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In JoVE (1)
Other Publications (28)
- Environmental Management
- The Protein Journal
- Journal of Huazhong University of Science and Technology. Medical Sciences = Hua Zhong Ke Ji Da Xue Xue Bao. Yi Xue Ying De Wen Ban = Huazhong Keji Daxue Xuebao. Yixue Yingdewen Ban
- Journal of Fluorescence
- Clinical Rheumatology
- Biopolymers
- Journal of Fluorescence
- Clinical Rheumatology
- Biochimie
- Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry
- Molecular BioSystems
- The Journal of Organic Chemistry
- Biochemical Pharmacology
- International Journal of Food Microbiology
- Toxicon : Official Journal of the International Society on Toxinology
- Chemistry (Weinheim an Der Bergstrasse, Germany)
- Protein and Peptide Letters
- Metallomics : Integrated Biometal Science
- Organic Letters
- American Journal of Hypertension
- BMC Immunology
- Cellular & Molecular Immunology
- BMC Developmental Biology
- Amino Acids
- Biochemical and Biophysical Research Communications
- The Journal of Biological Chemistry
- World Journal of Surgical Oncology
- Metallomics : Integrated Biometal Science
Articles by Liyun Zhang in JoVE
JoVE General
Microdissection of Zebrafish Embryonic Eye Tissues
Department of Biological Sciences, Purdue University
This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.
Other articles by Liyun Zhang on PubMed
Eco-environmental Evolution, Control, and Adjustment for Aibi Lake Catchment
Aibi Lake in north Xinjiang is a typical lake of the arid area, but with a peculiar wetland-arid area ecosystem. Due to the climate becoming drier and the disturbance of human activities, the eco-environment of Aibi Lake catchment has degraded. It was found in our study that there were spatial-temporal changes of vegetation cover, plant species, and soil physical and chemical properties in the catchment. In the upper section of alluvial-fluvial plains, the desertified steppe of Stipa and Artemisia spp. is developed with vegetation cover of some 50%. Haloxylon ammodendron desert occupies the lower section with vegetation cover of some 60%. In these regions with an intensive human disturbance, vegetation has degraded into herb vegetation of annual plant complexes. On the margins of the alluvial-fluvial fans, the lakeshore, and the surrounding regions where the river mouths join the lake, different azonal vegetation-Phragmites communis marsh, Phragmites communis meadow, and Tamarix shrubs-have developed with a vegetation cover of some 80%. On heavier, salinized land, succulent halophyte desert vegetation dominated by Halocnemum strobilaceum has formed with a fractional canopy cover of 10-15%. Haloxylon persicum, Aristida pennata, and other species with a vegetation cover of 30-50% grow in the sand desert zone on the periphery in the lake. In contrast with the 1950s, the vegetation cover around the lakebed and at the river deltas has slightly increased; however, the vegetation cover around the periphery of the lake has decreased and the plant species have still degraded. The surface soils on the windward area and the dried lakebed that have lost vegetation protection have become coarser, whereas the land on the leeward side of the lake has accumulated fine particles. In contrast with the 1980s, soil organic matter has declined markedly. The analyses of climatic data show that the number of days of drifting dust in Jinghe County and Bole City increased in the last 20 years. In the investigation, we found that intensively developed land, the bare lakebed, and abandoned cultivated land provided a great deal of material for drifting dust. In conclusion, we consider the eco-environmental degradation resulting from the inappropriate human activities and put forward recommendations for land-use adjustment and dust control.
Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom
Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A (1%) (280)) of acutolyisn D have been determined to be 47,850 +/- 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca(2+)-binding sites and two Zn(2+)-binding sites determined by atomic absorption spectrophotometer. Zn(2+) is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn(2+) significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)(2) precipitate formation. Ca(2+) is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca(2+) markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu(2+), Co(2+), Mn(2+) or Tb(3+) and inhibited completely by Cd(2+), is enhanced by Mg(2+). The fluorescence intensity of the protein decreases in the presence of Cu(2+), Co(2+), Cd(2+) or Mn(2+), but neither for Ca(2+), Mg(2+) nor for Tb(3+). Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+), Co(2+ )and Tb(3+) have slight effects on its secondary structure contents. In addition, Cd(2+) causes a marked increase of antiparallel beta-sheet content from 45.5% to 60.2%.
ANP T2238C, C-664G Gene Polymorphism and Coronary Heart Diseasein Chinese Population
The association between atrial natriuretic peptide (ANP) polymorphism and coronary heart disease (CHD) was studied in Chinese population. The genotypes of ANP T2238C and ANP C-664G were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) methods in 158 consecutive CHD patients and 165 controls. It was found that the distribution of A2A2 genotype in CHD group was significantly higher than that in control group (P<0.05). Stepwise Logistic regression analysis revealed that male, smoking, history of hypertension, history of diabetes, family history of hypertension, high level of serum cholesterol, and ANP T2238C polymorphism were the possible risk factors in patients with CHD (P<0.05). However, there was no significant difference between the patients with CHD and the control group in the distribution of ANP C-664G polymorphism (P>0.05). The results suggest that A2A2 T2238C genotype could be one of the risk factors for CHD (P<0.05, OR: 1.80, 95 % CI: 1.03-3.15).
Calcium Ion-induced Stabilization and Refolding of Agkisacutacin from Agkistrodon Acutus Venom Studied by Fluorescent Spectroscopy
Agkisacutacin isolated from the venom of Agkistrodon acutus is a coagulation factor IX / coagulation factor X-binding protein with marked anticoagulant- and platelet-modulating activities. Ca(2+) ion-induced stabilization and refolding of Agkisacutacin have been studied by following fluorescent measurements. Ca(2+) ions not only increase the structural stability of agkisacutacin against GdnHCl denaturation, but also induce its refolding. The GdnHCl-induced unfolding of the apo-agkisacutacin and the purified agkisacutacin is a single-step process with no detectable intermediate state. Ca(2+) ions play an important role in the stabilization of the structure of agkisacutacin. Ca(2+)-stabilized agkisacutacin exhibits higher resistance to GdnHCl denaturation than the apo-agkisacutacin. It is possible to induce refolding of the unfolded apo-agkisacutacin merely by adding 1 mM Ca(2+) ions without changing the concentration of the denaturant. The kinetic result of Ca(2+)-induced refolding provides evidences for that agkisacutacin consists of at least two refolding phases and the first phase of Ca(2+)-induced refolding should involve the formation of the compact Ca(2+)-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.
Response to One Infusion Predicts Subsequent Improvement As Well As the Rate of Relapse of Ankylosing Spondylitis Infused with Three Pulses of Infliximab
The objective of this study was to evaluate if there are early clinical parameters in AS patients treated with three standard infusions of infliximab, which would predict whether a patient will derive a significant improvement at the tenth week, or whether there will be a lengthy clinical response after discontinuing the infusions. Sixty three AS patients were given three infusions of 5 mg/kg of infliximab at weeks 0, 2, and 6 and evaluated serially before each infusion and also at week 10. Afterwards, patients were followed up by telephone interview until their disease activities were >60% of the baseline values. At that point, disease was considered to have relapsed. Clinical parameters at baseline as well as at week 2 were used for analysis to identify factors that might predict an improvement at week 10, or predict a delayed relapse. A predictor is regarded as being useful if the area under the curve (AUC) when analyzed by receiver operator calculations (ROC) exceeds 0.75. No parameters at baseline have sufficient predictive values. However, ASAS20 (Assessment in Ankylosing Spondylitis International Working Group criteria) at week 2 predicts improvement at week 10, and also duration of clinical response after discontinuing the infliximab at week 6. The response to one pulse of infliximab is the best predictor of subsequent response as well as rate of relapse after discontinuing the infliximab.
Metal Ions- and PH-induced Conformational Changes of Acutolysin A from Agkistrodon Acutus Venom Probed by Fluorescent Spectroscopy
Acutolysin A isolated from the venom of Agkistrodon acutus is a protein of 22 kDa with marked haemorrhagic and proteolytic activities. The metal ions- and pH-induced conformational changes of acutolysin A have been studied by following fluorescence and activity measurements. Here, we provide evidence for the fact that native holo-acutolysin A adopts two subtly different conformations, native state a (Na) stable in the weak acidic pH range from 6.0 to 7.0 with low activity and native state b (Nb) stable in the weak alkaline pH range from 7.5 to 9.0 with high activity. Holo-acutolysin A has an optimum pH of 8.5 for caseinolytic activity, and the protein adopts the most stable conformation with the maximum fluorescence at pH 8.5. The Ca2+ and Zn2+ ions have significant effects on both the pH-induced denaturing transition curve and the pH-dependent activity curve. Addition of 1 mM Ca2+ to holo-acutolysin A shifts both the acid-induced denaturing transition curve and the end zone of acid-induced inactivation curve towards lower pH value, and shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value. Addition of 1 mM Zn2+ also shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value and shifts the acid-induced denaturing transition curve to lower pH value, but has little effect on the acid-induced inactivation. Removal of Ca2+ and Zn2+ from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. It is also evident from the present work that the free Zn2+ -induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)2 precipitation on the protein.
Oxygen-dependent Oxidation of Fe(II) to Fe(III) and Interaction of Fe(III) with Bovine Serum Albumin, Leading to a Hysteretic Effect on the Fluorescence of Bovine Serum Albumin
The serum albumin is the most abundant protein in blood plasma and the iron is essential for many cellular processes. However, the interaction between Fe(3+) and haem-free serum albumin remains unclear. Here we provide evidence for the fact that haem-free BSA possesses one specific Fe(3+)-binding site. The binding of Fe(3+) to BSA results in a significant quenching of the Trp fluorescence of BSA. The average apparent dissociation constant value for the interaction of Fe(3+) and BSA is 3.46 x 10(-8)+/-3 x 10(-10) M at 37 degrees C and 3.30 x 10(-8)+/-5 x 10(-10) M at 25 degrees C, respectively, as determined by fluorescence titration. Addition of 50 microM Fe(2+) to 1 microM BSA results in an obvious hysteretic effect on the fluorescence of BSA. The time-dependent fluorescence quenching of BSA by Fe(2+) is not caused by the Fe(2+)-induced conformational change of BSA, but the oxygen-dependent oxidation of Fe(2+) to Fe(3+). Fe(2+) undergoes an oxygen-dependent oxidation to Fe(3+) under aerobic conditions, which is accelerated by the interaction of BSA with Fe(3+) and extensively inhibited under anaerobic conditions. The results suggest that BSA may take part in non-transferrin bound iron transfer.
Identification of Acute Phase Reactants and Cytokines Useful for Monitoring Infliximab Therapy in Ankylosing Spondylitis
Although most ankylosing spondylitis patients show an apparent clinical response to infliximab therapy, there is considerable individual variation. Because current clinical assessment relies heavily on subjective patient self-evaluation, biomarkers of high sensitivity and specificity are much needed. Here, we assessed potential biomarkers in 47 ankylosing spondylitis patients who received three standard pulses of infliximab. Before each infusion and at week 10, the following were measured: erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), platelet count, serum levels of metalloproteinase-3 (MMP-3), and 22 different cytokines. We discovered that, 2 weeks after the first infusion, the combination of ESR, CRP, and platelet count distinguished responders from non-responders with 81.3% sensitivity and 72.7% specificity. The distinguishing power was much less when each acute phase reactant was used alone. Among the 22 cytokines, serum IL-1alpha was able to distinguish responders from non-responders at week 6, with sensitivity of 84.9% and specificity of 53.8%. Serum IL-1alpha was probably generated from the joint compartments, as synovial fluid levels were much higher than corresponding serum levels. Although infliximab infusions led to rapid and significant suppression of serum MMP-3 levels, serum MMP-3 levels did not distinguish responders from non-responders. Besides identifying potential biomarkers, our results also demonstrate the usefulness of using sensitivity and specificity to assess usefulness of potential biomarkers.
Identification of an Unusual AT(D)Pase-like Activity in Multifunctional NAD Glycohydrolase from the Venom of Agkistrodon Acutus
NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of C-N glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu(2+) ion. Here, we show that AA-NADase is not only able to cleave the C-N glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the P-O-P bond of ATP, ADP and AMP-PNP without the cleavage of P-O-P bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant k(cat)/K(m) of 293+/-7mM(-1)s(-1). AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.
Effect of Metal Ion Substitutions in Anticoagulation Factor I from the Venom of Agkistrodon Acutus on the Binding of Activated Coagulation Factor X and on Structural Stability
Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca(2+)-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca(2+)-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr(2+) and Ba(2+), with ionic radii larger than the ionic radius of Ca(2+), can bind to Ca(2+)-free ACF I (apo-ACF I), while Mg(2+), with an ionic radius smaller than that of Ca(2+), shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr(2+)-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca(2+)-ACF I, while Ba(2+)-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca(2+)-ACF I. Mg(2+) and Sr(2+), with ionic radii close to that of Ca(2+), can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba(2+), with a much larger ionic radius than Ca(2+), cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg(2+), Ca(2+), and Sr(2+) ions to FXa may be essential for the recognition between FXa and ACF I.
Metal Ions Binding to RecA Inteins from Mycobacterium Tuberculosis
Zinc has been found in the crystal structures of inteins and the zinc ion can inhibit intein splicing both in vitro and in vivo. The interactions between metal ions and three minimized recA inteins have been studied in this work. Isothermal titration calorimetry (ITC) results show that the zinc binding affinity to three inteins is in the order of DeltaI-SM > DeltaDeltaI(hh)-SM approximately DeltaDeltaI(hh)-CM, but is much weaker than to EDTA. These data explain the reversible inhibition and the presence of zinc only in the crystal structure of DeltaI-SM of recA intein. A positive correlation between binding constants and inhibition efficiency was observed upon the titration of different metal ions. Single-site binding modes were detected in all interactions, except DeltaDeltaI(hh)-CM which has two Zn sites. Zinc binding sites on DeltaDeltaI(hh)-CM were analyzed by NMR spectroscopy and ITC titration on inteins with chemical modifications. Results indicate that the Cys1 and His73 are the second zinc binding sites in DeltaDeltaI(hh)-CM. CD studies show the metal coordinations have negligible influence on protein structure. This work suggests that the mobility restriction of key residues from metal coordination is likely the key cause of metal inhibition of intein splicing.
A General Copper-catalyzed Coupling of Azoles with Vinyl Bromides
A copper-catalyzed methodology for the coupling of vinyl bromides with azoles has been developed. This protocol uses a combination of 10 mol % of copper iodide and 20 mol % of ethylenediamine as catalyst. The reaction proceeds with various azoles and vinyl bromides, and the double bond geometry of vinyl bromides is retained under the reaction conditions.
Identification of a Nitric Oxide-dependent Hypotensive Effect of Anticoagulation Factor II from the Venom of Agkistrodon Acutus
Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is a member of the coagulation factor IX/coagulation factor X-binding protein (IX/X-bp) family. ACF II forms a 1:1 complex with activated coagulation factor X in a Ca(2+)-dependent fashion and thereby blocks the amplification of the coagulation cascade. In the present study, we have investigated the effect of ACF II on the mean arterial blood pressure (MABP) and heart rate (HR) in anaesthetized rats. The results indicate that ACF II induces a dose-dependent response in rats with a short fast drop of MABP followed by an increase and then a longer lasting slight decrease in MABP, but does not obviously affect HR. ACF II-induced hypotension is significantly blocked by the nitric oxide (NO) synthase inhibitor N-omega-L-arginine methyl ester (L-NAME). ACF II produces a concentration-dependent relaxation of rat aortic rings with functional-endothelium. The ACF II-induced vasodilatation is completely inhibited by removal of endothelium and significantly inhibited by pretreatment with L-NAME. These observations demonstrate that ACF II induces hypotension through an endothelium-dependent vasodilation, which is strongly mediated by the release of NO from endothelium. ACF II exhibits high anticoagulation activity in vivo based on activated partial thromboplastin time assay. Therefore, ACF II is so far identified as the first unique bifunctional protein in the IX/X-bp family that has both anticoagulant and hypotensive effects on the blood of rats through different pathways.
Effect of High Pressure CO2 and Mild Heat Processing on Natural Microorganisms in Apple Juice
Apple juice was pasteurized by high pressure carbon dioxide (HPCD) at 20MPa with CO(2) concentration of 4.5-5.3% and mild heat (MH) at atmospheric pressure. Microbial inactivation and stability of natural microorganisms in apple juice were investigated. The temperatures were 37, 42, 47, 52, 57 and 62 degrees C, treatment time was 30min, and storage temperatures of pasteurized apple juice were 2 and 28 degrees C. The aerobic bacteria (AB) treated by MH at 62 degrees C and by HPCD at > or =52 degrees C were almost totally inactivated, the microbial counts were<10CFU/mL. The yeasts and moulds (Y&M) treated by MH at > or =57 degrees C and by HPCD at > or =42 degrees C were totally inactivated. HPCD increased the susceptibility of these natural microorganisms to temperature and enhanced their microbial inactivation. The AB in apple juice treated by HPCD at > or =52 degrees C and the Y&M treated by HPCD at > or =57 degrees C, the AB and the Y&M treated by MH at 62 degrees C showed a better stability during storage at 2 and 28 degrees C, but apple juice treated by HPCD at < or =47 degrees C was characterized with high microbial counts of the AB> or =2.75x10(3)CFU/ml. A viable but non-culturable (VBNC) state of the Y&M treated by MH at 57 degrees C and by HPCD at 42, 47 and 52 degrees C was observed during storage at 28 degrees C. Apparently the proper temperature of HPCD treatment of apple juice at 20MPa and stored at 2 and 28 degrees C was greater than or equal to 52 degrees C, while for MH treatment the proper temperature increased to 62 degrees C in this study.
Mg(II)-induced Binding of Factor IX-binding Protein from the Venom of Agkistrodon Halys Pallas with Factor Xa
Factor IX-binding protein (AHP IX-bp), a Ca2+- and Zn2+-binding protein from the venom of Agkistrodon Halys Pallas was reported to bind specifically with factor IX in a Zn2+-dependent manner. Here we have purified AHP IX-bp by a simple two-step of chromatography procedure and found that AHP IX-bp also binds factor Xa (FXa) with high binding-affinity in a Mg2+-dependent manner. Although Mg2+ ions have a significantly low binding-affinity for apo-AHP IX-bp as determined by isothermal titration calorimetry, they can induce the binding of apo-AHP IX-bp with FXa even in the absence of Ca2+ as determined by native PAGE and surface plasmon resonance. Mg2+ ions are required to maintain in vivo function of FX Gla domain for its recognition of AHP IX-bp. Both Ca2+ and Zn2+ ions fail to induce the binding between apo-AHP IX-bp and FXa. The abundant Mg2+ ions in blood play an important role in the anticoagulation of AHP IX-bp.
Binding and Inhibition of Copper Ions to RecA Inteins from Mycobacterium Tuberculosis
Protein splicing is a unique post-translational process in which an intein excises itself from a precursor with the concomitant ligation of flanking sequences. The binding of zinc to intein inhibits protein splicing reversibly and EDTA relieves the inhibition. Copper was found to inhibit protein trans splicing; however, the recovery of intein splicing required both EDTA and TCEP, suggesting a different inhibition mechanism for copper compared to zinc. In this work, we have investigated the binding properties and inhibition effects of copper ions on the RecA intein from Mycobacterium tuberculosis. Both Cu(+) and Cu(2+) exhibited high binding affinity to inteins, while different binding sites were identified. Cu(2+) coordinates to Cys1, the key residue involved in the mechanism of protein splicing, however, Cu(+) does not coordinate to cysteine. An in vitro inhibition assay indicated that monovalent Cu(+) demonstrates reversible inhibition to protein splicing, and the inhibitory efficiency is comparable to Zn(2+). Redox reaction between Cu(2+) and cysteine in inteins were observed and the rate constants were determined. The results suggested a dual role for Cu(2+) in the inhibition of intein splicing: strong coordination of Cu(2+) to key residues (including Cys1) in the intein, and subsequent oxidation of Cys1, the residue required for the N-->S acyl shift step in protein splicing. A kinetic study suggested that the coordination could be the major cause of inhibition effect of Cu(2+) initially, whereas the redox reaction could play an additional role in inhibition at a later stage.
Location of MBL-associated Serine Proteases Binding Motifs on Human Mannan-binding Lectin (MBL)
The lectin pathway provides an antibody independent route of complement activation. Mannan-binding lectin (MBL) can form compound with MBL-associated serine proteases (MASPs) through its collagen-like region (CLR) to initiate complement fixation. In this study, we designed and synthesized a range of peptides according to the sequence of CLR in human MBL, which were assumed to block the MBL-MASP interaction, in order to locate the serine protease binding motifs on human MBL. It was demonstrated that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. In addition, Arg32Cys, Gly35Asp and Gly37Glu mutant proteins have the similar serine protease binding characteristic with wild type MBL, but the binding between mutated MBL proteins and MASPs is much weaker than that between wild type MBL protein and MASPs.
Metal Ions Binding to NAD-glycohydrolase from the Venom of Agkistrodon Acutus: Regulation of Multicatalytic Activity
AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.
Domino N-H/C-H Bond Activation: Copper-catalyzed Synthesis of Nitrogen-bridgehead Heterocycles Using Azoles and 1,4-dihalo-1,3-dienes
Copper-catalyzed tandem coupling of 1,4-dihalo-1,3-dienes with azoles via an N-H bond and its adjacent C-H bond activation has been described. The reaction exhibits good regioselectivity when unsymmetrical 1,4-dihalo-1,3-diene is employed. This method provided a novel route to the synthesis of nitrogen-bridgehead azolopyridine derivatives.
Arterial Stiffness and Asymptomatic Intracranial Large Arterial Stenosis and Calcification in Hypertensive Chinese
Intracranial large artery disease (ICLAD), such as stenosis and calcification, is common in Chinese patients with stroke. However, little is known about ICLAD and its association with large arterial stiffness in hypertensive patients.
Mannan-binding Lectin Regulates Dendritic Cell Maturation and Cytokine Production Induced by Lipopolysaccharide
Mannan-binding lectin (MBL) is a pattern-recognition molecule present in serum, which is involved in the innate immune defense by activating complement and promoting opsonophagocytosis. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are crucial for the initiation of adaptive immunity. Lipopolysaccharide (LPS) has been shown to be a strong activator of the inflammatory response and immune regulation. We first examined whether MBL modulated LPS-induced cellular responses, then investigated possible mechanisms of its inhibitory effect.
Mannan-binding Lectin Directly Interacts with Toll-like Receptor 4 and Suppresses Lipopolysaccharide-induced Inflammatory Cytokine Secretion from THP-1 Cells
Mannan-binding lectin (MBL) plays a key role in the lectin pathway of complement activation and can influence cytokine expression. Toll-like receptor 4 (TLR4) is expressed extensively and has been demonstrated to be involved in lipopolysaccharide (LPS)-induced signaling. We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-κB (NF-κB) activity by using the monocytoid cell line THP-1. We then investigated the possible mechanisms underlying any observed regulatory effect. Using ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we found that at both the protein and mRNA levels, treatment with MBL suppresses LPS-induced tumor-necrosis factor (TNF)-α and IL-12 production in THP-1 cells. An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-κB DNA binding and translocation in THP-1 cells. While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations, this binding occurred optimally in response to supraphysiological calcium concentrations. This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody (HTA125). Activation of THP-1 cells by LPS treatment resulted in increased MBL binding. We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner, and this interaction could attenuate the binding of LPS to cell surfaces. Taken together, these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways. These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.
Cellular Expression of Smarca4 (Brg1)-regulated Genes in Zebrafish Retinas
In a recent genomic study, Leung et al. used a factorial microarray analysis to identify Smarca4 (Brg1)-regulated genes in micro-dissected zebrafish retinas. Two hundred and fifty nine genes were grouped in three-way ANOVA models which carried the most specific retinal change. To validate the microarray results and to elucidate cellular expression patterns of the significant genes for further characterization, 32 known genes were randomly selected from this group. In situ hybridization of these genes was performed on the same types of samples (wild-type (WT) and smarca4a50/a50 (yng) mutant) at the same stages (36 and 52 hours post-fertilization (hpf)) as in the microarray study.
Synchrotron Vacuum Ultraviolet (VUV) Photo-induced Fragmentation of Cyclic Dipeptides Radical Cations
Cyclic dipeptides, due to their chemical properties and various bioactivities, are very attractive for medicinal chemistry. Fragmentations of three simple cyclic dipeptides including cyclo(Gly-Gly), cyclo(Ala-Ala) and cyclo(Gly-Val) in the gas-phase are determined with synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (VUV PIMS) and theoretical calculations. Cyclo(Gly-Gly) and cyclo(Ala-Ala) show the similar fragmentation pathways. The primary decomposition reactions of cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations are found to be HNCO loss and CO elimination. The appearance energies (AEs) of fragment ions [CH(2)NHCOCH(2)](+•) and [CH(3)CHNHCOCHCH(3)](+•) are measured to be 10.21 and 9.66 ± 0.05 eV, respectively, which are formed from cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations with HNCO elimination. Due to the stabilization of the radical cation of cyclo(Gly-Val) with isopropyl side group, the dominant fragment ion m/z 114 assigned as [C(4)H(6)N(2)O(2)](+•) is produced by γ-H migration and i cleavage to lose propylene. The ionization energies (IEs) of three cyclic dipeptides decrease in the order cyclo(Gly-Gly) (9.33 ± 0.05 eV) > cyclo(Ala-Ala) (9.21 ± 0.05 eV) > cyclo(Gly-Val) (9.09 ± 0.05 eV) from measurements of photoionization efficiency spectra. It implies that IEs of cyclic dipeptides are affected by substituent groups and symmetrical characterization of molecular structures. These observations of the chemical properties of cyclic dipeptides radical ion (M(+•)) may be important for understanding gas-phase molecular reactivity of 2,5-diketopiperazines and guiding diketopiperazine-based drug design.
Transcriptional Up-regulation of RhoE by Hypoxia-inducible Factor (HIF)-1 Promotes Epithelial to Mesenchymal Transition of Gastric Cancer Cells During Hypoxia
Epithelial-mesenchymal transition (EMT) is a key process that drives cancer invasion. Recently, hypoxia has been reported to induce EMT, accompanied by cytoskeleton remodeling. As RhoE is a key regulator in cytoskeleton formation, we hypothesized that RhoE may play a role in hypoxia-induced EMT. For the first time, we report that RhoE protein levels increase in gastric cancer cells under hypoxic conditions. Rigorous analysis revealed that RhoE up-regulation is at the transcriptional levels and requires hypoxia-inducible factor (HIF)-1α induction, and that HIF-1α binds a hypoxia-responsive element (HRE) on the RhoE promoter. Additionally, we discovered that hypoxia or overexpression of RhoE in normoxia up-regulates the mesenchymal marker Vimentin, down-regulates the epithelial marker E-cadherin, and significantly increases cell invasion in vitro. Silencing of HIF-1α or RhoE by specific siRNAs rescued these hypoxia-induced effects. Ectopic expression of RhoE also induced up-regulation of MMP2/MMP-9 in gastric cancer cells. This study identifies RhoE as a direct target for HIF-1 in gastric cancer cells. In addition, RhoE up-regulation represents a pivotal cellular adaptive response to hypoxia with implications in gastric cancer cell EMT and invasion. We propose that RhoE-targeted therapy might inhibit the high invasive potential of gastric cancer cells in hypoxic regions.
Cisplatin Inhibits Protein Splicing, Suggesting Inteins As Therapeutic Targets in Mycobacteria
Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ∼2 μM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ∼40 μM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.
Dynamic Expression of CEACAM7 in Precursor Lesions of Gastric Carcinoma and Its Prognostic Value in Combination with CEA
ABSTRACT:
Cu(ii)- and Disulfide Bonds-induced Stabilization During the Guanidine Hydrochloride- and Thermal-induced Denaturation of NAD-glycohydrolase from the Venom of Agkistrodon Acutus
NAD-glycohydrolase (AA-NADase) from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. Among all identified NADases, only AA-NADase is a disulfide-linked dimer and contains Cu(2+). Cu(2+) and disulfide bonds are essential for its multicatalytic activity. In this study, the effects of Cu(2+) and disulfide-bonds on guanidine hydrochloride (GdnHCl)- and thermal-induced unfolding of AA-NADase have been investigated by fluorescence, circular dichroism (CD) and differential scanning calorimetry (DSC). Cu(2+) and disulfide bonds not only increase the free energy change during the GdnHCl-induced unfolding as determined by fluorescence, but also increase the overall enthalpy change and the transition temperature during the thermal-induced unfolding as determined by CD and DSC. The slope of the GdnHCl-induced unfolding curve at its midpoint and the heat capacity of thermal-induced unfolding are slightly affected by Cu(2+) but significantly decrease after reduction of three disulfide-bonds. This work suggests that Cu(2+) stabilizes the folded state by increasing the enthalpy of unfolding, while disulfide-bonds stabilize the folded state by increasing the enthalpy of unfolding and stabilizing the packing of hydrophobic residues. Thus both Cu(2+) and disulfide bonds play a structural role in its multicatalytic activity.
