Human uroporphyrinogen III synthase: NMR-based mapping of the active site

Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.     

A novel synthetic oleanolic acid derivative (CPU-II<Subscript>2</Subscript>) attenuates liver fibrosis in mice through regulating the function of hepatic stellate cells

Regulation on the function of the hepatic stellate cells (HSCs) is one of the proposed therapeutic approaches to liver fibrosis. In the present study, we examined the in vitro and in vivo effects of CPU-II₂, a novel synthetic oleanolic acid (OLA) derivative with nitrate, on hepatic fibrosis. This compound alleviated CCl₄-induced hepatic fibrosis in mice with a decrease in hepatic hydroxyproline (Hyp) content and histological changes. CPU-II₂ also attenuated the mRNA expression of α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) induced by CCl₄ in mice and reduced both mRNA and protein levels of α-SMA in HSC-T6 cells. Interestingly, CPU-II₂ did not affect the survival of HSC-T6 cells but decreased the expression of procollagen-α1 (I) in HSC-T6 cells through down-regulating the phosphorylation of p38 MAPK. Conclusion: CPU-II₂ attenuates the development of liver fibrosis rather by regulating the function of HSCs through p38 MAPK pathway than by damaging the stellate cells.     

油菜光合器官面积与导数光谱特征的相关关系

 运用导数光谱分析技术,研究了不同氮肥水平下不同品种油菜(Brassica napus)的 叶面积指数（Leaf area index, LAI）及角果皮面积指数 （Pod area index,PAI ）与冠层导数光谱及其衍生参数的定量关系。结果表明,油菜导数光谱与花前LAI和花后PAI均有良好的相关关系,在 750 nm附近相关关系最好,相关系数达到0.9左右。三边参数与油菜LAI和PAI的相关性顺序为：红边＞黄边＞蓝边,面积参数＞振幅参数＞位置 参数。油菜红边导数光谱的双峰现象降低了红边位置对油菜LAI和PAI的敏感程度,利用线性外推法拟合红边位置能 提高其对油菜LAI和PAI的敏 感程度。在三边参数及其衍生参数中,红边面积及其与蓝边面积的差与LAI及PAI的相关性最好,且适用于该研究中使用的不同品种。因此,750 nm 处的一阶导数光谱、红边面积及其与蓝边面积的差可用于有效地监测油菜的光合器官面积。     

MafG controls the hypoxic response of cells by accumulating HIF-1alpha in the nuclei

We identified MafG as a protein that interacts with HIF-1alpha, a key factor in hypoxic response, using the yeast two-hybrid system. Interaction between MafG and HIF-1alpha was confirmed by surface plasmon resonance and by translocation to the nucleolus with the NoLS signal. A knockdown of MafG reduced erythropoietin (EPO) mRNA levels as well as luciferase reporter activity with the hypoxia response element. The knockdown of MafG did not change total HIF-1alpha protein, but reduced the accumulation of HIF-1alpha in the nuclei. These results suggest that MafG regulates the hypoxic response of cells by detaining HIF-1alpha in the nuclei.     

X-ray crystallographic study of several 2'-deoxy-beta-D-ribonucleosides with 1-deazapurine-derived aglycones

The 2'-deoxy-beta-D-ribonucleosides of 1,3-deazapurine (benzimidazole (1)), 1-deazapurine (both 1H-imidazo[4,5-b]pyridine (2) and 3H-imidazo[4,5-b]pyridine (3)), and 6-benzoylamino-1-deazapurine (7-benzoylamino-3H-imidazo[4,5-b]pyridine (4)) have been prepared and structurally characterized by X-ray crystallography. Especially compounds 1-3 can serve as artificial nucleosides that may substitute 2'-deoxy adenosine because they lack the exocyclic amino group and one or two of the endocyclic nitrogen atoms and hence have a much smaller potential to engage in hydrogen bonds. In the latter respect, they are candidates for nucleosides in metal-ion mediated base pairs. The unit cell of compound 3 contains two crystallographically independent molecules. Compound 4 was crystallized from methanol and water, respectively, giving rise to two different solvates. Despite the closely related aglycones, the sugar conformations in 1-4 are found to be highly variable (1: (2)T(1); 2: (3)T(2); 3: (3)E and E(4); 4: (2)E and (2)T(3)). The structures reported here confirm that there is no simple correlation between the sugar conformation and the character of the nucleoside, and they will hopefully contribute to a better understanding of the complex interplay of different effects that are in control of the conformational equilibrium.     

Derivative polarography of normal and cancerous tissue extracts and urines

Animal tissue extracts show a derivative (alternating-current) polarographic waves at ?1.7 V (B-wave) and frequently a second wave at ?1.4 V (A-wave) against S.C.E.A third wave around ?1.9 V (C-wave) vs S.C.E. was found in the extracts of malignant mammalian tumors and in noncancerous tissues of tumor bearing animals. The ?1.9 V wave was also frequently present in normal (noncancerous)-human urine. The same signal was always missing from the tissue extracts of animals without cancer and urine of human cancer patients.The formation of C-wave is dependent upon the simultaneous presence of three factors: the C-substances, which seems to be nitrogen containing carbohydrate, halogen ions and certain hydrogen ion concentration. A possible relation between the C-wave producing compounds and the immunological specificity of the cell surface is discussed.     

Phytochrome chromophore-deficient mutants

Phytochrome chromophore-deficient mutants have been used as phytochrome-deficient plants to study many aspects of plant development. However, there are still a number of important questions to be resolved concerning both the targets and the phenotypic consequences of these mutations. Recently, progress has been made in our understanding of the molecular basis of the chromophore deficiency in these mutants. Biochemical assays for the committed steps of chromophore synthesis have been developed and used to demonstrate that the pcd1 and yellow-green-2 mutants of pea and tomato, respectively, are unable to synthesize biliverdin IXα from heme while pcd2 and aurea are deficient in phytochromobilin synthase activity. This review focuses on how this information can be used to help understand the basis of other chromophore-deficient mutants, such as the hy1 and hy2 mutants of Arabidopsis, and discusses how the phenotype of chromophore-deficient mutants is related to lesions in the chromophore biosynthesis pathway.