Transferrin Modifications and Lipid Peroxidation: Implications in Diabetes Mellitus

Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at <formula>≥2 mg/ml</formula> to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 μmol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p<0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio=2.4) also decreased from 0.726 to 0.696 and 0.585 mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p<0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.     

Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation

Many clinical trials have demonstrated the beneficial effects of soybean (Glycine max) on general cardiovascular health. Among a variety of soybeans, black soybean is known to display diverse biological activities superior to those of yellow and green soybeans, such as in antioxidant, anti-inflammatory and anticancer activities. However, few studies have been directed on the effect of black soybean on cardiovascular function. In this study, we aimed to investigate the effect of black soybean extract (BB) on platelet activation, a key contributor to thrombotic diseases. In freshly isolated human platelets, BB has shown potent inhibitory activity on collagen-induced platelet aggregation, while yellow soybean extract had marginal activity only. BB also attenuated serotonin secretion and P-selectin expression, which are important factors for the platelet–tissue interaction along with thromboxane A₂ formation. These in vitro results were further confirmed in an ex vivo platelet aggregation measurement and in vivo venous thrombosis model where oral administration of BB reduced collagen-induced platelet aggregation and FeCl₃-induced thrombus formation significantly. A potential active ingredient for antiplatelet effects of BB was isolated and identified to be adenosine through bioassay-directed fractionation and NMR and ESI-MS analyses. These results indicate that black soybean can be a novel dietary supplement for the prevention of cardiovascular risks and the improvement of blood circulation.     

贵州鼠尾草化学成分的研究

从贵州鼠尾草95%乙醇提取物分离得到8个化合物,通过理化性质及波谱方法分别鉴定为正三十三烷(1),十二烷酸十四烷酯(2),β-谷甾醇(3),胡萝卜苷(4),乌苏酸(5),2 a,3β-二羟基乌苏酸(6),白桦酸(7)和迷迭香酸(8)。以上化合物均首次从该植物中分离得到。     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.     

Identification of amino acid residues essential for heparin binding by the A1 domain of human von Willebrand factor

Platelet adhesion is mediated by von Willebrand factor (VWF) that binds platelet glycoprotein Ib (GPIb). Previous observations suggested that heparin competitively inhibits the binding of VWF to GPIb and may down-regulate platelet adhesion. We performed charged-to-alanine scanning mutagenesis of domain A1 and studied dose-dependent binding to heparin-Sepharose beads. Mutations at Lys1362 and Arg1395, at which the GPIb binding was markedly decreased, showed 41% and 42% binding, respectively. Clustered mutations in the segments 1332KDRKR1336 and 1405KKKK1408, which have been proposed as heparin binding sequences, showed 72% and 52% binding, respectively. However, single alanine substitutions within these clusters showed normal binding. Our findings suggest that heparin may inhibit the binding of VWF to GPIb by interacting with GPIb binding and interpret why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.     

中国大戟科一新归化种——硬毛巴豆

首次报道了大戟科(Euphorbiaceae)巴豆属硬毛巴豆(Croton hirtus L'Hér.)在中国的归化新记录,并提供该种的特征描述和形态图。凭证标本保存于中国科学院华南植物园标本馆(IBSC)。     

Chronology of the Neolithic Necropolis at Camí de Can Grau (NE-Iberian Peninsula). Funerary pattern changes and long-distance raw material exchanges

Camí de Can Grau is one of the most important Neolithic necropolises of the “Pit Burials” horizon (North-East of the Iberian Peninsula. Late fifth–early fourth millennia cal BC), because of its large number of graves. However, the number of buried individuals and the type of grave goods of the site have some peculiarities suggesting that could be one of the last manifestations of this horizon. For proving that, a radiocarbon dating programme and some statistical analysis were carried out so as to determine its chronology. The results are discussed regarding the duration of the necropolis, the degree of contemporaneity between graves and grave goods and the number of buried individuals. Moreover, chronological relationships with other similar contexts of the same horizon and located in other regions are presented. This study goes beyond a purely local research, as it proposes a method for addressing the chronology of funerary contexts.     

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ibα

A fragment (residues His1-Val289) of the chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, and inhibitor of N-linked glycosylation. The recombinant GP Ib fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 g of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib fragment.Abbreviations ABTS 2.2-azino-di-(3-ethylbenzthiazoline sulphonate) - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - GP Ib-IX glycoprotein Ib-IX complex - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - IEF isoelectric focusing - Ig immunoglobulin - mAb monoclonal antibody - MEM minimum essential medium - PMSF phenyl-methylsulfonyl fluoride - SDS sodium dodecyl sulfate - vWF von Willebrand factor     

麦红吸浆虫滞育习性研究

1986～1991年研究了麦红吸浆虫的滞育习性,提出了该由主动延长滞育和被动延长滞育的两种形式,探讨了土壤湿度、温度对延长滞育的影响,从体重、羽化率、卵胚数、死亡率等方面测定了延长滞育幼虫的生殖势能,文章最后讨论了麦红吸浆虫多峰羽化的特点和延长滞育的生物学意义。