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Angiogenesis plays a vital role in tumor progression and metastasis. To better understand the role of anesthesia in tumor biology, we previously reported that bupivacaine displayed the inhibitory effects in endothelial cells. In this work, we demonstrated that fentanyl, an opioid medication commonly used in cancer patients, stimulated tumor angiogenesis. We found that fentanyl at nanomolar concentrations significantly stimulated capillary network formation of human lung tumor-associated endothelial cell (HLT-EC) in a similar manner as vascular endothelial growth factor (VEGF), and furthermore that the stimulatory effect of fentanyl was mainly involved in early stage of HLT-EC vascular structure assembly. Particularly, fentanyl significantly increased HLT-EC growth and migration. Fentanyl also protected HLT-EC from apoptosis induced by growth factor withdrawal. In contrast, the same concentrations of fentanyl did not affect human lung cancer cell growth and survival. Fentanyl stimulated migration of some but not all tested human lung cancer cells. Mechanism analysis suggested that fentanyl activates multiple pro-angiogenic signaling pathways, including VEGFR2/FAK/PI3K/Akt and small GTPases. Our work systematically demonstrates that fentanyl stimulates tumor angiogenesis via activating multiple pro-angiogenic signaling pathways. Our findings highlight the potential adverse effect of fentanyl in cancer patients.

Supplemental Material, sj-pdf-1-iji-10.1177_03946320221084835 for Roles of C-reactive protein polymorphisms and life event changes on cognitive function in bipolar patients receiving valproate by Po See Chen, Li-Yi Tang and Hui Hua Chang in International Journal of Immunopathology and Pharmacology

A practical procedure for the addition of arylboronic acids to aromatic aldehydes has been developed in the presence of NiCl2(PPh3)2/1,3-bis-(2,6-diisopropylphenyl)imidazolium chloride (IPr·HCl) system with good yield. Generally, electron-rich and -neutral aryl aldehydes showed excellent reactivity and provided desired products in high yields. This procedure will provide new way to the synthesis of diarylmethanols.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis. There is a necessary search for improvement in diagnosis and treatment methods to improve the prognosis. Some useful prognostic markers of HCC are still lacking. Pyroptosis is a type of programmed cell death caused by the inflammasome. It is still unknown whether pyroptosis-related genes (PRGs) are involved in the prognosis in HCC. The gene expression and clinical data of LIHC (liver hepatocellular carcinoma) patients were downloaded from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium database (ICGC). In this study, we identified 40 PRGs that were differentially expressed between LIHC and normal liver tissues. Based on the TCGA-LIHC cohort, a 9-gene prediction model was established with the Least absolute shrinkage and selection operator (LASSO)-penalized Cox regression. The risk score was calculated according to the model in the TCGA-LIHC cohort and the ICGC-LIHC cohort. Utilizing the median risk score from the TCGA cohort, LIHC patients from the ICGC-LIHC cohort were divided into two risk subgroups. The Kaplan–Meier (KM) survival curves demonstrated that patients with lower risk scores had significantly favorable overall survival (OS). Combined with the clinical characteristics, the risk score was an independent factor for predicting the OS of LIHC patients in both the TCGA-LIHC cohort and the ICGC-LIHC cohort. Functional enrichment and immune function analysis were carried out. Furthermore, a nomogram based on risk score, age, gender, and tumor stage was used to predict mortality of patients with LIHC. Moreover, KM survival analysis was performed for 9 genes in the risk model, among which CHMP4A, SCAF11, and GSDMC had significantly different results and the ceRNA network was constructed. Based on the core role of SCAF11, we performed loss-of-function experiments to explore the function of SCAF11 in vitro. Suppression of SCAF11 expression inhibited the proliferation, attenuated the migration and invasion, and induced apoptosis of liver cancer cell lines. In conclusion, the pyroptosis-related model and nomogram can be utilized for the clinical prognostic prediction in LIHC. This study has demonstrated for the first time that SCAF11 promotes the progression of liver cancer.

Objective This study aimed to clarify the mechanism by which the long non-coding RNA cancer susceptibility candidate 9 (CASC9) alleviates sepsis-related acute kidney injury (S-AKI). Methods A lipopolysaccharide (LPS)-induced AKI model was established to simulate S-AKI. HK-2 human renal tubular epithelial cells were treated with LPS to establish an in vitro model, and mice were intraperitoneally injected with LPS to generate an in vivo model. Subsequently, the mRNA expression of inflammatory and antioxidant factors was validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) production was assessed using an assay kit. Apoptosis was detected by western blotting and fluorescence-activated cell sorting. Results CASC9 was significantly downregulated in the LPS-induced AKI model. CASC9 attenuated cell inflammation and apoptosis and enhanced the antioxidant capacity of cells. Regarding the mechanism, miR-424-5p was identified as the downstream target of CASC9, and the interaction between CASC9 and miR-424-5p promoted thioredoxin-interacting protein (TXNIP) expression. Conclusions CASC9 alleviates LPS-induced AKI in vivo and in vitro, and CASC9 directly targets miR-424-5p and further promotes the expression of TXNIP. We have provided a possible reference strategy for the treatment of S-AKI.

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds, and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than the diphenolase activity. In the present study, a novel tyrosinase with comparable monophenolase and diphenolase activities was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residues 262 and 266 in the substrate inhibition and activity modulation of the enzyme, and the D262N variant showed an enhanced monophenolase/diphenolase catalytic efficiency ratio compared to the wild-type enzyme.

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17β-HSD members, 17β-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17β-HSD1 in complex with estrone (E1) and cofactor analog NADP+ , demonstrating critical enzyme-substrate-cofactor interactions. These complexes revealed a reversely bound E1 in 17β-HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His221 is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead-end complex, which exists widely in NADP(H)-preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17β-HSD1 through the formation of a dead-end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17β-HSD1 based on the dead-end inhibition model for efficiently targeting EDDs. DATABASES: Coordinates and structure factors of 17β-HSD1-E1 and 17β-HSD1-E1-NADP+ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively. ENZYMES: 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) EC 1.1.1.62.

Uric acid (UA) is a major antioxidant molecule in the human blood, and it has been linked with cell longevity. However, it is unclear whether serum UA levels are associated with red blood cell (RBC) indexes. This cross-sectional study included 10,759 Chinese subjects, recruited from the Shanghai Xuhui Central Hospital from January 2014 to December 2017. The participants were categorized into gender groups and then further divided into three different subgroups according to their UA reference range as follows: low (male (UA<0.202 mmol/l), female (UA<0.143 mmol/l)), normal (male (0.417 mmol/l>UA≥0.202 mmol/l), female (0.339 mmol/l>UA≥0.143 mmol/l)), and high (male (UA≥0.417 mmol/l), female (UA≥0.339 mmol/l)). The associations of UA levels with RBC parameters were analyzed using 1-way ANOVA, Pearson correlations, and multivariate linear regression. The levels of mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, RBCs, and hemoglobin were lowest in the low UA group, followed by the normal UA group and high UA group (p<0.001). Pearson analysis showed that there was a statistically significant correlation between UA levels with mean corpuscular hemoglobin, mean corpuscular hemoglobin concentrations, mean corpuscular volumes, RBC counts, and hemoglobin (p<0.05). Multiple linear regression analysis suggested that there were statistically significant positive correlations between UA levels and RBC counts (B=0.245, p<0.001, 95% CI=0.003 to 0.092), as well as UA levels and hemoglobin concentrations (B=0.138, p<0.001, 95% CI=0.002 to 0.082). Furthermore, similar results were observed in both the male and female subgroups. The serum UA levels may be independently associated with RBC parameters, regardless of sex, and UA may protect RBCs owing to its antioxidant effect.

Cyclin-dependent kinases (CDKs) are historic therapeutic targets implicated in tumorigenic events due to their critical involvement in the cell cycle phase. However, selectivity has proven to be a bottleneck, causing repeated failures. Previously, we reported CR6-interacting factor 1 (CRIF1), acting as a cell cycle negative regulator through interaction with CDK2. In the current report, we identified the CRIF1-CDK2 interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on CRIF1 using 40 678 drug-like compounds. These compounds were tested by cell proliferation assay, and four of these molecules were found to selectively inhibit the proliferation of osteosarcoma (OS) cell lines, but do not affect normal bone mesenchymal stem cells (BMSC). A binding study reveals significant affinities of the inhibitors on CRIF1. More importantly, treatment of the OS cells with a combination of ionizing radiation (IR) and the best-performing inhibitors remarkably increased IR inhibition potential from 19.9% to 59.6%. This occurred by selectively promoting G2/M arrest and apoptosis related to CDK2 overactivation in OS cells but not in BMSC and was supported by significant CDK2 phosphorylation modifications. Knocking down of CRIF1 by siRNA treatment showed similar effects to the interface inhibitors. Together we substantiate the identification of novel lead molecules, which may provide a new treatment to overcome selectivity issues and enhance the radiosensitivity of tumor cells, opening a conceptually novel strategy of CDK-targeting for different cancer types.

With increasing application of PEGylated products, drawbacks are beginning to emerge such as the "PEG dilemma". Other promising materials may need to be found in the current situation. Endogenous polysialic acid (PSA), which is highly expressed on mammalian, bacterial, and malignant surface, may be a promising material in oncology. In this study, a dual-responsive amphiphilic PSA cholesterol derivative (PSA-CS-CH) was synthesized to explore the opportunity of PSA in targeted drug delivery systems. PSA-CS-CH, F127 mixed micelles (PF-M), and pure F127 micelles (F-M) were prepared for comparative antitumor experiments. The in vitro experiments showed that modification of PSA-CS-CH significantly increased cytotoxicity and cellular uptake. PF-M had excellent tumor microenvironment response release behavior on acidic media with high GSH levels. The in vivo fluorescence imaging and antitumor experiments showed that PF-M had excellent tumor targeting ability and great tumor suppression ability. In summary, biodegradable PSA may contribute to cancer therapy.

Because of the lack of anatomical landmarks during reduction of multiple articular surfaces and fragments in comminuted patellar fractures, loss of bone fragments or aggravation of soft tissue and ligament injuries readily occurs. In the present case, we used multiple three-dimensional (3D)-printed guide plates to reduce and fix a comminuted patellar fracture. A 22-year-old man was hospitalized for 2 days because of left knee joint pain and limited movement caused by a traffic accident. Preoperative imaging revealed a comminuted fracture of the left patella (type 34-C3 according to the AO/OTA classification). Throughout a 2-year follow-up, the patient remained in generally good condition with no significant limitation of his left knee joint activity. Application of multiple 3D-printed guide plates is a safe and effective auxiliary technique for the treatment of comminuted patellar fractures. This novel technique can shorten the operation time, reduce the number of fluoroscopic procedures, and ensure fracture healing and recovery of knee joint function through reliable reduction of the articular surface.

Inulinase is a member of the glycoside hydrolase family 32 (GH32). It catalyzes the randomly hydrolyzation of 2,1-β-D-fructosidic linkages in inulin and plays a role in the production of high-fructose syrup. In this study, detailed roles of the conserved residues W79, F113, M117, R181, C239, and W334 of the exo-inulinase from Kluyveromyces cicerisporus CBS4857 (KcINU1) in substrate binding and stabilization were evaluated by in silico analysis and site-directed mutagenesis. These residues belong to the conserved WG, FSGSMV, RDP, ECP, and WQY regions of the GH32 and are located around the catalytic pocket of KcINU1. Zymogram assay showed relatively weaker band for F113W and similar band for M117A compared to the wild-type enzyme toward inulin and sucrose, whereas all other variants showed no observable stain on the native polyacrylamide gel electrophoresis. These results were further confirmed with the dinitrosalicylic acid colorimetric method. It showed that the residual activities of F113W toward inulin and sucrose were 33.8 ± 3.3% and 96.2 ± 5.5%, respectively, and that of M117A were 103.8 ± 1.3% and 166.5 ± 12%, respectively. Results from fluorescence spectra indicated that there is a significant conformational change that happened in F113W compared to the wild-type enzyme, while M117A exhibited limited impact although the quenching effect was increased.

A distinct interaction pattern of lytic polysaccharide monooxygenases (LPMOs) with their insoluble substrate, cellulose, was revealed through the combination of computational and biochemical approaches. The results indicated that the enzymes can stably bind on the flat hydrophobic surface of cellulose via the interactions of the key residues located in the axis across the conserved distal tyrosine residue and copper ion with two adjacent cellulose chains. Further studies on the correlation of substrate binding and H

Alginate is a family of industrially important linear polymers consisting of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The function of alginate is closely related to the ratio of M/G. Mannuronan C-5 epimerase, which converts M to G, is a key enzyme involved in the biosynthesis of alginate. A new mannuronan C-5 epimerase isolated from Pseudomonas mendocina. sp. DICP-70 named PmC5A was characterized in this study. From the 1H NMR analysis of the products, we have found that PmC5A possesses alginate lyase function in addition to mannuronan C-5-epimerase. The optimal pH and temperature of lyase and epimerase were found to be 8.0, 9.0 and 40 °C, 30 °C, respectively. PmC5A also shows lyase activity toward PolyMG and G-blocks.

There is much interest in using chickens as “bioreactors” to produce large quantities of biopharmaceuticals. However, transient expression of foreign genes have been known to cause low efficiency of obtaining transgenic offspring, especially when using nonviral vectors. In present study, a transgenic chicken model was investigated to determine whether an exogenous gene can be expressed stably and transferred to its offspring through a matrix attachment region (MAR)-mediated non-viral vector using the eGFP marker gene. The eukaryotic expression vector pEGFP-N1-MAR, which contains the eGFP gene and MAR, was constructed and transfected into a chicken stage-X blastoderm to produce a G0 generation of transgenic chickens. The hatchabilities of different injection regions were tested; 18 of the 40 eggs injected with pEGFPN1- MAR in the area opaca hatched after 21 days of incubation, and had a hatchability rate of 45%. By contrast, eggs injected at the area pellucida did not hatch. Results from the fluorescence signal detection and polymerase chain reaction (PCR) verified that four hatched chicks from the G0 generation expressed the eGFP gene. Furthermore, fluorescence signal detection results indicated that 2 of the 65 chicks from the G1 generation expressed the eGFP gene. We conclude that MAR facilitates the production of transgenic chickens; pEGFP-N1-MAR application is a novel approach that can produce transgenic chicken offspring.

Type 1 diabetes (T1D) is an autoimmune disease characterized by a selective destruction of insulin-secreting β-cells. Both T cells and B cells serve a crucial role in pathogenesis and development of T1D. CD20 is a specific membrane antigen of B lymphocytes, while interleukin (IL)-10 is an important cytokine secreted by T helper 2 cells and has a short half-life in vivo. The combined effect of anti-CD20 and IL-10 on immune function of mice with T1D remains unknown. In the present study, 30 non-obese diabetic (NOD) mice were treated with anti-CD20 and adenoviral vector-mediated interleukin-10 (Ad-mIL-10) therapy. Alterations in CD4+, CD8+, CD4+CD25+Foxp3+ T cells, T-box expressed in T-cells (T-bet), GATA-binding protein-3 (GATA-3) interferon-γ (IFN-γ) and IL-4 were detected by flow cytometry, reverse transcription-quantitative polymerase chain reaction in NOD mice spleen tissue. The present results suggested that anti-CD20 and IL-10 treatment in NOD mice can modulate the immune functions by upregulating GATA-3 and IL-4 expression as well as downregulating T-bet and IFN-γ expression, which are involved in the pathogenesis of T1D. The current findings may provide a potential method for T1D treatment and a novel preventive therapy for T1D. Combination of anti-CD20 and Ad-mIL-10 treatment had not only immune regulatory effects but also protective effects on islet β-cells in NOD mice with T1DM at the early stages, by regulating T-bet/GATA-3 expression and Th1/Th2 cell differentiation, which has the potential for diabetes prevention and therapy.

In spite of the significant progress of estrogen-dependent breast cancer (BC) treatment, aromatase inhibitor resistance is a major problem limiting the clinical benefit of this frontier endocrine-therapy. The aim of this study was to determine the differential expression of steroid-converting enzymes between tumor and adjacent normal tissues, as well as their correlation in modulating intratumoral steroid-hormone levels in post-menopausal estrogen-dependent BC. RNA sequencing dataset (n = 1097) of The-Cancer-Genome-Atlas (Breast Invasive Carcinoma) retrieved through the data portal of Genomic Data Commons was used for differential expressions and expression correlation analyses by Mann-Whitney U and Spearman's rank test, respectively. The results showed significant up-regulation of 17β-HSD7 (2.50-fold, p 0.0001) in BC, supporting its effect in sex-hormone control. Besides, suppression of 11β-HSD1 expression (-8.29-fold, p 0.0001) and elevation of 11β-HSD2 expression (2.04-fold, p 0.0001) provide a low glucocorticoid environment diminishing BC anti-proliferation. Furthermore, 3α-HSDs were down-regulated (-1.59-fold, p 0.01; -8.18-fold, p 0.0001; -33.96-fold, p 0.0001; -31.85-fold, p 0.0001 for type 1-4, respectively), while 5α-reductases were up-regulated (1.41-fold, p 0.0001; 2.85-fold, p 0.0001; 1.70-fold, p 0.0001 for type 1-3, respectively) in BC, reducing cell proliferation suppressers 4-pregnenes, increasing cell proliferation stimulators 5α-pregnanes. Expression analysis indicates significant correlations between 11β-HSD1 with 3α-HSD4 (r = 0.605, p 0.0001) and 3α-HSD3 (r = 0.537, p 0.0001). Significant expression correlations between 3α-HSDs were also observed. Our results systematically present the regulation of steroid-converting enzymes and their roles in modulating the intratumoral steroid-hormone levels in BC with a vivid 3D-schema, supporting novel therapy targeting the reductive 17β-HSD7 and proposing a new combined therapy targeting 11β-HSD2 and 17β-HSD7.

The obesity-asthma phenotype is characterized by increased asthma severity and decreased glucocorticoid responsiveness. To date, the mechanism underlying the association between obesity and asthma remain to be fully elucidated. The present study investigated the correlation between oxidative stress and the nuclear factor (NF)-κB pathway in obese asthmatic mice. The animals were divided into the following groups: Control (n=8), comprising C57BL/6J mice without exposure to a high-fat diet; non-obese asthma group (n=8), comprising mice of a normal weight subjected to the induction of asthma; obese control group (n=8), comprising C57BL/6J mice subjected to a high-fat diet; and obese asthmatic group (n=8), comprising obese mice subject to the induction of asthma. The levels of the malondialdehyde (MDA) oxidant and glutathione (GSH) antioxidant in the lungs and bronchoalveolar lavage fluid (BALF) were measured using ELISA. The expression levels of inhibitory κB kinase-β (IKK-β) and the inhibitor of κBα (IκB-α) in the pulmonary tissues was determined using western blot analysis. An electrophoretic mobility shift assay was performed to determine the transcription activity of NF-κB. The levels of MDA in the BALF and lung tissues increased significantly in the two asthmatic groups, compared with the control groups (P

Androgen receptor (AR) signaling is essential for prostate cancer (PC) progression and treatment. Experiments have demonstrated that the intratumoral androgen levels are not affected by circulating androgen levels, but rather modulated by local steroid-converting enzyme activities. The expression modulation status of human steroid-converting enzymes and nuclear receptors are of great promise to identify novel therapeutic targets. Meta-analysis was performed with 9 cohorts (1093 specimens) from Gene Expression Omnibus, 16 cohorts (933 specimens) from Oncomine and the TCGA cohort (550 specimens). We found significant up regulation of 5α-reductase type 1 and type 3 in both primary and metastatic PC, together with the down regulation of AKR1C2 in primary PC, contributing to the high intratumoral DHT levels. The expression of AR in metastatic PC was up regulated, indicating the importance of AR signaling in the progression of this cancer. The down regulations of HSD11B1 and NR3C1 in primary and metastatic PC may diminish the anti-inflammation and anti-proliferation effects of glucocorticoids signaling. Furthermore, the decrease of progesterone receptor (PGR) expression in primary and metastatic PC was also observed, relieving the suppression effect of PGR on PC proliferation. The clinical evidences of the remarkable expression modulation of steroid-converting enzymes and receptors in PC may indicate novel combined treatment against this highly incident cancer.

Konjac glucomannan is safely and widely used in the functional food industry. Compared with glucomannan, glucomannan oligosaccharides have higher water solubility making them easier to absorb and utilize. These oligosaccharides are also associated with many biological activities. A novel glucomannan hydrolase PpGluA with high activity and substrate specificity which releases a series of konjac glucomannan oligosaccharides having DP 2–9 is described here. Selenium is one of the essential trace elements for humans. The selenium-modified glucomannan oligosaccharides were prepared by the sodium selenite-nitric acid method for the first time. Antitumor activity studies demonstrated that selenium-modified glucomannan oligosaccharides had better activity than inorganic sodium selenite and glucomannan oligosaccharides and inhibited tumor by inducing apoptosis through the mitochondrial pathway. This study suggests that selenium-modified glucomannan oligosaccharides may be have potential as a new functional food for inhibition of tumors.

Acetoin (3-hydroxy-2-butanone) is an important four-carbon compound widely used in the food industry and other industrial applications. This study aimed to identify a novel butanediol dehydrogenase that can efficiently catalyze the formation of acetoin. A novel butanediol dehydrogenase, BtBDH, was identified from Bacillus thuringiensis subsp. Kurstaki ACCC 10066, and its enzymatic properties were characterized. The optimum pH and temperature for the oxidation activity of BtBDH were 10.0 and 50 °C, respectively, and those for the reduction activities were 7.5 and 35 °C, respectively. In addition, it exhibits stability over a wide pH range (6–10) and temperatures up to 70 °C. BtBDH showed good stability after storage for 3 months at 4 °C. Moreover, ethylenediaminetetracetic acid (EDTA) inhibits the enzymatic activity of BtBDH, indicating that the enzyme is metal-dependent. This study characterized a novel (2R,3R) −2,3-butanediol dehydrogenase. Its excellent oxidation activity and stability ensure its great industrial application potential in the production of acetoin.

A convenient and environmental benign synthesis of biaryls has been demonstrated by a straightforward reaction catalyzed by the palladium-containing metal-organic framework (Pd-MOF) [Pd (2-pymo)2]n (2-pymo = 2-pyrimidinolate). A series of functionalized biaryl derivatives have been synthesized in good to excellent yields by the Suzuki-miyaura cross-couplings of sustainable arenediazonium salts with a variety of arylboronic acids and the reactions were catalyzed by the Pd-MOF using methanol as a benign solvent. Those base- and additive-free catalytic reactions proceeded smoothly under non-anhydrous and non-degassed condition. Such transformation avoided high reaction temperature, tolerated many functional groups and presented a wide substrate scope. The catalyst could be recovered by filtration and reused for four successive cycles before collapse of the MOF structure.

Supplementary_Tables for Association of Single-Nucleotide Polymorphisms in Monoubiquitinated FANCD2-DNA Damage Repair Pathway Genes With Breast Cancer in the Chinese Population by Fei-Yu Chen, Hao Wang, Hui Li, Xue-Li Hu, Xu Dai, Shou-Man Wang, Guo-Jiao Yan, Ping-Lan Jiang, Yuan-Ping Hu, Juan Huang, and Li-Li Tang in Technology in Cancer Research & Treatment

Motivation: Recovering the gene structures is one of the important goals of genome assembly. In low-quality assemblies, and even some high-quality assemblies, certain gene regions are still incomplete; thus, novel scaffolding approaches are required to complete gene regions. Results: We developed an efficient and fast genome scaffolding method called PEP_scaffolder, using proteins to scaffold genomes. The pipeline aims to recover protein-coding gene structures. We tested the method on human contigs; using human UniProt proteins as guides, the improvement on N50 size was 17% increase with an accuracy of ∼97%. PEP_scaffolder improved the proportion of fully covered proteins among all proteins, which was close to the proportion in the finished genome. The method provided a high accuracy of 91% using orthologs of distant species. Tested on simulated fly contigs, PEP_scaffolder outperformed other scaffolders, with the shortest running time and the highest accuracy. Availability and Implementation: The software is freely available at http://www.fishbrowser.org/software/PEP_scaffolder/ Contact: lijt@cafs.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Under LC-TOF-MS fraction-guided analysis, the n-butanol was extracted from Bidens pilosa. Three bioactivity isomers compounds, including methyl 3,4-dicaffeoyl quinate (1), methyl 3,5-dicaffeoyl quinate (2), and methyl 4,5-dicaffeoyl quinate (3) were successfully isolated by countercurrent chromatography (CCC) with two phase solvent system composed of chloroform-methanol-water-n-buthanol (4:3:2:0.5, v/v) elution. Their purities were 93.7%, 82.4%, and 91.8% determined by HPLC. HR-ESI-MS and NMR was used to confirm their structures. This is the first time the isolation of geometrical isomers of methyl dicaffeoyl quinates by CCC has been reported.

17beta-hydroxysteroid dehydrogenase type 7 (17β-HSD7) promotes breast cancer cell growth via dual-catalytic activity by modulating estradiol and DHT. Here, we clarified the expression pattern of 17β-HSD7 in postmenopausal luminal A type breast cancer with The Cancer Genome Atlas (TCGA) cohort. The impact of 17β-HSD7 inhibition on the proteome of MCF-7 cells was investigated and on cell apoptosis was revealed. MCF-7 cells were treated with an efficient inhibitor of 17β-HSD7 (INH7) or with vehicle, and a differential proteomics study was performed using two-dimensional (2D) gel electrophoresis followed by mass spectrometry and ingenuity pathway analysis (IPA). Cell apoptosis was analyzed by flow cytometry, followed by reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot to investigate the expression of apoptosis-related genes. Our data showed 17β-HSD7 is amplified in primary and progressive breast cancer, inhibition of 17β-HSD7 in MCF-7 cells modulated 104 proteins primarily involved in cell death/survival, cell growth and DNA processing. The expression of 78 kDa glucose-regulated protein (GRP78) and anti-apoptosis factor Bcl-2 were significantly suppressed via 17β-HSD7 inhibition with INH7, consequently induced MCF-7 cell apoptosis. However, INH7 treatment of T47D, another widely used epithelial ER+ breast cancer cell line, led to an up-regulation of GRP78 expression, resulting in a limited increase in apoptosis. These results suggest cell-specific effects of INH7 in the breast cancer, which is interesting for further study. An combinatory effect on apoptosis by INH7 and Letrozole (aromatase inhibitor) was further demonstrated in MCF-7. Down-regulation of GRP78 via 17β-HSD7 inhibition enhances cell apoptosis in response to Letrozole. This study highlights GRP78 as a key regulator related to 17β-HSD7 inhibition and effect. Taken together, results from the present study suggest a hypothesis that inhibition of 17β-HSD7 would be a complementary strategy to Letrozole by suppression of GRP78 in ER+ breast cancer. However, from a research perspective, further studies have to be carried out with more breast cancer cell lines as well as in vivo model to assess the efficacy of inhibitor combination.

Water erosion is the major reason for the loss of soil organic carbon in the Northeast China, which leads to the soil quality deterioration and adjacent water pollution. In this study, the effect of extraction temperature, pH value, and salt on the water extractable organic matter (WEOM) was determined by means of the UV absorbance, fluorescence excitation-emission matrix, and derived fluorescence indexes. In general, the carbon content and aromaticity of WEOM increased with the increasing of extraction temperature, with the exception that there was no significant difference in the amount at 0 and 20°C. More fluorophores, especially microbially-derived organic matter were extracted at high temperature. The pH values of extractant, including 5, 7, and 10, showed no effect on the carbon amount of WEOM, whereas the aromaticity and microbially-derived component gradually increased with the increasing of pH values. The fluorescence intensity of humic acid-like fluorophore was stronger in neutral and alkali condition than that in acidic condition. The addition of 10 mmol L−1 CaCl2 significantly decreased the carbon amount of recovered WEOM. Moreover, it significantly decreased the aromaticity of WEOM and the quantity of fulvic acid-like and humic acid-like fluorophores, whereas increased the percentage of tyrosine-like and tryptophan-like fluorophores in the total fluorophores and the amount of microbially-derived organic matter. Generally, 10 mmol L−1 KCl showed the same influence trend, but with low influence degree.

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response., Author Summary Ebola virus (EBOV), a highly virulent pathogen, causes a severe hemorrhagic fever syndrome. The fatal infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is thought to contribute to disease pathogenesis, as high amounts of shed GP from virus-infected cells are detected in patients, and activate macrophages and dendritic cells (DCs) to produce proinflammatory cytokines. Here, we show that LSECtin plays an important role in GP-mediated inflammatory responses in human DCs. LSECtin is a DAP12-coupled receptor able to initiate specific signaling events in human DCs. LSECtin interacts with Ebola GP and results in DAP12 phosphorylation. LSECtin knockdown impairs the production of proinflammatory cytokines induced by Ebola GP. Thus, this study suggests that LSECtin may contribute to Ebola GP-mediated pathogenicity.

The protein elicitor from Alternaria tenuissima (PeaT1) presented excellent thermotolerance and potential application in agriculture as a pesticide. Previous synchrotron radiation circular dichroism study demonstrated that the secondary structures in PeaT1 protein are reversible with temperature change. To further clarify the mechanism of its thermotolerance, synchrotron radiation small angle x-ray scattering (SAXS) technique was used to study the shape change of PeaT1 protein with temperature in this article. Ab initio structure restorations based on the SAXS data revealed that PeaT1 protein has a prolate shape with a P2 symmetry axis along the prolate anisometric direction. With temperature increase, a gooseneck vase-like (25 degrees C), to jug-like (55 degrees C), then to oval (85 degrees C) shape change can be found, and these shape changes are also approximately reversible with temperature decrease. PeaT1 protein contains two homogenous molecules, and each of them consists of F, NAC, T, and UBA domains. The structures of the four domains were predicted. Simulated annealing algorithm was used to superimpose the domain structures onto the SAXS shapes. It was found that all the structural domains have position rotation and translation with temperature change, but the NAC domains are relatively stable, playing a role of frame. This shape change information provides clues for further exploring its biological function and application. Proteins 2013. (c) 2012 Wiley Periodicals, Inc.

Accumulation of reserve materials in filling grains involves the coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice (Oryza sativa) is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 d after flowering (DAF); thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout eight sequential developmental stages from 6 to 20 DAF and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation, whereas the down-regulated proteins in the process were involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis, and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.

Common carp is a widely cultivated fish with longer than 2,000 years domestication history, due to its strong environmental adaptabilities, especially hypoxia tolerance. The common carp genome has experienced a very recent whole genome duplication (WGD) event. Among a large number of highly similar duplicated genes, a pair of Ras-associated binding-GTPase 1a (Rab1a) genes were found fast diverging. Four analogous Rab1a genes were identified in the common carp genome. Comparisons of gene structures and sequences indicated Rab1a-1 and Rab1a-2 was a pair of fast diverging duplicates, while Rab1a-3 and Rab1a-4 was a pair of less diverged duplicates. All putative Rab1a proteins shared conserved GTPase domain, which enabled the proteins serve as molecular switches for vesicular trafficking. Rab1a-1 and Rab1a-2 proteins varied in their C-terminal sequences, which were generally considered to encode the membrane localization signals. Differential expression patterns were observed between Rab1a-1 and Rab1a-2 genes. In blood, muscle, spleen, and heart, the mRNA level of Rab1a-1 was higher than that of Rab1a-2. In liver and intestine, the mRNA level of Rab1a-2 was higher. Expression of Rab1a-1 and Rab1a-2 showed distinct hypoxia responses. Under severe hypoxia, Rab1a-1 expression was down-regulated in blood, while Rab1a-2 expression was up-regulated in liver. Compared with the less diverged Rab1a-3/4 gene pair, common carp Rab1a-1/2 gene pair exhibited strong characteristics of sub-functionalization, which might contribute to a sophisticated and efficient Ras-dependent regulating network for the hypoxia-tolerant fish.

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate (Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502) and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography - mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae , Plasmodium ovale and Plasmodium vivax have been expressed in recombinant form, and properties of the active site of each enzyme characterized by kinetic analysis. A panel of chromogenic peptide substrates systematically substituted at the P3, P2, P2′ and P3′ positions was used to estimate enzyme/ligand interactions in the corresponding enzyme subsites based upon kinetic data. The kinetic parameters k cat , K m and k cat / K m were measured to identify optimal substrates for each enzyme and also sequences that were readily cleaved by the plasmepsins but poorly by host aspartic peptidases. Computer generated models were utilized to compare enzyme structures and interpret kinetic results. The orthologous plasmepsins share highly similar subsite specificities. In the S3 and S2 subsites, the plasmepsin 4 orthologs all preferred hydrophobic amino acid residues, Phe or Ile, but rejected charged residues such as Lys or Asp. In S2′ and S3′ subsites, these plasmepsins tolerated both hydrophobic and hydrophilic residues. Subsite specificities of the plasmepsin 4 family of orthologs are similar to those of human cathepsins D and E, except in S3′ where the plasmepsins accept substrates containing Ser significantly better than either of these human aspartic proteases. Peptidomimetic methyleneamino reduced-peptide inhibitors, which have inhibition constants in the picomolar range, were prepared for each plasmepsin 4 ortholog based upon substrate preferences. A peptidomimetic inhibitor designed for plasmepsin 4 from P. falciparum having Ser in P3′ had the lowest K i of the series of inhibitors prepared, but did not significantly improve the selectivity of the inhibitor for plasmepsin 4 versus human cathepsin D.

Type 1 diabetes is an autoimmune disorder, which occurs due to β cell damage. Interleukin (IL)-10, a pleotropic cytokine, has been reported to have anti‑inflammatory, immunosuppressive and immunostimulatory properties. Administration of IL‑10 is known to prevent autoimmune diabetes in non‑obese diabetic (NOD) mice. However, the mechanism of IL‑10‑induced protection in NOD mice requires further investigation. The aim of the present study was to evaluate the protective effect of transgenic IL‑10 expression in pancreatic β cells against autoimmune damage in NOD mice and to elucidate its mechanism of action. Female NOD mice (9 weeks old) were intraperitoneally injected with an adenovirus carrying either IL‑10 (Adv‑IL‑10) or green fluorescent protein (Adv‑GFP). Blood glucose was monitored weekly and the expression of IL‑10 was evaluated using reverse transcription quantitative polymerase chain reaction. IL‑10 and interferon (IFN)‑γ expression levels in serum and splenocytes were analyzed. CD4+CD25+FoxP3+ T regulatory (Treg) cells were determined by flow cytometry. Apoptosis of pancreatic β cells was determined using a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick‑end labeling assay and expression levels of Fas and caspase‑3 were estimated by immunohistochemistry analysis. The results revealed that mice treated with IL‑10 showed less severe insulitis and a lower incidence of diabetes compared with the saline control and Adv‑GFP groups. In addition, compared with the control group, IFN‑γ levels were decreased in sera and splenocytes, while IL‑10 expression was increased in sera only. The number of CD4+CD25+FoxP3+ Treg cells was increased in IL‑10‑injected mice. Furthermore, the expression levels of Fas and caspase‑3 were decreased in IL‑10‑injected mice compared with that of GFP‑injected and control mice, which was concomitant with a reduction in β cell apoptosis. In conclusion, the present study demonstrated that IL‑10 gene transfer reduced the expression of the inflammatory cytokines, attenuated pancreatic insulitis and inhibited β cell apoptosis. This therefore indicated that IL-10 reduced the incidence of diabetes in female NOD mice.

Objective To investigate the protective effects of combined intervention with adenovirus vector mediated interleukin 10 (IL-10) and insulin-like growth factor 1 (IGF-1) genes on islet β cells in nonobese diabetes (NOD) mice with type 1 diabetes mellitus (T1D) at early stage. Methods Twenty-four female NOD mice at onset of diabetes and aged 17–20 weeks old were randomly divided into four groups. Mouse 1, 2 and 3 groups were intraperitoneally injected 0.1 ml of Ad-mIGF-1, Ad-mIL-10, and combined Ad-mIGF-1 and Ad-mIL-10, respectively. Mouse 4 group were used as diabetes control. In addition, six age- and sex-matched non-diabetic NOD mice were intraperitoneally injected 0.1 ml of PBS and assigned 5 group as normal controls. All mice were weekly monitored for body weight, urine glucose and blood glycose, and sacrificed 3 weeks after injection. Their serum levels of IL-10, IGF-1, IFN-γ, IL-4 and C-peptide were measured and the degree of insulitis and the local expression of IGF-1 and IL-10 gene were observed. Results 1) IL-10 and IGF-1 levels in serum and pancreas were enhanced in 1, 2, and 3 groups; 2) serum INF-γ level was decreased while serum IL-10 and IL-4 levels were increased in 1, 2 and 3 groups, and these alterations were more significant in 3 group than 1 and 2 groups (P

1 Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China 2 Center for Bioinformatics, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China 3 Department of Neurology, Center for Genetic Medicine, Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, 303 E. Superior, Chicago, IL 60611, USA 4 Graduate University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100039,China ✉ Correspondence: jane-wu@northwestern.edu (J. Y. Wu), weilp@mail.cbi.pku.edu.cn (L. Wei)

Corynebacterium glutamicum uses 4-cresol as sole carbon source for growth. Protocatechuate 3,4-dioxygenase activity had been detected when C. glutamicum was grown with 4-cresol. In this work, we found that 4-cresol was catabolized via 4-hydroxybenzoate and protocatechuate as intermediate metabolites, and a genetic cluster called cre (designated for 4-cresol, creABCDEFGHIR, tagged as ncgl0521-ncgl0531 in NCBI) was identified. The cre gene cluster comprises of 11 genes, and six of them were experimentally confirmed to be involving in 4-cresol catabolism. The genes creD, creE, and creJ were involved in oxidation of 4-cresol into 4-hydroxybenzyl alcohol. The creD encoded a protein showing Mg(2+)-dependent phosphohydrolase activity. The genes creE, creF, creJ encoded a putative P450 system. The creG encoded a NAD(+)-dependent dehydrogenase and catalyzed 4-hydroxybenzyl alcohol to 4-hydroxybenzaldehyde. Two other genes creH and creI were involved in conversion of 4-hydroxybenzyl alcohol to 4-hydroxybenzoate, but their catalytic function is still unknown. Similar genetic clusters with high DNA sequence identity were identified in Arthrobacter and additional Corynebacterium species, suggesting that this genetic organization for 4-cresol catabolism might be more widely distributed in Gram-positive bacteria.

Genistein, a soybean-derived isoflavone, may contribute to the lower cancer incidence in South Asian countries. In this study, the effects and molecular mechanisms of genistein on growth and differentiation of B16-BL6 mouse melanoma cells were investigated. Genistein suppressed the growth of these melanoma cells. The IC50 value is 15.5 microM. On the other hand, genistein induced the changes of cell shape and cytoskeletal network. The cytoskeletal filaments were induced to form a bundle along the direction of elongation of the cells. Moreover, tyrosine phosphorylation levels of cytoskeleton-associated proteins decreased after the cells were exposed to 20 or 30 microM of genistein for 3 days. All these morphological and molecular changes were accompanied by appearance of the differentiated phenotypes. Genistein induced the increase of cellular melanin content, enhancement of tyrosinase activity, and decrease of colonization potentials in soft agar in a time-dependent and dose-dependent manner. The effective concentration was no more than 10 microM after 3 days' exposure. The tumorigenic potentials of B16-BL6 cells in C57BL/6 mouse also decreased after exposure to 20 or 30 microM of genistein for 3 days. When expressions of tumor-related genes were investigated in the differentiation-induced cells, the content of P53 dramatically increased while that of c-Myc protein decreased. Therefore, due to its ability to induce cellular and molecular changes, genistein suppressed the growth and induced differentiated phenotypes in B16-BL6 melanoma cells.

The rol (designated for r esorcin ol ) gene cluster rolRHMD is involved in resorcinol catabolism in Corynebacterium glutamicum , and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of the rol genes in C. glutamicum . The transcription start sites and promoters of rolR and rolHMD were identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription of rolHMD and of its own gene. Further, a 29-bp operator rolO was located at the intergenic region of rolR and rolHMD and was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR to rolO was affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR- rolO complex, thus releasing RolR from the complex and derepressing the transcription of rol genes in C. glutamicum . It is proposed that the binding of RolR to its operator rolO blocks the transcription of rolHMD and of its own gene, thus negatively regulated resorcinol degradation in C. glutamicum .

Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP(Cg), was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP (Cg), which is located upstream of the genetic cluster (ncgl1110 ~ ncgl1113) for resorcinol catabolism, was identified as a new L-Phe specific transporter from C. glutamicum RES167. The disruption of pheP (Cg) resulted in RES167∆ncgl1108, and this mutant showed decreased growth on L-Phe (as nitrogen source) but not on L-Tyr or L-Trp. Uptake assays with unlabeled and (14)C-labeled L-Phe and L-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in L-Phe uptake than RES167. Expression of pheP (Cg) in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 restored their ability to uptake for L-Phe and growth on L-Phe. The uptake of L-Phe was not inhibited by nine amino acids but by L-Tyr. The K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 for L-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min(-1) (mg DW)(-1), respectively, which are different from K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg)) for L-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min(-1) (mg DW)(-1)]. In conclusion, this PheP(Cg) is a new L-Phe transporter in C. glutamicum.

Rad21 and its meiotic counterpart Rec8, the key components of the cohesin complex, are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis, respectively. In contrast to yeast and vertebrates, which have only two RAD21/REC8 genes, the rice genome encodes four Rad21/Rec8 proteins. Here, we report on the cloning and characterization of OsRAD21-2 from rice (Oryza sativa L.). Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific Rad21 subfamily. Semi-quantitative reverse transcription-polymerase chain reaction revealed OsRAD21-2 preferentially expressed in premeiotic flowers. Further RNA in situ hybridization analysis and promoter::β-glucuronidase staining indicated that OsRAD21-2 was mainly expressed in actively dividing tissues including premeiotic stamen, stem intercalary meristem, leaf meristem, and root pericycle. Ectopic expression of OsRAD21-2 in fission yeast resulted in cell growth delay and morphological abnormality. Flow cytometric analysis revealed that the OsRAD21-2-expressed cells were arrested in G2 phase. Our results suggest that OsRad21-2 functions in regulation of cell division and growth.