A new crystal form of mouse thiamin pyrophosphokinase

Thiamin pyrophosphokinase (TPK) transfers a pyrophosphate group from ATP to the hydroxyl group of thiamin and produces thiamin pyrophosphate (TPP). TPP is the cofactor of metabolically important enzymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, transketolase and 2-hydroxyphytanoyl-CoA lyase. Thiamin deficiency results in Wernike-Korsakof Syndrome (WKS) due to neurological disorder and wet beriberi, a potentially fatal cardiovascular disease. Mouse TPK associates as a dimer revealed by previous solved crystallographic structures. In this study, we report mouse TPK complexed with TPP-Mg²⁺ and thiamin -Mg²⁺, respectively, in a new crystal form. In these two structures, four mouse TPK molecules were found in each asymmetric unit. Although we cannot rule out this tetramer form can be an artifact from crystal packing, mouse TPK tetramer has a more closed ATP binding pocket and has the potential to provide specific interactions between mouse TPK and ATP compared with the previous dimeric structure and is likely to be an active form.     

Crystal structures of dihydroxyacetone and its derivatives

The crystal and molecular structures of three crystalline forms of the dihydroxyacetone dimer, C6H12O6, DHA-dimer: alpha (1a), beta (1b), and gamma (1c), the hydrated calcium chloride complex of dihydroxyacetone monomer, CaCl2(C3H6O3)(2) x H2O, CaCl2(DHA)2 x H2O (2a), the tetrahydrated calcium chloride complex of dihydroxyacetone monomer, CaCl2(C3H6O3) x 4H2O, CaCl2(DHA) x 4H2O (2b), the dihydroxyacetone monomer, C3H6O3, DHA (2c), and dihydroxyacetone dimethyl acetal, C5H12O4, (MeO)2DHA (3) are described. Compounds 1a and 2b crystallize in the triclinic system, and 1b,c, 2a,c, and 3 are monoclinic. Molecules of all forms of dihydroxyacetone dimer 1a,b, and 1c are the trans isomers, with the 1,4-dioxane ring in the chair conformation and the hydroxyl and hydroxymethyl groups in axial and equatorial dispositions, respectively. The Ca2+ ions in 2a and 2b are bridged by the carbonyl O atoms from two symmetry-related DHA molecules to form centrosymmetric dimers with Ca...Ca distance of 4.307(2)A in 2a and 4.330(2) and 4.305(2)A in two crystallographically independent dimers in 2b. DHA molecules coordinate to the Ca2+ ions by hydroxyl and carbonyl oxygen atoms. The eight-coordinate polyhedra of Ca2+ are completed by water molecule and Cl- ion in 2a and by four water molecules in 2b. The dihydroxyacetone molecules in 2a,b, and 2c are in an extended conformation, with both hydroxyl groups being synperiplanar (sp) to the carbonyl O atom. All hydroxyl groups in 2c (along with water molecules in 2a and 2b) are involved as donors in medium strong and weak intermolecular O-H...O hydrogen bonding. Some of them, as well as carbonyl O atoms or Cl- ions in 2a and 2b, act as acceptors in C-H...O (and C-H...Cl) hydrogen interactions.     

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.     

醒脑静注射液对急性脑出血患者FIB和D-二聚体水平的影响

目的:研究醒脑静注射液对急性脑出血患者血浆纤维蛋白原和D-二聚体水平的影响。方法:选取我院神经内科收治的急性脑出血患者90例,随机分为两组,其中对照组45例,予降低颅内压、控制血压、抗感染、营养脑细胞等常规治疗。实验组45例,在常规治疗的基础上加用醒脑静注射液。对比治疗前后患者血浆纤维蛋白原(FIB)、D-二聚体(D1-D)的含量及临床疗效。结果:治疗后,两组患者病情均有所改善,实验组有效率(91.11%)与对照组(82.22%)比较明显较高,差异有统计学意义(P0.05);两组患者血浆FIB含量较治疗前均明显升高,但实验组高于对照组,差异有统计学意义(P0.05);实验组血浆D-D含量明显降低,对照组明显升高,实验组明显低于对照组,差异有统计学意义(P0.05)。结论:醒脑静注射液能够使急性脑出血患者血浆纤维蛋白原含量增加,D-二聚体水平下降,具有良好的临床疗效,值得进一步研究推广。     

Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase

More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6-4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle.Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme alone is not sufficient to fully understand the enzyme's action on UV-damaged DNA. Much effort has therefore been invested to learn more about, for example, the geometry of the enzyme-substrate complex, and the mechanism and pathways of intra-enzyme and enzyme ↔DNA electron transfer. Many of the key results from biochemical and molecular biology characterizations of the enzyme or the enzyme-substrate complex have been summarized in a number of reviews. Complementary to these articles, this review focuses on recent biophysical studies of photoreactivation comprising work performed from the early 1990s until the present.     

Cytotoxic sesquiterpenes from Ligularia platyglossa

Four sesquiterpene lactones including an eremophilenolide dimer, named as biligulaplenolide, 1, 8beta-hydroxy-1-oxo-(14alpha,15alpha eremophil-7(11),9(10)-dien-12,8alpha-olide, 2, 1-hydroxy-2-oxo-(14alpha,15alpha eremophil-1(10),7(11),8(9)-trien-12,8-olide, 3, 4alpha,8beta,9alpha-trihydroxy- 5alphaEta-7(11)-eudesmen-12,8alpha-olide, 4, along with two known ones, 10alpha-hydroxy-1-oxo-eremophil-7(11),8(9)-dien-12,8-olide, 5, and furanoeremophil-1(10)-ene-2,9-dione, 6, were isolated from the underground organs of Ligularia platyglossa (Franch.) Hand.-Mazz. Their structures were elucidated by spectroscopic methods including single-crystal X-ray diffraction analysis (2 and 3). Their in vitro cytotoxicities against seven cancer cell lines (BGC-823, A549, HL-60, B16, SMMC-7721, BEL7402, Hela) were evaluated. Compounds 2, 3, 5 showed cytotoxic activities on HL-60 cancer cells with IC50 in the range of 24.0 to 51.1 microM, whereas compound 3 exhibited only weak cytotoxic activity against the B16, BEL7402 and Hela cancer cells. Flow cytometric analysis indicated that compound 3 induces Hela cells to apoptotic death after 48 h treatment with 0.38 mM of this compound.     

Palladium(II) complexes of 1,10-phenanthroline: Synthesis and X-ray crystal structure determination

Two palladium(II) complexes, [Pd(phen)(NCCH₃)₂][O₃SCF₃]₂ (1) and [Pd(phen)(μ-OH)]₂[O₃SCF₃]₂ · 2H₂O (2) (where phen = 1,10-phenanthroline), have been crystallized following the reaction of Pd(phen)Cl₂ with silver triflate, Ag(O₃SCF₃), in acetonitrile and water, respectively. The structures of both complexes are based on a Pd(phen)²⁺ metal core, with two acetonitrile molecules binding in a monodentate fashion in complex 1 and two hydroxo bridges holding together two cores to form a dimer in complex 2. Additionally, both complexes present a hydrogen bonded 3-D network involving the triflate anions in 1, and water and triflate anions in 2. Both complexes have been characterized by infrared and ¹H NMR spectroscopy and their crystal structures determined by X-ray crystallography.     

Characterization of Inhibitor-Bound α-Synuclein Dimer: Role of α-Synuclein N-Terminal Region in Dimerization and Inhibitor Binding

α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC₅₀ values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.     

The binuclear iron site of membrane-bound methane hydroxylase from <Emphasis Type="Italic">Methylococcus capsulatus</Emphasis> (Strain M)

The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: at 47 (α), 27 (β), and 25 kDa (γ). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4°C exhibited, along with the copper signal of type II with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00 After incubation at ?30°C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at ?30°C. It was established that this intermediate is a high-spin dimer [Fe(III)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(III)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the β-subunit and cannot be detected by ESR.     

A dimeric structure of PD-L1: functional units or evolutionary relics?

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222₁ with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.