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Objectives To compare the levels of serum pepsinogen (PG) in patients with gastric cancer (GC), patients with atrophic gastritis (AG), and healthy donors. Also, we explored the clinical value of PG detection for the diagnosis and treatment of GC. Methods The PG level in peripheral blood from patients and heathy donors was determined using an Abbott automatic chemiluminescence instrument. The study included 117 patients with GC confirmed by gastroscopy and histopathology, of whom 13 patients had cancer at stage I, 47 at stage II, 41 at stage III, and 16 at stage IV. The AG group included 122 patients, and the control group had 120 healthy donors. The relationship between serum PG levels and the occurrence and development of GC, as well as the evaluation of the clinical value of diagnostic tests based on serum PG detection, were investigated by receiver operating characteristic (ROC) curve analyses. Results Pepsinogen I (PGI) levels gradually decreased from the control group, the AG group, and the GC group. PGI exhibited high diagnostic value for GC (area under the curve [AUC], 0.834; cutoff, 51.2 ng/mL, sensitivity, 81.7%; specificity, 68.4%), PGII (AUC, 0.587; cutoff value, 13.05 ng/mL; sensitivity, 65.8%; specificity, 53.8%), and PGR (AUC, 0.752; cutoff, 5.65; sensitivity, 54.2%; specificity, 87.2%). The occurrence of GC was negatively correlated with serum levels of PGI (B = −0.054; OR = 0.947; 95% confidence interval [CI], 0.925–0.970; P Conclusions The combined detection of PGI, PGII, and PGR has important clinical value for the screening, prevention, and diagnosis of GC and could allow for earlier detection, diagnosis, and treatment of GC.

Microeunicellols A (1) and B (2), two undescribed eunicellin diterpenoids, were isolated from the culture of a bacterial symbiont, Streptomyces albogriseolus SY67903. Their structures, including ab...

Two main problems in partial nitrification at low temperature are its difficult start-up and easy inhibition, and this issue significantly limits the broad application of advanced partial nitrification technology. To promote rapid culturing and stabilization of partial nitrification biofilms, two experiments were carried out to investigate the influence of C/N on partial nitrification through ratio control (R, dissolved oxygen (DO)/effluent total ammonia nitrogen (TANeff)) in a lab-scale moving bed biofilm reactor (MBBR) at 12 °C. One experiment looked at the effect of C/N on the stable operation of partial nitrification at 12 °C, which inoculated a mature biofilm with good partial nitrification performance; the other looked at the effect of C/N on the biofilm cultivation and start-up of partial nitrification at 12 °C, which inoculated activated sludge and Pall rings. As for the startup experiments, only the C/N ratio was investigated, as the DO/TANeff ratio was kept constant. The effect of C/N on the mature partial nitrification biofilm showed that the increase of C/N could increase the resistance of partial nitrification to high DO. The nitrite accumulation ratio (NAR, (NO2−-N/(NO2−-N + NO3−-N) × 100%)) increased from 40.41% to 98.78% when R was 0.25 and C/N increased from 0 to 1. Meanwhile, when C/N increased to 2 and the R value slightly increased to 0.3, partial nitrification failed. The penetration of DO in the biofilm indicated that the increase of C/N enhanced the mass transfer resistance of DO, but it was similar when C/N was 1 and 2. Adding an appropriate amount of C/N can reduce the negative effect of partial nitrification damage caused by DO/TAN enlargement. The start-up experiment showed that C/N = 1 and C/N = 2 were essential for culturing biofilms and achieving partial nitrification at 12 °C, which can be successfully achieved (NAR was 85% and 96%) on the 56th and 52nd days. Neither C/N = 0 nor C/N = 3 could effectively achieve the partial nitrification. The increase of C/N will reduce the proportion of nitrifying bacteria in the biofilm, which is harmful to the partial nitrification. This will also lead to the decrease of protein/polysaccharide (PN/PS) and affect the stability of the biofilm.

The effect of the novel composite material LiNbO3@Fe3O4 on the nitrate removal, and Mn2+ oxidation efficiency by autotrophic denitrification strain Achromobacter sp. A14 was investigated in this st...

This study investigated the effects of substrate shock on the release of N-acyl-homoserine lactones (AHLs) in ANAMMOX granules and the relationship between AHL release, extracellular polymeric substance (EPS) excretion and the properties of granules. In this study, a transient high total nitrogen (TN) concentration of 2100 mg L−1 for 24 h stimulated a rapid increase in N-hexanoyl-DL-homoserine lactone (C6-HSL) and N-octanoyl-DL-homoserine lactone (C8-HSL) in ANAMMOX granules resulting in abundant EPS excretion and deterioration of granule settleability. Substrate shock didn't affect the distribution of AHLs in the system. C8-HSL was dominant in the biomass phase without EPS and C6-HSL was dominant in the EPS phase and the biomass phase without EPS. The results of the batch tests also confirmed that the high substrate concentration can stimulate the release of abundant C8-HSL and C6-HSL. Loosely-bound EPS (LB-EPS) excretion was closely related to the content of C8-HSL. A significant increase in C8-HSL from 222.6 to 1124.7 ng L−1 occurred when the substrate concentration increased from 200 to 1200 mg L−1, leading to excessive accumulation of LB-EPS, which was considered the key factor for the deterioration of granule stability after substrate shock. C6-HSL could stimulate the granule activity. Substrate shock triggered the release of abundant C8-HSL, leading to excessive accumulation of EPS, in which C6-HSL was dominant. So C6-HSL increased significantly after substrate shock, serving to maintain bacteria activity. From the perspective of AHL-based quorum sensing, this study confirmed that excessive EPS are usually excreted under extreme environmental stress to protect bacteria activity while this self-defense behavior also results in unstable reactor operation performance.

Around 5–10% of healthy vaccinees lack or produce an inadequate antibody response following receipt of a standard hepatitis B vaccination regimen. Studying immune response to hepatitis B vaccination could promote researches of immunological events contributing to this poor response. To address this, we investigated follicular helper T (Tfh) cells and firstly demonstrated similar kinetics between circulating Tfh (cTfh) cells and Tfh cells derived from mice spleen after hepatitis B vaccination. And cTfh cells were positively associated with anti-HBs at one week after vaccination (D7). Furthermore, we found PBMCs stimulated by HBsAg showed preferential activation of CXCR3(−) Tfh cells subsets in vitro. The expression of transcription factor BCL6 in CD4(+) T cell significantly differed between D7 and four weeks after vaccination (D28). However, dynamic curve of CD19(+) B cells tended to rise then fall but no significant trends were observed. Our findings revealed a decrease in cTfh cells and subset skewing contribute to reduced antibody responses in immune response to hepatitis B vaccination, which indicated the importance of Tfh cell in facilitating the optimization of vaccine efficacy.

In this study, strain CC76, identified as Enterobacter sp., was tested for the reduction of Fe3+ and denitrification using immobilized pellets with strain CC76 as experimental group (IP) and immobilized pellets with strain CC76 and magnetite powder as experimental group (IPM) in the autotrophic denitrification immobilized systems (ADIS). Compared with IP, a higher nitrate removal rate was obtained with IPM by using three levels of influent Fe3+ (0, 5, and 10 mg/L), four levels of pH (5.0, 6.0, 7.0, and 8.0), and three levels of hydraulic retention time (HRT) (12, 14, and 16 h), respectively. Furthermore, response surface methodology (RSM) analysis demonstrated that the optimum removal ratios of nitrate of 87.21% (IP) and 96.27% (IPM) were observed under the following conditions: HRT of 12 h, pH of 7.0 and influent Fe3+ concentration of 5 mg/L (IP) and 1 mg/L (IPM).

The activation of Janus kinase 1 (JAK1) has been reported to occur in non-small cell lung cancer (NSCLC), activating the JAK/signal transducers and activators of transcription cascade. However, the association between JAK1 activation and the prognostic value in NSCLC remains unclear. The present study initially investigated the association between expression of the activated form of JAK1 (p-JAK1) and prognosis in patients with NSCLC. A cohort of 142 resected primary NSCLC tissue samples, including 74 adenocarcinoma (ADCC) and 68 squamous cell carcinoma samples, were analyzed. p-JAK1 expression status was determined by immunohistochemistry. Evaluation of epidermal growth factor receptor (EGFR) gene amplification by fluorescence in situ hybridization was subsequently performed in 74 ADCC samples. The prognostic significance of p-JAK1 expression and EGFR gene amplification were evaluated with univariate and multivariate survival analyses. Compared with normal lung tissue, p-JAK1 expression level was significantly increased in NSCLC (P

Harmful algal blooms (HABs) could be deemed hazardous materials in aquatic environments. In this study, we investigated aerobic denitrification and algicidal effects of the Raoultella sp. R11. Based on PCR amplification, the denitrification genes nap A was detected. A response surface methodology (RSM) analysis demonstrated that the maximum nitrate removal ratio was 96.06%, and optimal conditions occurred for inoculum of 13.02% (v/v), initial pH of 6.65, C/N ratio of 7.59 and temperature of 27.08 °C based on a ridge analysis. When the Microcystis aeruginosa flour as carbon and nitrogen source for denitrification in aerobic conditions, the NO 3 − -N and TOC removal ratios were 72.36% and 85.56%, respectively. Additionly, the denitrification rate was 0.0123 mg L −1 h −1 . Furthermore, the infrared spectrogram and SEM images showed the M. aeruginosa cell structure was destroyed, indicating that R11 could use algal death for denitrification. Therefore, Raoultella sp. R11 played a crucial role for simultaneous denitrification and algicidal effects in wastewater treatment.

In this study, immobilization technique was employed to improve simultaneous algicidal and denitrification of immobilized Acinetobacter sp. J25 with magnetic Fe3O4 in eutrophic landscape water. After 7 days of operation, the maximum superoxide dismutase (SOD) activity (54.43 U mg-1), nitrate removal efficiency (100% (0.2127 mg L-1 h-1)), and chlorophyll-a removal efficiency (89.71%) were obtained from the immobilized J25 with magnetic Fe3O4. The results suggest that immobilized J25 with magnetic Fe3O4 had better nitrogen removal efficiency and algicidal activity in eutrophic landscape water. High-throughput sequencing data profiled the strain J25 that was immobilized with magnetic Fe3O4 which changed the composition of the microbial community. The results indicated a novel concept of enhancing the algicidal and denitrification property of immobilized bacteria with magnetic Fe3O4 in eutrophic landscape water.

Background Sepsis causes nutritional substrate malabsorption; hence, preventing gut barrier problems and improving the nutritional status in sepsis is a compelling issue. Aims We tested whether ghrelin administration affects peptide transporter 1 (PepT1) activity in the intestinal epithelium of rats with sepsis. Methods Sixty male Sprague-Dawley rats were randomly divided into sham-operated, sepsis, and ghrelin-treated groups. The cecum of sham-operated rats was separated after laparotomy without ligation and perforation. Sepsis group rats underwent cecal ligation and puncture (CLP). Mucosal specimens were used for immunohistochemstry, real-time PCR, and western blotting to detect PepT1 distribution, and mRNA and protein expression levels, respectively. TNF-α, IL-1β, and ghrelin levels were estimated in serum and intestinal mucosal tissue by ELISA. High-performance liquid chromatography was used to measure PepT1 uptake by the epithelial cells. Moreover, survival, body weight, and food intake of the rats were recorded during the 7-day treatment period. Results All rats in the sham-operated group survived, and 80% of rats in the sepsis group died within 7d of CLP. Treatment with ghrelin attenuated the CLP-induced body weight loss, intestine mucosa damage, and the survival rate was better. In addition, ghrelin attenuated increases in TNF-α and IL-1β production. The expressions of PepT1 mRNA and protein were higher in ghrelin-treated group rats than in sepsis rats. Moreover, the uptake function of PepT1 was better in ghrelin-treated group rats. Conclusion Ghrelin treatment can reduce the inflammatory response and greatly upregulate the physiological function of PepT1 in intestinal epithelial cells of rats with sepsis.

A possible correlation between liver cancer pain and the hypoxia-inducible factor (HIF)-1 and vascular endothelial growth factor (VEGF) expression levels was examined. From January, 2015 to January, 2016, 30 patients suffering from liver cancer with pain, 30 patients with liver cancer without pain and 30 hepatitis patients with pain were enrolled in the study. Pain level was evaluated by visual analogue scale (VAS), the expression levels of HIF-1 and VEGF mRNA were determined by RT-PCR and the expression levels of HIF-1 and VEGF proteins were examined by ELISA. Before intervention, the VAS in the hepatitis group was significantly higher than that of the liver cancer pain group. However, after intervention the VAS in the two groups was reduced. HIF-1 and VEGF mRNA expression levels in the liver cancer pain group were significantly higher than those in the liver cancer group before and after intervention. The expression levels of HIF-1 and VEGF mRNA in the hepatitis group were the lowest. The expression levels of HIF-1 and VEGF mRNA in the liver cancer pain group considerably increased after intervention. The expression levels of HIF-1 and VEGF mRNA in the other two groups showed no changes before or after intervention. Before and after the intervention, VAS in the liver cancer pain group was positively correlated to the expression levels of HIF-1 and VEGF. Thus, pain occurrence and the pain level in liver cancer patients were correlated with the expression levels of HIF-1 and VEGF. As the regular three-step medicine analgesic ladder is ineffective in these cases, verification of HIF-1 and VEGF expression levels may be considered the new target for pain release.

An immobilized biofilm reactor (IBR) was established to treat nitrate using different electron donors. A novel material, Fe3O4@Cu/PVA, was synthesized as an adsorbent and bacterial immobilized carrier in the reactor. The optimum condition of nitrate removal were pH 7.0, hydraulic retention time (HRT) of 10 h under autotrophic and mixotrophic conditions. Strain H-117 in the mixotrophic reactor had better adaptability to changes in the initial pH. The metabolism in the mixotrophic reactor was more vigorous than that in autotrophic reactor. The microbial communities and structures were evaluated to determine the nitrate removal mechanisms in this system. Microbial analyses demonstrated that different electron donor could influence the bacterial abundance and species in the IBR system. Proteobacteria was the most dominant phylum in all IBRs and accounted for more than 50% of the total phyla. Pseudomonas and Rhizobium were the dominant contributor to the effective removal of nitrate in the IBRs.

This study investigated whether bioaugmentation improves sulfamethoxazole (SMX) degradation and nitrogen removal in the Moving Bed Biofilm Reactor (MBBR) system. The effects of the C/N ratio on SMX degradation and nitrogen removal were also evaluated. Using MBBR system operation experiments, the bioaugmented reactor was found to perform more effectively than the non-bioaugmentation reactor, with the highest SMX, nitrate-N, and ammonia-N removal efficiencies of 80.49, 94.70, and 96.09%, respectively. The changes in the sulfonamide resistance genes and bacterial communities were detected at various operating conditions. The results indicate that the diversity of the bacterial communities and the abundance of resistance genes were markedly influenced by bioaugmentation and the C/N ratio, with Achromobacter among the dominant genera in the MBBR system. The bio-toxicity of samples, calculated as the inhibition percentage (IP) toward Escherichia coli, was found to decrease to non-toxic ranges after treatment.

MicroRNAs (miRNAs or miRs) are small, non-coding RNA molecules that play significant roles in numerous diseases. However, there is limited information regarding the plasma expression of miRNAs in patients with primary biliary cirrhosis (PBC) as well as the potential role of miRNAs in the development of PBC. miRNA microarray analysis was performed using plasma obtaind from three patients with PBC and three healthy controls. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the differential expression of miRNAs in the plasma and peripheral blood mononuclear cells (PBMCs) isolated from 20 patients with PBC, 20 patients with chronic hepatitis B (CHB) and 20 healthy controls. These miRNAs in PBMCs and plasma were validated by linear regression analyses. The T cell subset frequency was analyzed by flow cytometry. Correlations between altered miRNA expression and the frequency of the T cell subsets were determined by linear regression analyses. The co-expression of miRNAs and IL-17A was examined using fluorescence in situ hybridization (FISH) and immunohistochemistry. The microarray analysis identified sixteen miRNAs that were differentially expressed. Four miRNAs were validated by RT-qPCR. The expression pattern of miR-572 and miR-92a in the PBMCs correlated with the expression pattern in plasma. We also found that miR-92a expression closely correlated with the frequency of a subset of IL-17-producing T helper cells (Th17), and that miR-92a was co-expressed with IL-17A in patients with PBC. Taken together, these findings revealed that plasma from patients with PBC has a unique miRNA expression profile. Moreover, miR-92a may play an important role in the pathogenesis of PBC by regulating Th17 cell differentiation.

Background HOXB9 is a homeobox transcription factor which plays an important role in carcinoma development. This protein has been shown to inhibit cancer cell proliferation. However, the mechanisms that underpin HOXB9-mediated inhibition of cellular proliferation remain to be elucidated. Methods In this study, two gastric cancer cell lines, SGC7901 and MKN45, were transfected with plasmids pLVX-HOXB9 and shHOXB9. These transfections resulted in the over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells and knockdown of the HOXB9 gene in the MKN45/shHOXB9 cells. Results Over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells caused an increase in the apoptotic rate and a concomitant reduction in metastatic ability compared with the knocked-down MKN45/shHOXB9 cells. Moreover, a reduction in the expression of the phosphorylated-Akt protein was observed in the SGC7901/HOXB9 cells, while an increase in expression of the same protein was observed in the MKN45/shHOXB9 cells. We also observed that HOXB9 mediated a reduction in both NF-κB and N-cadherin and Snail protein expression. Conversely, HOXB9 caused an increase in the expression of E-cadherin. Conclusions In summary, this study reports that HOXB9 can suppress both phosphorylated-Akt expression and NF-κB activity. The latter phenomenon affects Snail protein expression and the inhibition of gastric carcinoma proliferation.

The aim of this study was to identify algicidal bacteria J25 against the Microcystis aeruginosa (90.14%), Chlorella (78.75%), Scenedesmus (not inhibited), and Oscillatoria (90.12%). Meanwhile, we evaluate the SOD activity and efficiency of denitrification characteristics with Acinetobacter sp. J25. A novel hybrid bioreactor combined biological floating bed with bio-contact oxidation (BFBO) was designed for treating the landscape water, and the average removal efficiencies of nitrate-N, ammonia-N, nitrite-N, TN, TP, TOC, and algal cells were 91.14, 50, 87.86, 88.83, 33.07, 53.95, and 53.43%, respectively. A 454-pyrosequencing technology was employed to investigate the microbial communities of the BFBO reactor samples. The results showed that Acinetobacter sp. J25 was the dominant contributor for effective removal of N, algal cells, and TOC in the BFBO reactor. And the relative abundance of Acinetobacter showed increase trend with the delay of reaction time. Graphical abstract Biological floating bed and bio-contact oxidation (BFBO) as a novel hybrid bioreactor designed for simultaneous removal Microcystis aeruginosa, TOC, nitrogen, and phosphorus. And high-throughput sequencing data demonstrated that Acinetobacter sp. J25 was the dominate species in the reactor and played key roles in the removal of N, TOC, and M. aeruginosa. Proposed reaction mechanism of the BFBO.

PepT1 can transport bacterial oligopeptide products and induce intestinal inflammation. Our aim was to investigate the mechanism of the small intestine injury induced by bacterial oligopeptide product muramyl dipeptide (MDP) which is transported by PepT1. We perfused the jejunum with a solution with or without MDP, or with a solution of MDP + Gly-Gly and explored the degree of inflammation to determine the role of PepT1–Nod2 signaling pathway in small intestine mucosa. MDP perfusion induced inflammatory cell accumulation and intestinal damage, accompanied by an increase in mucosal Nod2 and Rip2 transcript expression. NFκB activity and inflammatory cytokine expression, including serum levels of TNF-α, IL-1β, and IL-6, increased in the MDP group compared to the controls; these effects were reversed by perfusion of the nutritional dipeptide Gly-Gly. MDP can be transported through PepT1, causing inflammatory damage in the rat small intestine. Nod2–Rip2–NFκB signaling involved in the small intestinal inflammatory injury caused by MDP which is transported through PepT1.

Lanostane-type triterpenoids, inotolactones A and B, a drimane-type sesquiterpenoid, inotolactone C, and five known terpenoids 6β-hydroxy-trans-dihydroconfertifolin, inotodiol, 3β,22-dihydroxyanosta-7,9(11),24-triene, 3β-hydroxycinnamolide, and 17-hydroxy-ent-atisan-19-oic acid, were isolated from the submerged culture of chaga mushroom, Inonotus obliquus. Their structures were characterized by spectroscopic methods, including MS and NMR (1D and 2D) spectroscopic techniques. Inotolactones A and B, examples of lanostane-type triterpenoids bearing α,β-dimethyl, α,β-unsaturated δ-lactone side chains, exhibited more potent alpha-glucosidase inhibitory activities than the positive control acarbose. This finding might be related to the anti-hyperglycemic properties of the fungus and to its popular role as a diabetes treatment. In addition, a drimane-type sesquiterpenoid and an atisane-type diterpenoid were isolated from I. obliquus.

Chronic infection with hepatitis B virus (HBV) plays a significant role in hepatocellular carcinoma development. To investigate the effect of hepatitis B virus X protein (HBx) on inflammatory cytokines of human T cell, a eukaryotic expression vector, HBx-pEGFP-C1, was constructed and transfected into the Jurkat human T-cell line. Jurkat cells were transfected transiently using Lipofectamine 2000 and activated by phytohemagglutinin (PHA). Interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-4, IL-10, IL-13, and IL-14 mRNA was measured. The results showed that the vector HBx-pEGFP-C1 was successfully constructed, and HBx was expressed in Jurkat cells. Compared with a control group, mRNA of IL-1β and TNF-α was significantly elevated in the HBx-pEGFP-C1 group (p < 0.05), while IL-4, IL-10, IL-13, and IL-14 mRNA was decreased (p < 0.05). Therefore, transient overexpression of HBx promoted PHA-induced pro-inflammatory cytokine secretion and repressed anti-inflammatory cytokine secretion in human T cells.

Two new polyketide derivatives, huaspenones C and D (1 and 2, resp.), were isolated from the cultures of an endophytic fungus Peyronellaea sp. HS-12, derived from the stems of Huperzia serrata. They share N-bearing furan-3(2H)-one backbone, and 2 has an unprecedented furo[3,2-c]pyridine skeleton. Their structures including the absolute configuration were elucidated by extensive spectroscopic analysis combined with quantum-chemical calculations. (2E,4E)-6-hydroxy-2-methylocta-2,4-dienoic acid (3), a key intermediate of the biosynthesis of 1 and 2, was also obtained from the endophyte.

Inflammatory and immune responses, as well as melatonin secretion, are affected by circadian regulation. Abnormal circadian rhythm of melatonin release has been reported to be associated with the later stages of sepsis; however, its role in the early stages of sepsis is unclear. We studied 11 septic and 11 non-septic patients in our intensive care unit (ICU). Peripheral blood was drawn at 4-h intervals on the first day, beginning at 2:00 p.m., over a total period of 24 h. Plasma levels of melatonin, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were measured by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA). Messenger RNA levels of circadian genes Cry-1 and Per-2 were analyzed using quantitative real-time PCR. Results show the circadian rhythm of melatonin secretion was altered in the early stages of sepsis. The melatonin secretion acrophase occurred earlier in septic patients at 6:00 p.m., compared with at 2:00 a.m. in non-septic ICU patients. Compared with the non-septic group, both Cry-1 and Per-2 expression were significantly decreased while TNF-α and IL-6 expression were significantly increased in septic patients [TNF-α, 64.1 (43.6-89.1) vs. 11.4 (10.4-12.5) ng/ml; IL-6, 41.2 (35.7-50.8) vs. 19.1 (16-136.7) ng/ml; median (range), both P=0.04]. The peak concentrations of TNF-α and IL-6 were shown to be in concordance with the rhythm of melatonin secretion. The circadian rhythm of melatonin secretion and circadian gene expression were altered in the early stages of sepsis, which likely led to the changes in pro-inflammatory cytokine release. These findings shed light on the potential link between circadian rhythm and the progression of early-stage sepsis.

Background Ghrelin is a hormone that protects against hypoxic injury of cardiac cells by inducing autophagy, but the role of autophagy in sepsis remains unclear. This study aimed to evaluate whether ghrelin could enhance autophagy in rats with intestinal sepsis. Methods The cecal ligation and perforation (CLP) method was used to induce sepsis in Sprague-Dawley rats. The rats were assigned to four groups: normal group, sham-operated group, sepsis group, and Ghrelin-treated group. Sera and small intestinal tissues were collected from all groups. The sepsis was evaluated by histological analysis, and autophagy of small intestinal epithelial cells was assessed by electron microscopy, immunofluorescence, and biochemical methods. Results The expression of autophagy-associated proteins such as LC3, Atg 7 and Beclin 1 increased by 8 h post-CLP and declined to basal levels by 12 h post-CLP. The expression of LC3, Atg 7 and Beclin 1 in Ghrelin-treated rats was higher than that in rats with sepsis. Furthermore, compared to rats with sepsis, Ghrelin-treated rats showed significantly reduced intestinal mucosa injury at 20 h post-CLP. Conclusion Autophagy is induced in the early stages of sepsis. Ghrelin could enhance the autophagy of intestinal epithelial cells in rats with sepsis and protect the small intestinal epithelium against sepsis-induced injury.

CD36, a scavenger receptor, plays an important role in the progression of atherosclerosis through its interaction with oxidized low-density lipoprotein (ox-LDL). Porphyromonas gingivalis (P. gingivalis, Pg) has been shown to promote macrophage-derived foam cell formation by affecting the expression of CD36. However, the regulatory role of CD36 in macrophages infected with Pg remains largely unknown. Therefore, the aim of the present study was to explore the molecular mechanism of Pg induced CD36 expression in macrophages. Our results showed that Pg promoted ox-LDL uptake by macrophages and the formation of foam cells. Pg infection increased CD36 mRNA and protein levels in ox-LDL-untreated macrophages. Moreover, small interferon RNA (siRNA) targeting CD36 significantly reduced foam cell formation induced by Pg. Additionally, Pg stimulated nuclear translocation of p65, which directly bound to the promoters of CD36 to facilitate its transcription. Inhibition of p65, NF-κB or ERK1/2 blocked Pg-induced CD36 production; whereas, overexpression of NF-κB subunits p65 and p50 upregulated CD36. Furthermore, Ras inhibitors significantly attenuated ERK1/2 activation and CD36 expression. Taken together, the data indicated that stimulation of the ERK/NF-κB pathway by Pg led to transactivation of the CD36 promoters, thereby upregulating CD36 expression in the infected macrophages. These findings may help design new treatment strategies in atherosclerosis.

Purpose Unbalanced inflammatory response and lymphocyte apoptosis is associated with high mortality in septic patients. Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, is an anti-inflammatory and anti-apoptotic factor. Recently, DcR3 expression was found to be increased in septic patients. This study evaluated the therapeutic effect and mechanisms of DcR3 on cecal ligation and puncture (CLP)-induced sepsis in mice. Methods C57BL/6 mice were subjected to CLP-induced polymicrobial sepsis. DcR3 Fc was intravenously injected 30 min before and 6 h after CLP. Bacterial clearance, cytokine production, histology, lymphocyte apoptosis and survival were evaluated. Furthermore, we investigated the systemic effects of DcR3 in in vitro lymphocyte apoptosis regulation. Results Our results demonstrated that DcR3 protein treatments significantly improved survival in septic mice (p

International audience; Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B 6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9 A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked α/β domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.

Rubrerythrin (Rr) is a non-heme iron protein isolated from anaerobic sulfate-reducing bacteria. Rr is a dimeric molecule, each monomer contains a Fe(SCys)4 center in the C-terminal domain and a binuclear metal center in the N-terminal domain. Rr structures with different protein sources and/or preparation procedures have been studied. Two Rr crystal structures have been solved with significant differences in their binuclear metal centers. The first structure, which was obtained from expressed protein under aerobic conditions, has a diiron–oxo center. The second structure, which was obtained from native protein of Desulfovibrio vulgaris under aerobic conditions, has an Fe–Zn center with the zinc position differing from the corresponding iron position in the former structure by approximately 2 A. The crystal structures of Rr isolated from D. vulgaris (Hildenborough, NCIB 8303), the same as the second structured but prepared under anaerobic conditions, are reported in this paper. The binuclear metal center in these structures is an Fe–Zn center. When the crystal was exposed to air, the zinc atom moved gradually, approximately 2 A, accompanied by the entrance of a water molecule (or hydroxyl group) and changes in the binuclear metal center microenvironment. This finding can explain the differences between the two different structures. The results suggest that the zinc movement may be related to the enzymatic activity of Rr. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0400-0. On that page (frame on the left side), a link takes you directly to the supplementary material.

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.

Zinc finger motif widely exists in protein structure, which can play different roles in different proteins. RecR is an important recombination mediator protein (RMP) in the RecFOR pathway and zinc finger motif is the most conserved domain in RecR protein. However, the function of this zinc finger motif in RecR is unclear. Here, we have studied the structures of the single cysteine and double cysteines mutation within the zinc finger motif in Thermoanaerobacter tengcongensis RecR (TTERecR). We have also studied the DNA binding ability as well as TTERecO protein binding ability of single, double and even triple cysteines mutation of the zinc finger motif, and the mutants do not alter DNA binding by RecR nor the interaction between RecR and RecO. The function of TTERecR zinc finger motif is to maintain the stability of the three-dimensional structure.

In the present study, we aimed to develop and validate a rapid and sensitive, Alu-based real-time PCR method for the detection of circulating cell-free DNA (cfDNA). This method targeted repetitive elements of the Alu reduplicative elements in the human genome, followed by signal amplification using fluorescence quantification. Standard Alu-puc57 vectors were constructed and 5 pairs of specific primers were designed. Valuation was conducted concerning linearity, variation and recovery. We found 5 linear responses (R1–5=0.998–0.999). The average intra- and inter-assay coefficients of variance were 12.98 and 10.75%, respectively. The recovery was 82.33–114.01%, with a mean recovery index of 101.26%. This Alu-based assay was reliable, accurate and sensitive for the quantitative detection of cfDNA. Plasma from normal controls and patients with myocardial infarction (MI) were analyzed, and the baseline levels of cfDNA were higher in the MI group. The area under the receiver operating characteristic (ROC) curve for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu (Alu1 + Alu2 + Alu3 + Alu4 + Alu5) was 0.887, 0.758, 0.857, 0.940, 0.968 and 0.933, respectively. The optimal cut-off value for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu to predict MI was 3.71, 1.93, 0.22, 3.73, 6.13 and 6.40 log copies/ml. We demonstrate that this new method is a reliable, accurate and sensitive method for the quantitative detection of cfDNA and that it is useful for studying the regulation of cfDNA in certain pathological conditions. Alu4, Alu5 and Alu showed better sensitivity and specificity for the diagnosis of MI compared with cardiac troponin I (cTnI), creatine kinase MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH). Alu5 had the best prognostic ability.

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2(1) and cell parameters a =108.0 A, b= 117.0 A, c = 184.0 A, beta= 90.2 degrees and 12(alphabeta) units in the asymmetric unit was obtained by using (NH(4))(2)SO(4) as precipitant. These crystals diffract up to 2.8 A. Form II crystal obtained by using PEG4000 as precipitant belongs to space group P6(3) with unit cell constants a = 155.0 A, c = 40.3 A, gamma =120.0 degrees and one(alphabeta) unit in the asymmetric unit. The crystals diffract beyond 2.9 A. The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.

Using the crystal structure of allophycocyanin from cyanobacteriumSpirulina platensis (APC-SP) as a search model, the crystal structure of allophycocyanin from red algaePorphyra yezoensis (APC-PY) has been studied by molecular replacement methods. The APC-PY crystals (Form 3) belong to the space group of R32, cell dimensions a =b = 10.53 nm,c = 18.94 nm, alpha= beta = 90 degrees , gamma= 120 degrees ; there is one alphabeta monomer in each crystallographic asymmetric unit in the cell. The translation function search gave a unique peak with a correlation coefficient (Cc) of 67.0% and an R-factor of 36.1 % for reflection data from 1.0 to 0.4 nm. Using the results by molecular replacement, the initial model of APC-PY was built, and the coincidence of the chromophore in APC-PY initial model with its2F (o)- F(c) OMIT map further confirms the results by molecular replacement.

Anaplastic lymphoma kinase (ALK) translocation-positive adenocarcinoma of the lung is a newly recognized molecular subgroup. Limited data on the clinicopathological features of this entity in the Chinese population are available. We performed immunohistochemical staining for the ALK protein and fluorescence in situ hybridization detection of the ALK translocation. We enrolled 793 Chinese patients with lung adenocarcinoma and identified 54 ALK translocation-positive patients (6.8%) in the group. Compared with the entire group of patients, ALK translocation-positive patients were younger (P < .01) and more likely to be nonsmokers (P = .017), but presented with a higher percentage of advanced-stage disease (P = .022) and lymph node metastases (P = .006). ALK translocation-positive patients more commonly exhibited poorly differentiated tumor histology and a predominantly solid tumor growth pattern relative to the ALK translocation-negative patients. Morphologically, ALK translocation was associated with extracellular mucus secretion, a mucinous cribriform structure, and signet ring cell (SRC) components. ALK translocation was present in 42.5% and 34.0% of adenocarcinomas with SRC components or wild-type EGFR, respectively. ALK translocation, occurring at a frequency of 6.8% in Chinese patients, defines a unique molecular subgroup of lung tumors. Fluorescence in situ hybridization should be performed in each case of lung adenocarcinoma with SRC components or wild-type EGFR to identify ALK translocation-positive patients.

Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine673. Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme. OA Förderung 2013

RecR is an important recombination mediator protein in the RecFOR pathway. RecR together with RecO and RecF facilitates RecA nucleoprotein filament formation and homologous pairing. Structural and biochemical studies of Thermoanaerobacter tengcongensis RecR (TTERecR) and its series mutants revealed that TTERecR uses the N-N dimer as a basic functional unit to interact with TTERecO monomer. Two TTERecR N-N dimers form a ring-shaped tetramer via an interaction between their C-terminal regions. The tetramer is a result of crystallization only. Hydrophobic interactions between the entire helix-hairpin-helix domains within the N-terminal regions of two TTERecR monomers are necessary for formation of a RecR functional N-N dimer. The TTERecR N-N dimer conformation also affects formation of a hydrophobic patch, which creates a binding site for TTERecO in the TTERecR Toprim domain. In addition, we demonstrate that TTERecR does not bind single-stranded DNA (ssDNA) and binds double-stranded DNA very weakly, whereas TTERecOR complex can stably bind DNA, with a higher affinity for ssDNA than double-stranded DNA. Based on these results, we propose an interaction model for the RecOR:ssDNA complex.

Crystal structures of the AHL-lactonase AidH in complex with substrate and product are reported at high resolution and a catalytic mechanism is proposed for the metal-independent AHL-lactonase., Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell–cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-­hexanoyl homoserine lactone, N-hexanoyl homoserine and N-­butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid–base covalent catalysis.

Pyridoxal kinase (ATP:pyridoxal 5'-phosphotransferase; EC 2.7.1.35) is a key enzyme in the transformation of vitamin B(6) to pyridoxal-5'-phosphate. Pyridoxal-5'-phosphate is the crucial cofactor required by numerous enzymes involved in the metabolism of amino acids and the synthesis of many neurotransmitters. Pyridoxal kinase from sheep brain was crystallized in an orthorhombic form using the hanging-drop vapour-diffusion method with sodium citrate as the precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 59.8, b = 94.4, c = 128.2 A, and diffract to a resolution of 2.1 A. Crystals were transferred into a soaking liquid without citrate and two heavy-atom derivatives were prepared.

Manganese superoxide dismutase (GP-MnSOD), a component of the so-called `green protein' (green protein complex) from the facultative anaerobic halodenitrifier Bacillus halodenitrificans , has been crystallized using the hanging-drop vapor diffusion method. Crystals have unit-cell parameters a = b =93.4 A, c =65.0 A, and belong to the space group P4 3 2 1 2. Preliminary analysis indicates there is one monomer in each asymmetric unit. The structural information from this enzyme will enrich our knowledge on its high catalytic activity and its possible role in green protein complex.

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 A crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

Methylmalonyl-CoA is an important metabolic intermediate that exists in several key degradative and biosynthetic pathways.1 The pathway responsible for the degradation of branched amino acids and odd chain fatty acids involves three steps. In the first step, a biotindependent carboxylation produces the S-epimer of methylmalonyl-CoA. The third step, the B12-dependent methylmalonyl-CoA mutase converts the R-epimer of methylmalonyl-CoA to succinyl-CoA.1 This key epimerization between the two steps is carried out by the essential enzyme methylmalonyl-CoA epimerase (MMCE: EC number 5.1.99.1), which catalyzes the conversion of (2S)methylmalonyl-CoA to (2R)-methylmalonyl-CoA. In prokaryotes, MMCE participated in autotrophic CO2 fixation via the 3-hydroxypropionate pathway, in propionate fermentation, the regeneration of glyoxylate and in the biosynthesis of polyketide antibiotics.2–6 In animals, defects of this pathway can result in a buildup of methylmalonic acid, which causes severe acidosis and also damages the central nervous system.7,8 In this study, we determined the crystal structure of TTE0360 from Thermoanaerobacter tengcongensis to 2.0 A resolution using single-wavelength anomalous dispersion (SAD) by short cryo-soaking with a high concentration of potassium iodide.9,10 In GenBank database, the gene tte0360 is predicted to be glyoxalase I, a member of glyoxalase superfamily. However, the TTE0360 structure is very different than the released structures of glyoxalase I. A search for the 3D homologs using DALI11 shows that TTE0360 has significant structural similarity with the MMCE from Propionibacterium shermanii,12 which is also a member of glyoxalase superfamily. The fold type of the TTE0360 molecule belongs to the vicinal-oxygen-chelate superfamily (VOC).13 The similarities of structure folds, dimer formation, and active sites between TTE0360 and MMCE, suggest that the protein TTE0360 may be the MMCE from T. tengcongensis.

Tetrahydropalmatine in Rhizoma corydalis and its preparations were separated and determined with no pretreatment in the buffer solution of 50 mmol/L of sodium acetate in methanol containing 2 mol/L acetic acid.

The urotensin II (UII)/UII receptor (UT) system is closely related to immune inflammation. In acute liver failure (ALF), the UII/UT system can promote the production and release of proinflammatory cytokines, inducing an inflammatory injury response in liver tissue. However, the mechanism by which the hepatic UII/UT system promotes proinflammatory cytokine production and release is not clear. To solve this problem, we used primary Kupffer cells (KCs) as the model system in the current study. The results showed that after lipopolysaccharide (LPS) stimulation, KCs showed significantly increased expression and release of UII/UT and proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Pretreatment with urantide, which is a UT receptor antagonist, significantly inhibited the LPS-stimulated expression and release of UII/UT, TNF-α, and IL-1β by KCs. In addition, LPS stimulation induced nuclear p38 mitogen-activated protein kinase (MAPK) protein phosphorylation and expression of the nuclear nuclear factor κB (NF-κB) p65 subunit in KCs and enhanced the binding activity of NF-κB to DNA molecules, whereas urantide pretreatment significantly inhibited the LPS-stimulated nuclear expression and activity of these molecules in KCs. Therefore, our conclusion is that the UII/UT system mediates LPS-stimulated production and release of proinflammatory cytokine by KCs, and this mediating effect at least partially relies on the inflammatory signaling pathway molecules p38 MAPK and NF-κB.

Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn2+. This constitutes an essential step in the synthesis of pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, a cofactor for over 140 enzymes. (R)-Roscovitine (CYC202, Seliciclib) is a relatively selective inhibitor of cyclin-dependent kinases (CDKs), currently evaluated for the treatment of cancers, neurodegenerative disorders, renal diseases, and several viral infections. Affinity chromatography investigations have shown that (R)-roscovitine also interacts with PDXK. To understand this interaction, we determined the crystal structure of PDXK in complex with (R)-roscovitine, N6-methyl-(R)-roscovitine, and O6-(R)-roscovitine, the two latter derivatives being designed to bind to PDXK but not to CDKs. Structural analysis revealed that these three roscovitines bind similarly in the pyridoxal-binding site of PDXK rather than in the anticipated ATP-binding site. The pyridoxal pocket has thus an unexpected ability to accommodate molecules different from and larger than pyridoxal. This work provides detailed structural information on the interactions between PDXK and roscovitine and analogs. It could also aid in the design of roscovitine derivatives displaying strict selectivity for either PDXK or CDKs.

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis–trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a β-barrel covered by two α-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes . The mutant has no enzyme activity. Its crystal structure determined at 1.65 A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the Ne atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.