions were decreased, ace-p53 and ace-NF-κB were rasied, and cell apoptosis, proliferation, and migration were enhanced after caerulein induced in each group. Conclusion: When AP happened, expressions of p-AMPKα and SIRT1 were reduced, resulting in up-regulation of acetylation levels of p53 and NF-κB, acceleration of cell apoptosis, proliferation and migration. It hinted that AMPK-SIRT1 pathway could modulate the apoptosis, proliferation, migration and inflammation reaction of AR42J cells by regulating p53 and NF-κB.

The development of hemoglobin-based oxygen carriers (HBOCs) is a persistent and urgent need in biomedicine. As a potential HBOCs, Dextran-hemoglobin (Dex-bHb) has been developed over the past years. The novel Dex-bHb, whose thiol group of Cys-93(β) was reversibly protected, was produced and the characteristics were evaluated in our previous study. Herein, blood compatibility was characterized in terms of the red blood cell aggregation and hemolysis rate in vitro, and Dex-bHb showed no obvious effects. After intravenous administration of Dex-bHb to golden Syrian hamsters with hemorrhages shock, it showed mean arterial pressure recovery, blood flow increase and the organ protection from serious hemorrhage injury. Consequently, Dex-bHb is hopeful to be a safe and available blood substitute.

Polymer dielectric composites with high dielectric permittivity and low dielectric loss play an increasing important role for modern electronic and electric industry. In this study, polyvinylidene fluoride (PVDF) incorporated with expanded graphite (EG) or reduced graphene oxide (rGO) was prepared by solution casting and the following hot pressing. The structure, morphology, and properties of the obtained products were characterized. In comparison with EG, rGO exhibits well dispersion and strong interaction with PVDF matrix. With the increasing content of conductive fillers, the dielectric permittivity of both PVDF/EG and PVDF/rGO composites shows an increasing trend whereas the dielectric loss keeps at a low level. The dielectric permittivity of PVDF filled with 4 wt% EG and 8 wt% rGO reaches 420 and 38 at 100 Hz, respectively. Moreover, the introduction of EG and rGO improves the mechanical strength and the thermal stability of PVDF matrix.

Aims Acute pancreatitis (AP) is a common inflammatory disorder with high incidence and mortality. AMPK-SIRT1 pathway is involved in a variety of diseases, but its role in AP remains elusive. This study was aimed to explore the role of AMPK-SIRT1 pathway in AP. Main methods AP models in vivo and vitro were constructed by intraperitoneal administration of L-arginine and caerulein-stimulated respectively. Rat serum amylase, IL-6 and TNF-α were determined by ELISA. The expression levels of AMPK, SIRT1, Beclin-1, LC3 and p62 were determined by qRT-PCR and western blot. The number of autophagosome was checked by transmission electron microscope. Key findings Compared with NC rats, serum amylase, IL-6 and TNF-α were increased in AP rats. The expressions of AMPK and SIRT1 were decreased, while Beclin-1, LC3II/Iratio and p62 were markedly increased in AP rats. After activation of AMPK by metformin, expressions of p-AMPKα, SIRT1 were significantly raised, while expressions of Beclin-1, LC3 II/I, p62, TNF-α, IL-6 were reduced, and the number of autophagosome was decreased significantly in caerulein-stimulated AR42J cells. The inhibition of AMPK by compound C obtained opposite results. Significance During AP occurrence, p-AMPK and SIRT1 were down-regulated, leading to the accumulation of p62, increase of autophagic vacuoles, damage of autophagy, and the occurrence of inflammation. It hinted that activation of AMPK restored impaired autophagy and inhibited inflammation reaction by up-regulating SIRT1. Our findings might provide important theoretical basis for explaining the pathogenesis of AP and investigating therapeutic target to treat and prevent AP.

Polymer hollow microspheres were prepared by performing alkali treatment on the multilayer core/shell polymer latex particles containing carboxyl groups. Effects of the shell composition and dosage as well as alkali type on the morphology of the microspheres were investigated. Results showed that in comparison with acrylonitrile (AN) and methacrylic acid (MAA), using butyl acrylate (BA) as the shell co-monomer decreased the glass transition temperature (T g) of shell effectively and was beneficial to the formation of uniform and big hollow structure. Along with the increase of the shell dosage, the alkali-treated microspheres sequentially presented porous and hollow morphology, and the size of microspheres increased, while the hollow diameter increased first and then decreased, and the maximum hollow ratio reached 39.5%. Furthermore, the multilayer core/shell microspheres had better tolerance to NH3·H2O than to NaOH. When the molar ratio of alkali to methacrylic acid (MRalkali/acid) for NaOH ranged from 1.15 to 1.30 or MRalkali/acid for NH3·H2O ranged from 1.30 to 2.00, the regular polymer hollow microspheres could be obtained.

Haemoglobin-based oxygen carrier (HBOC) is highly susceptible to autoxidation that renders a series of tissue and cellular toxicities. HBOC is prepared by chemical modification of haemoglobin (Hb), which typically increases the autoxidation rate of Hb. Thus, it is necessary to decrease the autoxidation of HBOC. In the present study, two dextran-bHb conjugates (dex20-bHb and dex40-bHb) were prepared by conjugation with 20 kDa or 40 kDa dextrans, where the thiol group of Cys-93(β) was reversibly protected. The autoxidation rate of bHb was decreased by conjugation with 20 kDa dextran and maintained by conjugation with 40 kDa dextran. In order to understand the low autoxidation rate of dex20-bHb, the effects of aldehyde modification and dextran on the autoxidation rate of dex20-bHb were investigated. The high oxygen affinity, high tetramer stability and dextran itself were found to decrease the autoxidation rate of dex20-bHb. The conjugated dextran had a predominant effect on decreasing the autoxidation rate of bHb, which was particularly promising for the potential development of safe HBOCs. Conjugation with dextran is of general significance to decrease the oxidation process of the heme-containing proteins, such as Hb and myoglobin.

As a hemoglobin (Hb)-based oxygen carrier (HBOC), Hb suffers from the disadvantages of short half-life, renal toxicity and vasoactivity. Because dextran is a macromolecule that can be easily derivatized with various chemical moieties, conjugation of Hb with dextran can effectively increase the size of Hb and overcome the disadvantages of Hb. Thus, a dextran-bovine Hb (bHb) conjugate (dex-bHb) was prepared by conjugation of bHb with periodate-oxidized dextran here. As an important functional amino acid residue of bHb, Cys-93(β) was reversibly protected by 4,4′-dithiodipyridine to avoid reaction with periodate-oxidized dextran. Dex-bHb showed significantly higher hydrodynamic volume and higher viscosity than bHb. Conjugation with dextran stabilized the R state of bHb and slightly altered the heme environment of bHb. Conjugation with dextran decreased the P50 of bHb, lowered the sensitivity to the allosteric effectors and slightly decreased the autoxidation rate of bHb. Thus, dex-bHb was expected to act as a potent HBOC with low oxidative toxicity.

PEGylation has successfully improved the pharmacological properties of therapeutic proteins. However, polyethylene glycol (PEG) has been burdened by immunogenicity that renders a negative clinical effect on therapeutic proteins. The anti-PEG immune response to PEGylated proteins possibly depends on the nature of proteins and the conjugated methoxy PEG (mPEG). Thus, it is necessary to investigate the effects of protein immunogenicity, the extent of PEGylation, the molecular weight (Mw), and the branching of mPEG on the anti-PEG immune response. Ovalbumin, tetanus toxoid (TT), TT–TT conjugate, and TT–bovine serum albumin conjugate were used as target proteins. PEGylated proteins with different extents of PEGylation were obtained by fractionation of the PEGylated TT with size exclusion chromatography. The PEGylated proteins with different Mw and branching of mPEG were obtained by modification of TT with linear mPEG (5 kDa and 20 kDa) and branched mPEG (20 kDa). The PEGylated proteins elicited high levels of anti-PEG antibodies (predominantly IgM and IgG1). The anti-PEG immune response depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of mPEG. In contrast, branching of mPEG had an insignificant effect on the anti-PEG immune response to the PEGylated proteins.

To meet the challenge of new military change in world and the demand across centuries to adapt modernization of national defense and air force, numbers of flight trainees with high quality, multi-structure of knowledge and comprehensive ability, and enterprising spirit and ability to innovate are trained, which is also the target of training of bright people at air force flight university. The comprehensive quality evaluation of Air force's flight trainees is the basis of scientific management, by which advantages and disadvantages can be analyzed in training of bright people, whether they can satisfy the demand of the information-based war in the future.

This paper analyzes the failure phenomenon and adjustment skill through the basic introduces of the principle of Bourdon tube Type pressure gauge. The detect of bourdon tube type pressure gauge was discussed to make sure the accuracy of the data measured by bourdon tube type pressure gauge to colleagues as reference. Bourdon tube type pressure gauge has already widely used in the maintenance of the ground equipment, and pressure gauge performance stable and reliable quality will influence aircraft maintain quality. The correct use and debug the bourdon tube type pressure gauge determine to the measure of pressure gauge. We should analysis on the structure and principles of bourdon tube type pressure gauge and the cause of the problem, to give the repair method.

Military academy, troops are two important stages of shaping military talents. Army colleges and universities is the main channel, joint operations command training forces is that they eventually exercise mature stage. Military academy and troops only work together, doing things that more closely, to ensure the quality of the talents cultivation. Therefore to strengthen military academy and forces combination education, set up scientific training system, pays special attention to the joint operations command personnel training.

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1 M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.

In recently years, in order to exactly judge artillery capability, we should measure important parts of distortion. In this article, we research and design a non-contact three –dimension measure system of artillery parts distortion, and it can get exactly measure results.

The article introduce the mechanical structure and the principles of 80000 N·m torsion testing machine, the structure and the principles of checking power unit of this kind of testing machines and the control manner and the principles of the electronic control system, than expound the benefits of the equipment and its application.

Aiming at the problems existing NC machining, combining with the existing advanced manufacturing mode, and cloud computing, application integration, new technologies such as service-oriented technology, based on cloud resources in manufacturing, manufacturing services in the cloud, the relationship between manufacturing cloud are expounded. On this basis, the design of the NC machining of cloud services management prototype system function structure, based on whole life cycle of application integration operation of the cloud service portfolio requirements are expounded, and the implementation is based on application integration and cloud service platform of CNC processing key technology are studied, for the future to realize efficient intelligent cloud manufacturing services management to provide theoretical support.

In the machine manufacture industry, the on-line test technique applies very extensively, the particularly automatic test technique is more outstanding. This article with the form error margin for example, designed a set of simple on-line test system to raise the accuracy of the machine product examination, assurance product quality, decline low cost depletion.

The analysis of all kinds of selected chemicals, mixed starch was donethrough a specific process and orthogonal experiments. The best proportion ofthe new type of surface sizing agent was prepared through the glue applicationexperiments, testing paper physical properties. The results showed that thebest formula of surface sizing agent including borax, sodium silicate,anhydrous sodium sulfate, ferrous chloride which ratio is 3:4:2:1. At the sametime modifying agent was added so that the stable performance can be obtainedfrom the solid surface sizing agent. Homemade solid surface sizing agent canmake paper greatly improve the sizing degree, increased by 50.9%, the stiffnessof the tear strength increased by 56.7%, folding degrees increased by 53.0%.Compared with the surface sizing agent market, various aspects index improvedsignificantly.

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.

Background: The well-established scientific observation has shown that the process of sensing and making adaptive response to external stimuli in bacteria is regulated by the two-component signal-transduction systems (TCSTSs). Typical TCSTSs are composed of two different types of proteins, a histidine kinase (HK) and a response regulator (RR). This review focuses on the influence of TCSTSs on the virulence, competence, biofilm formation and antibiotic resistance, and further discusses the value of TCSTSs for the new drug targets. Methods and findings: The HK protein is responsible for response to external stimuli by autophosphorylation, subsequently transfering this phosphate group to the RR protein, so then activating a series of biological processes. Loss of function or genetic mutation of these systems has been reported to study the function of the two component systems. It suggests that this system can regulate the expression of virulence factors, control the competence for transformation, modulate biofilm formation and cell envelope stress response, and influence the susceptibility to antibiotics. Conclusion: The homologies of HK and RR proteins in different genera of bacteria are highly conserved respectively, especially in the residues located near active sites. Moreover the signal transduction in mammals has an entirely different mechanism compared with bacteria. Altogether, this system may be very suited as a good potential drug target for the development of novel antibacterial.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro-Hb, and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA. In contrast, bovine Hb (bHb) has no quantity constraints for its ample resource. Thus, bHb is of potential to function as an alternative substrate to obtain a PEGylated bHb (bHb-PEG). bHb-PEG was prepared under the same reaction condition as HbA-PEG, using maleimide chemistry. The structural, functional, solution and physiological properties of bHb-PEG were determined and compared with those of HbA-PEG. bHb-PEG showed higher hydrodynamic volume, colloidal osmotic pressure, viscosity and P50 than HbA-PEG. The high P50 of bHb can partially compensate the PEGylation-induced perturbation in the R to T state transition of HbA. bHb-PEG was non-vasoactive and could efficiently recover the mean arterial pressure of mice suffering from hemorrhagic shock. Thus, bHb-PEG is expected to function as a potent HBOC for its high oxygen delivery and strong plasma expanding ability. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:252-260, 2017.

We study the early stages of flocculation in suspensions of charged colloidal particles in the presence of oppositely charged polyelectrolytes. Absolute aggregation rate constants are determined by...

By the methods of fumigation and contact assay, this paper determined the toxicity of four kinds of soil fumigants and six kinds of non-fumigants against Ditylenchus destructor. In the meantime, the bioactivity of the agents was evaluated by the corrected mortality rate of D. destructor after treated with the LC50 dose of each agent for different time. The LC50 values of soil fumigants dazomet, 1, 3-dichloropropene, metham-sodium, and chloropicrin were 0.49, 0.89, 0.91, and 3.60 mg x L(-1), and those of non-fumigants emamectin benzoate, abamectin, ethoprophos, fosthiazate, aldicarb, and carbosulfan were 31.2, 48.1, 224.3, 288.4, 632.3, and 823.9 mg x L(-1), respectively. The corrected mortality rate increased with treating time. After treated with LC50 dose, the inhibitory effects of dazomet, 1,3-dichloropropene, abamectin, and emamectin benzoate kept on a higher level at the time, and the corrected mortality rate did not decline after breaking away from the treatment, but the D. destructor recovered when exposed to natural state after treated with ethoprophos and aldicarb for 48h. It was suggested that dazomet, 1,3-dichloropropene, abamectin, and emamectin benzoate were the potential alternative nematicides used for the control of D. destructor.

The β-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the β-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of β(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and β-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 Å crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (β/α)(8) TIM barrel with the active site residing at the center of the β-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-β-d-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k(cat)/K(m)) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAGβ(1,2)Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the β(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the β(1,2)-linked β-N-acetylglucosides.

Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.

Ceramide kinase (CerK) catalyzes the generation of ceramide-1-phosphate which may regulate various cellular functions, including inflammatory reactions and cell growth. Here, we studied the effect of a recently developed CerK inhibitor, NVP-231, on cancer cell proliferation and viability and investigated the role of cell cycle regulators implicated in these responses.The breast and lung cancer cell lines MCF-7 and NCI-H358 were treated with increasing concentrations of NVP-231 and DNA synthesis, colony formation and cell death were determined. Flow cytometry was performed to analyse cell cycle distribution of cells and Western blot analysis was used to detect changes in cell cycle regulator expression and activation.In both cell lines, NVP-231 concentration-dependently reduced cell viability, DNA synthesis and colony formation. Moreover it induced apoptosis, as measured by increased DNA fragmentation and caspase-3 and caspase-9 cleavage. Cell cycle analysis revealed that NVP-231 decreased the number of cells in S phase and induced M phase arrest with an increased mitotic index, as determined by increased histone H3 phosphorylation. The effect on the cell cycle was even more pronounced when NVP-231 treatment was combined with staurosporine. Finally, overexpression of CerK protected, whereas down-regulation of CerK with siRNA sensitized, cells for staurosporine-induced apoptosis.Our data demonstrate for the first time a crucial role for CerK in the M phase control in cancer cells and suggest its targeted inhibition, using drugs such as NVP-231, in combination with conventional pro-apoptotic chemotherapy.

Glucose-regulated protein 78 (GRP78) is a stress protein and molecular chaperone. It usually is located in the endoplasmic reticulum and can be translocated to the cell surface as a receptor for a variety of ligands. GRP78 is implicated in a series of biological processes, such as protein folding and assembly, angiogenesis, metastasis, and regulation of endoplasmic reticulum stress signaling. However, the molecular mechanism of GRP78 remains unclear. Recently, …

Ischemia-reperfusion (IR) injury is significantly worse in aged hearts, but the underlying mechanisms are poorly understood. Age-related damage to mitochondria may be a critical feature, which manifests in an exacerbation of IR injury. Silent information regulator of transcription 3 (SIRT3), the major mitochondrial NAD+-dependent lysine deacetylase, regulates a variety of functions, and its inhibition may disrupt mitochondrial function to impact recovery from IR injury. In this study, the role of SIRT3 in mediating the response to cardiac IR injury was examined using an in vitro model of SIRT3 knockdown (SIRT3kd) in H9c2 cardiac-derived cells and in Langendorff preparations from adult (7 mo old) wild-type (WT) and SIRT3+/− hearts and aged (18 mo old) WT hearts. SIRT3kd cells were more vulnerable to simulated IR injury and exhibited a 46% decrease in mitochondrial complex I (Cx I) activity with low O2 consumption rates compared with controls. In the Langendorff model, SIRT3+/− adult hearts showed less functional recovery and greater infarct vs. WT, which recapitulates the in vitro results. In WT aged hearts, recovery from IR injury was similar to SIRT3+/− adult hearts. Mitochondrial protein acetylation was increased in both SIRT3+/− adult and WT aged hearts (relative to WT adult), suggesting similar activities of SIRT3. Also, enzymatic activities of two SIRT3 targets, Cx I and MnSOD, were similarly and significantly inhibited in SIRT3+/− adult and WT aged cardiac mitochondria. In conclusion, decreased SIRT3 may increase the susceptibility of cardiac-derived cells and adult hearts to IR injury and may contribute to a greater level of IR injury in the aged heart.

[1] Wang PX, Zhang R, Huang L, Zhu LH, Jiang DS, Chen HZ, et al. Interferon regulatory factor 9 is a key mediator of hepatic ischemia/reperfusion injury. J Hepatol 2014;62:111–120. [2] Zhang SM, Zhu LH, Chen HZ, Zhang R, Zhang P, Jiang DS, et al. Interferon regulatory factor 9 is critical for neointima formation following vascular injury. Nat Commun 2014;5:5160. [3] Chen HZ, Guo S, Li ZZ, Lu Y, Jiang DS, Zhang R, et al. A critical role for interferon regulatory factor 9 in cerebral ischemic stroke. J Neurosci 2014;34:11897–11912. [4] Zhang Y, Liu X, She ZG, Jiang DS, Wan N, Xia H, et al. Interferon regulatory factor 9 is an essential mediator of heart dysfunction and cell death following myocardial ischemia/reperfusion injury. Basic Res Cardiol 2014;109:434. [5] Jiang DS, Luo YX, Zhang R, Zhang XD, Chen HZ, Zhang Y, et al. Interferon regulatory factor 9 protects against cardiac hypertrophy by targeting myocardin. Hypertension 2014;63:119–127.