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2.  Skyrmion-skyrmion and skyrmion-edge repulsions in skyrmion-based racetrack memory 
Scientific Reports  2015;5:7643.
Magnetic skyrmions are promising for building next-generation magnetic memories and spintronic devices due to their stability, small size and the extremely low currents needed to move them. In particular, skyrmion-based racetrack memory is attractive for information technology, where skyrmions are used to store information as data bits instead of traditional domain walls. Here we numerically demonstrate the impacts of skyrmion-skyrmion and skyrmion-edge repulsions on the feasibility of skyrmion-based racetrack memory. The reliable and practicable spacing between consecutive skyrmionic bits on the racetrack as well as the ability to adjust it are investigated. Clogging of skyrmionic bits is found at the end of the racetrack, leading to the reduction of skyrmion size. Further, we demonstrate an effective and simple method to avoid the clogging of skyrmionic bits, which ensures the elimination of skyrmionic bits beyond the reading element. Our results give guidance for the design and development of future skyrmion-based racetrack memory.
doi:10.1038/srep07643
PMCID: PMC4284505  PMID: 25560935
3.  Association between Genetic Polymorphism in the Swine Leukocyte Antigen-DRA Gene and Piglet Diarrhea in Three Chinese Pig Breeds 
The swine leukocyte antigen (SLA)-DRA locus is noteworthy among other SLA class II loci for its limited variation and has not been investigated in depth. This study was investigated to detect polymorphisms of four exons of SLA-DRA gene and its association with piglet diarrhea in Landrace, Large White and Duroc pigs. No polymorphisms were detected in exon 3, while 2 SNPs (c.178G>A and c.211T>C), 2 SNPs (c.3093A>C and c.3104C>T) and 5 SNPs (c.4167A>G, c.4184A>G, c.4194A>G, c.4246A>G and c.4293G>A) were detected in exon 1, exon 2 and exon 4 respectively, and 1 SNP (c.4081T>C) in intron 3. Statistical results showed that genotype had significant effect on piglet diarrhea, individuals with genotype BC had a higher diarrhea score when compared with the genotypes AA, AB, AC and CC. Futhermore, genotype AC had a higher diarrhea score than the genotype CC in exon 1 (p<0.05); diarrhea scores of genotype AA and BB were higher than those of genotypes AC and CC in exon 2 (p<0.05); individuals with genotype AA had a higher diarrhea score than individuals with genotype AB and BB in exon 4 (p<0.05). Fourteen common haplotypes were founded by haplotype constructing of all SNPs in the three exons, its association with piglet diarrhea appeared that Hap2, 5, 8, 10, and 14 may be the susceptible haplotypes and Hap9 may be the resistant haplotype to piglet diarrhea. The genetic variations identified of the SLA-DRA gene may potentially be functional mutations related to piglet diarrhea.
doi:10.5713/ajas.2013.13567
PMCID: PMC4150187  PMID: 25178364
Swine Leukocyte Antigen; DRA Gene; Association; Genetic Polymorphism; Haplotype; Piglet Diarrhea
5.  dCTP Pyrophosphohydrase Exhibits Nucleic Accumulation in Multiple Carcinomas 
Nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) functions as one of the mechanisms to guarantee the fidelity of DNA replication through the cleavage of non-canonical nucleotides into di- or monophosphates. Human NTP-PPase is poorly understood and investigated. In the present study, by using tissue microarrays with the paired cancer and adjacent regions, we found that with the prevalent expression of dCTP pyrophosphohydrase (DCTPP1) in the cytosol and nucleus in tumors investigated, DCTPP1 was inclined to accumulate in the nucleus of cancer cells compared to the paired adjacent tissue cells in multiple carcinomas including lung, breast, liver, cervical, gastric and esophagus cancer. More significantly, the higher DCTPP1 expression in the nucleus of lung, gastric and esophagus cancer cells was associated with histological subtypes. The nucleic accumulation of DCTPP1 was apparently observed as well when tumor cell line MCF-7 was treated with H2O2 in vitro. Considering the roles of DCTPP1 on restricting the concentration of non-canonical nucleotides in the nucleotide pool, accumulation of DCTPP1 in the nucleus of tumor cells might suffice for maintaining the proper DNA replication in order to fulfill the requirement for the survival and proliferation of tumor cells.
doi:10.4081/ejh.2013.e29
PMCID: PMC3794360  PMID: 24085278
dCTP pyrophosphohydrase; carcinomas; nucleic accumulation; immunohistochemistry; tissue microarrays
6.  Swine Leukocyte Antigen-DQA Gene Variation and Its Association with Piglet Diarrhea in Large White, Landrace and Duroc 
The swine leukocyte antigen class II molecules are possibly associated with the induction of protective immunity. The study described here was to investigate the relationship between polymorphisms in exon 2 of the swine DQA gene and piglet diarrhea. This study was carried out on 425 suckling piglets from three purebred pig strains (Large White, Landrace and Duroc). The genetic diversity of exon 2 in swine DQA was detected by PCR-SSCP and sequencing analysis, eight unique SSCP patterns (AB, BB, BC, CC, CD, BD, BE and DD) representing five specific allele (A to E) sequences were detected. Sequence analysis revealed 21 nucleotide variable sites and resulting in 12 amino acid substitutions in the populations. A moderate level polymorphism and significant deviations from Hardy-Weinberg equilibrium of the genotypes distribution were observed in the populations (p<0.01). The association analysis indicated that there was a statistically significant difference in the score of piglet diarrhea between different genotypes, individuals with genotype CC showed a lower diarrhea score than genotypes AB (0.98±0.09), BB (0.85±0.77) and BC (1.25±0.23) (p<0.05), and significantly low than genotype BE (1.19±0.19) (p<0.01), CC genotype may be a most resistance genotype for piglet diarrhea.
doi:10.5713/ajas.2013.13067
PMCID: PMC4093232  PMID: 25049886
SLA; DQA Gene; Genetic Polymorphism; Piglet Diarrhea
7.  PPARgamma inhibits hepatocellular carcinoma metastases in vitro and in mice 
British Journal of Cancer  2012;106(9):1486-1494.
Background:
We have previously demonstrated that peroxisome proliferator-activated receptor (PPARγ) activation inhibits hepatocarcinogenesis. We aim to investigate the effect of PPARγ on hepatocellular carcinoma (HCC) metastatic potential and explore its underlying mechanisms.
Methods:
Human HCC cells (MHCC97L, BEL-7404) were infected with adenovirus-expressing PPARγ (Ad-PPARγ) or Ad-lacZ and treated with or without PPARγ agonist (rosiglitazone). The effects of PPARγ on cell migration and invasive activity were determined by wound healing assay and Matrigel invasive model in vitro, and in an orthotopic liver tumour metastatic model in mice.
Results:
Pronounced expression of PPARγ was demonstrated in HCC cells (MHCC97L, BEL-7404) treated with Ad-PPARγ, rosiglitazone or Ad-PPARγ plus rosiglitazone, compared with control (Ad-LacZ). Such induction markedly suppressed HCC cell migration. Moreover, the invasiveness of MHCC97L and BEL-7404 cells infected with Ad-PPARγ, or treated with rosiglitazone was significantly diminished up to 60%. Combination of Ad-PPARγ and rosiglitazone showed an additive effect. Activation of PPARγ by rosiglitazone significantly reduced the incidence and severity of lung metastasis in an orthotopic HCC mouse model. Key mechanisms underlying the effect of PPARγ in HCC include upregulation of cell adhesion genes, E-cadherin and SYK (spleen tyrosine kinase), extracellular matrix regulator tissue inhibitors of metalloproteinase (TIMP) 3, tumour suppressor gene retinoblastoma 1, and downregulation of pro-metastatic genes MMP9 (matrix metallopeptidase 9), MMP13, HPSE (heparanase), and Hepatocyte growth factor (HGF). Direct transcriptional regulation of TIMP3, MMP9, MMP13, and HPSE by PPARγ was shown by ChIP-PCR.
Conclusion:
Peroxisome proliferator-activated receptor-gamma exerts an inhibitory effect on the invasive and metastatic potential of HCC in vitro and in vivo, and is thus, a target for the prevention and treatment of HCC metastases.
doi:10.1038/bjc.2012.130
PMCID: PMC3341869  PMID: 22472882
PPARγ; hepatocellular carcinoma metastasis; heparanase; matrix metallopeptidase; tissue inhibitors of metalloproteinase
8.  Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy 
British Journal of Cancer  2011;104(5):798-807.
Background:
Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues.
Methods:
To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines.
Results:
Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells.
Conclusion:
These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.
doi:10.1038/bjc.2011.12
PMCID: PMC3048207  PMID: 21304523
glioma; ferrochelatase; 5-aminolevulinic acid; fluorescence; photodynamic therapy
9.  Nucleolar protein CSIG is required for p33ING1 function in UV-induced apoptosis 
Li, N | Zhao, G | Chen, T | Xue, L | Ma, L | Niu, J | Tong, T
Cell Death & Disease  2012;3(3):e283-.
Cellular senescence-inhibited gene (CSIG) protein, a nucleolar protein with a ribosomal L1 domain in its N-terminus, can exert non-ribosomal functions to regulate biological processes, such as cellular senescence. Here, we describe a previously unknown function for CSIG: promotion of apoptosis in response to ultraviolet (UV) irradiation-induced CSIG upregulation. We identified p33ING1 as a binding partner that interacts with CSIG. After UV irradiation, p33ING1 increases its protein expression, translocates into the nucleolus and binds CSIG. p33ING1 requires its nucleolar targeting sequence region to interact with CSIG and enhance CSIG protein stability, which is essential for activation of downstream effectors, Bcl-2-associated X protein, to promote apoptosis. Thus, our data imply that p33ING1–CSIG axis functions as a novel pro-apoptotic regulator in response to DNA damage.
doi:10.1038/cddis.2012.22
PMCID: PMC3317352  PMID: 22419112
apoptosis; CSIG; p33ING1; Bax; nucleolar protein; UV irradiation
10.  Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters 
Journal of structural biology  2009;169(3):450-454.
Human leukotriene C4 synthase (LTC4S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions.
It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC4S. Here we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC4S.
Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.
doi:10.1016/j.jsb.2009.11.002
PMCID: PMC2826519  PMID: 19903529
Two-dimensional crystallization; membrane protein; eukaryotic; structure; electron crystallography; cryo-EM
11.  An Unexpected Counter-Regulatory Role of IL-10 in B-Lymphocyte-Mediated Transplantation Tolerance 
Monoclonal antibody against the CD45RB protein induces stable transplantation tolerance to multiple types of allograft. We have previously established that this tolerance protocol relies on the regulatory function of B lymphocytes for its effect. B lymphocytes have also been reported to participate in immune regulation in several other settings. In most of these systems, the regulatory function of B lymphocytes depends on the production of IL-10. Therefore, we investigated the role of IL-10 in the anti-CD45RB model of B-cell-mediated transplantation tolerance. Surprisingly, using antibody-mediated neutralization of IL-10, IL-10-deficient recipients and adoptive transfer of IL-10-deficient B lymphocytes, we found that IL-10 actually counter-regulates tolerance induced by anti-CD45RB. Furthermore, neutralization of IL-10 reduced the development of chronic allograft vasculopathy compared to anti-CD45RB alone and reduced the production of graft reactive alloantibodies. These data suggest that the participation of regulatory B lymphocytes in transplantation tolerance may be distinct from how they operate in other systems. Identifying the specific B lymphocytes that mediate transplantation tolerance and defining their mechanism of action may yield new insights into the complex cellular network through which antigen-specific tolerance is established and maintained.
doi:10.1111/j.1600-6143.2010.03027.x
PMCID: PMC2934759  PMID: 20199511
B lymphocyte; IL-10; regulation; tolerance; transplantation
12.  Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect 
Yang, G | Wu, L | Chen, S | Zhu, L | Huang, P | Tong, L | Zhao, Y | Zhao, G | Wang, J | Mei, T | Xu, A | Wang, Y
British Journal of Cancer  2009;100(12):1912-1916.
Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect.
doi:10.1038/sj.bjc.6605087
PMCID: PMC2714242  PMID: 19455142
radiation-induced bystander effect; mitochondrial dysfunction; cytochrome c
13.  Requirement for Both Micron and Submicron Scale Structure for Synergistic Responses of Osteoblasts to Substrate Surface Energy and Topography 
Biomaterials  2007;28(18):2821-2829.
Objective
Surface roughness and surface free energy are two important factors that regulate cell responses to biomaterials. Previous studies established that titanium substrates with micron-scale and submicron scale topographies promote osteoblast differentiation and osteogenic local factor production and that there is a synergistic response to microrough Ti surfaces that have retained their high surface energy via processing that limits hydrocarbon contamination. This study tested the hypothesis that the synergistic response of osteoblasts to these modified surfaces depends on both surface microstructure and surface energy.
Methods
Ti disks were manufactured to present three different surface structures: smooth pretreatment surfaces (PT) with Ra of 0.2 µm; acid-etched surfaces (A) with a submicron roughness Ra of 0.83 µm; and sandblasted/acid-etched surfaces (SLA) with Ra of 3–4 µm. Modified acid-etched (modA) and modified sandblasted/acid-etched (modSLA) titanium substrates, which have low contamination and present a hydroxylated/hydrated surface layer to retain high surface energy, were compared with regular low surface energy A and SLA surfaces. Human osteoblast-like MG63 cells were cultured on these substrates and their responses, including cell shape, growth, differentiation (alkaline phosphatase, osteocalcin), and local factor production (TGF-β1, PGE2, osteoprotegerin [OPG]) were analyzed (N=6 per variable). Data were normalized to cell number.
Results
There were no significant differences between smooth PT and A surfaces except for a small increase in OPG. Compared to A surfaces, MG63 cells produced 30% more osteocalcin on modA, and 70% more on SLA. However, growth on modSLA increased osteocalcin by more than 250%, which exceeded the sum of independent effects of surface energy and topography. Similar effects were noted when levels of latent TGF-β1, PGE2 and OPG were measured in the conditioned media.
Conclusions
The results demonstrate a synergistic effect between high surface energy and topography of Ti substrates and show that both micron scale and submicron scale structural features are necessary.
doi:10.1016/j.biomaterials.2007.02.024
PMCID: PMC2754822  PMID: 17368532
Titanium; Surface energy; Microstructure; Submicron roughness; Osteoblast differentiation
14.  Support for smoke-free policies among smokers and non-smokers in six cities in China: ITC China Survey 
Tobacco Control  2009;19(Suppl_2):i40-i46.
Objective
To examine levels of support for comprehensive smoke-free policies in six large Chinese cities.
Methods
Data from Wave 1 of the International Tobacco Control (ITC) China Survey (April–August 2006) were analysed. The ITC China Survey employed a multistage sampling design in Beijing, Shenyang, Shanghai, Changsha, Guangzhou and Yinchuan (none of which has comprehensive smoke-free policies in place). Face-to-face interviews were conducted with 4815 smokers and 1270 non-smokers. Multivariate logistic regression models were used to identify factors associated with support for comprehensive smoke-free policies.
Results
About one in two Chinese urban smokers and four in five non-smokers believed that secondhand smoke (SHS) causes lung cancer. The majority of respondents supported comprehensive smoke-free policies in hospitals, schools and public transport vehicles while support for smoke-free workplaces, restaurants and bars was lower. Levels of support were generally comparable between smokers and non-smokers. Support for comprehensive smoke-free policies was positively associated with knowledge about the harm of SHS. Respondents who worked in a smoke-free worksite or who frequented smoke-free indoor entertainment places were more likely to support comprehensive smoking restriction in bars and restaurants.
Conclusion
Considerable support for smoke-free policies exists in these six large cities in China. Greater public education about the dangers of SHS may further increase support. Experiencing the benefits of smoke-free indoor entertainment places and/or workplaces increases support for these policies and suggests that some initial smoke-free policy implementation may hasten the diffusion of these public health policies.
doi:10.1136/tc.2009.029850
PMCID: PMC2976027  PMID: 19679889
15.  Mitochondria-dependent signalling pathway are involved in the early process of radiation-induced bystander effects 
Chen, S | Zhao, Y | Han, W | Zhao, G | Zhu, L | Wang, J | Bao, L | Jiang, E | Xu, A | Hei, T K | Yu, Z | Wu, L
British Journal of Cancer  2008;98(11):1839-1844.
Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (ρ0) AL or normal (ρ+) AL cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal AL cells treated with mitochondrial respiratory chain function inhibitors had an attenuated γ-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal AL donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased γ-H2AX induction and that radiation could stimulate cellular NO and O2•− production in irradiated ρ+ AL cells, but not in ρ0 AL cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O2•− levels in irradiated ρ+ AL cells, suggest that radiation-induced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process.
doi:10.1038/sj.bjc.6604358
PMCID: PMC2410123  PMID: 18475304
radiation-induced bystander effects; signalling pathway; mitochondrion; nitric oxide synthase
16.  Animal food intake and cooking methods in relation to endometrial cancer risk in Shanghai 
British Journal of Cancer  2006;95(11):1586-1592.
We evaluated animal food intake and cooking methods in relation to endometrial cancer risk in a population-based case–control study in Shanghai, China. A validated food frequency questionnaire was used to collect the usual dietary habits of 1204 cases and 1212 controls aged 30–69 years between 1997 and 2003. Statistical analyses were based on an unconditional logistic regression model adjusting for potential confounders. High intake of meat and fish was associated with an increased risk of endometrial cancer, with adjusted odds ratios for the highest vs the lowest quartile groups being 1.7 (95% confidence interval: 1.3–2.2) and 2.4 (1.8–3.1), respectively. The elevated risk was observed for all types of meat and fish intake. Intake of eggs and milk was not related to risk. Cooking methods and doneness levels for meat and fish were not associated with risk, nor did they modify the association with meat and fish consumption. Our study suggests that animal food consumption may play an important role in the aetiology of endometrial cancer, but cooking methods have minimal influence on risk among Chinese women.
doi:10.1038/sj.bjc.6603458
PMCID: PMC2360744  PMID: 17060930
endometrial cancer; dietary factor; case–control study
17.  Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice. 
Journal of Clinical Investigation  1998;102(8):1498-1505.
Hypertrophic cardiomyopathy (HCM) is a disease of sarcomeric proteins. The mechanism by which mutant sarcomeric proteins cause HCM is unknown. The leading hypothesis proposes that mutant sarcomeric proteins exert a dominant-negative effect on myocyte structure and function. To test this, we produced transgenic mice expressing low levels of normal or mutant human cardiac troponin T (cTnT). We constructed normal (cTnT-Arg92) and mutant (cTnT-Gln92) transgenes, driven by a murine cTnT promoter, and produced three normal and five mutant transgenic lines, which were identified by PCR and Southern blotting. Expression levels of the transgene proteins, detected using a specific antibody, ranged from 1 to 10% of the total cTnT pool. M-mode and Doppler echocardiography showed normal left ventricular dimensions and systolic function, but diastolic dysfunction in the mutant mice evidenced by a 50% reduction in the E/A ratio of mitral inflow velocities. Histological examination showed cardiac myocyte disarray in the mutant mice, which amounted to 1-15% of the total myocardium, and a twofold increase in the myocardial interstitial collagen content. Thus, the mutant cTnT-Gln92, responsible for human HCM, exerted a dominant-negative effect on cardiac structure and function leading to disarray, increased collagen synthesis, and diastolic dysfunction in transgenic mice.
PMCID: PMC508999  PMID: 9788962
18.  Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics. 
Journal of Bacteriology  1997;179(15):4901-4908.
To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.
PMCID: PMC179340  PMID: 9244281
19.  Identification, sequencing, and enzymatic activity of the erythrose-4-phosphate dehydrogenase gene of Vibrio cholerae. 
Journal of Bacteriology  1997;179(1):293-296.
We have identified a gene in Vibrio cholerae (epd) which encodes an erythrose-4-phosphate dehydrogenase activity and is located immediately downstream of an iron-regulated virulence gene, irgA, and immediately upstream of a gene encoding phosphoglycerate kinase (pgk). Expression of epd in V. cholerae is not regulated by iron, nor is it required for virulence in an infant mouse model.
PMCID: PMC178695  PMID: 8982014
20.  Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K-12. 
Journal of Bacteriology  1996;178(8):2445-2449.
We isolated 26 suppressor mutations that allowed growth of a delta pdxH::omega null mutant in the absence of pyridoxal. Each suppressor mapped to pdxJ, and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide. This bypass suppression suggests that PdxJ may participate in formation of the pyridine ring of pyridoxine 5'-phosphate.
PMCID: PMC177961  PMID: 8636054
21.  A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria. 
Journal of Bacteriology  1996;178(1):232-239.
Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis. Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps. In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction. Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA). Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations. HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction. Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant [Km(app)] = 88 microM; apparent catalytic constant [kcat(app)] = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme. The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly. The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed. Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively.
PMCID: PMC177644  PMID: 8550422
22.  Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis. 
Journal of Bacteriology  1995;177(10):2804-2812.
One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).
PMCID: PMC176952  PMID: 7751290
23.  Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida. 
The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.
Images
PMCID: PMC1263733  PMID: 7704842
24.  Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses. 
Journal of Bacteriology  1995;177(6):1576-1584.
hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.
PMCID: PMC176775  PMID: 7883715
25.  Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12. 
Journal of Bacteriology  1995;177(4):883-891.
We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP). The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography. The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN). In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (Km = 2 and 105 microM and kcat = 0.76 and 1.72 s-1 for PNP and PMP, respectively) and produces hydrogen peroxide. Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low kcat turnover number. Comparison of kcat/Km values suggests that PNP rather than PMP is the in vivo substrate of E. coli PdxH oxidase. In contrast, the eukaryotic enzyme has similar kcat/Km values for PNP and PMP and seems to act as a scavenger. E. coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 microM, respectively. Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence. Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E. coli growing exponentially in minimal medium plus glucose at 37 degrees C. Thus, E. coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis.
PMCID: PMC176679  PMID: 7860596

Results 1-25 (31)