Free factor Xa is on the main pathway of thrombin generation in clotting plasma

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).     

Vitamin K3 (menadione) and related quinones, like tumor-promoting phorbol esters, alter the affinity of epidermal growth factor for its membrane receptors

The effects of vitamin K3, quinones, fat-soluble vitamins, and various naturally occurring and synthetic compounds on the binding of 125I-epidermal growth factor (EGF) to mink lung cells or murine 3T3 cells in culture were studied. Vitamin K3, but not other fat-soluble vitamins, markeldy inhibits the binding of 125I-labeled EGF to treated cells, but does not affect the binding of insulin, concanavalin A, alpha-2-macroglobulin, and murine leukemia virus glycoprotein, gp70, to their membrane receptors. The binding of multiplication stimulating activity to treated cells is also reduced to some extent. Vitamin K3 alters the affinity of the receptors for EGF without changing the total number of available receptors per cell. Vitamin K3 modulation of EGF-receptor interaction is a temperature- and time-dependent phenomenon. EGF-receptor interaction is also significantly modulated by 1,4-naphthoquinone, 1,4-benzoquinone, and phenanthrenequinone but not by other quinones of anthracyclic antibiotics.     

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.     

Vitamin‐regulated cytokines and growth factors in the CNS and elsewhere

There is a growing awareness that natural vitamins (with the only exception of pantothenic acid) positively or negatively modulate the synthesis of some cytokines and growth factors in the CNS, and various mammalian cells and organs. As natural vitamins are micronutrients in the human diet, studying their effects can be considered a part of nutritional genomics or nutrigenomics. A given vitamin selectively modifies the synthesis of only a few cytokines and/or growth factors, although the same cytokine and/or growth factor may be regulated by more than one vitamin. These effects seem to be independent of the effects of vitamins as coenzymes and/or reducing agents, and seem to occur mainly at genomic and/or epigenetic level, and/or by modulating NF‐κB activity. Although most of the studies reviewed here have been based on cultured cell lines, but their findings have been confirmed by some key in vivo studies. The CNS seems to be particularly involved and is severely affected by most avitaminoses, especially in the case of vitamin B₁₂. However, the vitamin‐induced changes in cytokine and growth factor synthesis may initiate a cascade of events that can affect the function, differentiation, and morphology of the cells and/or structures not only in the CNS, but also elsewhere because most natural vitamins, cytokines, and growth factors cross the blood–brain barrier. As cytokines are essential to CNS‐immune and CNS‐hormone system communications, natural vitamins also interact with these circuits. Further studies of such vitamin‐mediated effects could lead to vitamins being used for the treatment of diseases which, although not true avitaminoses, involve an imbalance in cytokine and/or growth factor synthesis.     

A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.     

Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X

We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.     

Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos (Forsskal) to formalin-killed Vibrio vulnificus

Juveniles of milkfish, Chanos chanos (Forsskal), were fed two independent supra dietary levels of vitamins C (500 and 1500 mg kg(-1) feed, T1 and T2) and E (50 and 150 mg kg(-1), T3 and T4). Milkfish fed diets with supra (in addition to the vitamins present in the control diet) and normal levels (T5 containing 90 and 1.2mg of vitamins C and E, respectively, kg(-1) of feed) of vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV). Priming and booster antibody responses to the injected bacterin were significantly (P<0.05) better in the milkfish juveniles fed supra dietary levels. Survival response of the experimental fish fed supra dietary levels of vitamins (T1, T2 and T3) was significantly (P<0.01) better than that of the control set. Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was better than the control (T5) and T1 and T4. Memory factor reflecting immunological memory was superior in the fish fed vitamin-supplemented diets. Diets supplemented with either 1500 mg of Vitamin C or 50mg of Vitamin E kg(-1) produced the best antibody responses, final survival and protective response upon challenge. No conclusive inferences could be drawn on the growth responses from the experiment.     

Serum levels of antioxidant vitamins, copper, zinc and magnesium in children with chronic rhinosinusitis

Reactive oxygen species including hydroxyl radicals, superoxide anions and hydrogen peroxide which are produced by activated granulocytes play an essential role in many biochemical processes and diseases. Oxidant-mediated tissue damage may be important in the development of chronic sinusitis. The aim of this study was to investigate the serum levels of antioxidant vitamins and elements in 24 children (14 boys and 10 girls, age range: 7-12 years, mean age: 9.2 years) with chronic rhinosinusitis, compared to 20 age and sex matched healthy children. Blood samples were collected in the morning before breakfast and prior to any medication. Vitamin A, E and C levels were determined using reagent kits for high performance liquid chromatography. Cu, Zn and Mg levels were analyzed by atomic absorption spectrometry. Vitamin E, vitamin C, Cu and Zn levels were significantly lower in the patients group than in the control group. However, vitamin A and Mg levels did not differ. In conclusion, serum levels of antioxidant vitamins and elements may be important in the pathogenesis and treatment of chronic rhinosinusitis in children.     

Homologous metabolic and gene activating routes for vitamins E and K

Vitamins E and K share structurally related side chains and are degraded to similar final products. For vitamin E the mechanism has been elucidated as initial ω-hydroxylation and subsequent β-oxidation. For vitamin K the same mechanism can be suggested analogously. ω-Hydroxylation of vitamin E is catalyzed by cytochrome P450 enzymes, which often are induced by their substrates themselves via the activation of the nuclear receptor PXR. Vitamin E is able to induce CYP3A-forms and to activate a PXR-driven reporter gene. It is shown here that K-type vitamins are also able to activate PXR. A ranking showed that compounds with an unsaturated side chain were most effective, as are tocotrienols and menaquinone-4 (vitamin K₂), which activated the reporter gene 8–10-fold. Vitamers with a saturated side chain, like tocopherols and phylloquinone were less active (2–5-fold activation). From the fact that CYPs commonly responsible for the elimination of xenobiotics are involved in the metabolism of fat-soluble vitamins and the ability of the vitamins to activate PXR it can be concluded that supranutritional amounts of these vitamins might be considered as foreign.     

Effects of vitamins, coenzymes and amino acids on reactions of homolytic cleavage of the O-glycoside bond in carbohydrates

It has been established that vitamins B1, K3 and C, coenzyme Q0 and amino acids cysteine and histidine effectively inhibit reactions of homolytic cleavage of the O-glycoside bond, which are responsible for the destruction of di- and polysaccharides on gamma-irradiation or the action of other reactive radical initiators. This effect was shown to originate from either oxidation or reduction of the radicals of carbohydrates undergoing destruction.     

代谢工程在维生素生产中的应用及研究进展

维生素是维持人体生命活动必需的一类有机物质,机体本身一般不能合成或合成量不足,因此需经食物或其他强化产品获取。目前,维生素产品已广泛应用于医药、食品添加剂、饲料添加剂、化妆品等领域,而且全球对维生素的需求也是呈逐年增长态势。维生素的生产方法主要包括化学合成法和生物合成法。化学合成法通常安全隐患大、反应条件严苛、废物污染严重,相比之下,代谢工程生产维生素绿色环保安全、能耗低,因此建立微生物细胞工厂具有重大的科学意义和应用需求。文中回顾了近30年来代谢工程在维生素生产领域的研究进展,详细阐述了水溶性维生素(维生素B1、B2、B3、B5、B6、B7、B9、B12和维生素C的前体)和脂溶性维生素(维生素A、维生素D的前体、维生素E和维生素K)的生物合成研究现状,并对其发酵生产的瓶颈进行了探讨,最后对合成生物技术创建维生素生产菌种进行了展望。     

Effects of mutagenesis of Gln97 in the switch II region of Escherichia coli elongation factor Tu on its interaction with guanine nucleotides, elongation factor Ts, and aminoacyl-tRNA

Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to the A-site of the ribosome. In a multiple-sequence alignment of prokaryotic EF-Tu's, Gln97 is nearly 100% conserved. In contrast, in mammalian mitochondrial EF-Tu's, the corresponding position is occupied by a conserved proline residue. Gln97 is located in the switch II region in the GDP/GTP binding domain of EF-Tu. This domain undergoes a significant structural rearrangement upon GDP/GTP exchange. To investigate the role of Gln97 in bacterial EF-Tu, the E. coli EF-Tu variant Q97P was prepared. The Q97P variant displayed no activity in the incorporation of [(14)C]Phe on poly(U)-programmed E. coli ribosomes. The Q97P variant bound GDP more tightly than the wild-type EF-Tu with K(d) values of 7.5 and 12 nM, respectively. The intrinsic rate of GDP exchange was 2-3-fold lower for the Q97P variant than for wild-type EF-Tu in the absence of elongation factor Ts (EF-Ts). Addition of EF-Ts equalized the GDP exchange rate between the variant and wild-type EF-Tu. The variant bound GTP at 3-fold lower levels than the wild-type EF-Tu. Strikingly, the Q97P variant was completely inactive in ternary complex formation, accounting for its inability to function in polymerization. The structural basis of these observations is discussed.     

Mitomycin C-activity effected by vitamins B1, C,E and beta-carotene under irradiation with gamma-rays

Vitamin B1 (thiamine) can essentially effect the activity of mitomycin C (MMC), added individually or in combination with antioxidant vitamins (C, E-acetate, beta-carotene) as found in experiments in vitro (Escherichia coli bacteria, AB 1157) under irradiation with gamma-rays. The environment plays a crucial role. In airfree media vitamin B1 leads to a 2-fold increase of the MMC-efficiency, but adding vitamin C it decreases. In the presence of all vitamins (B1, C, E-ac., and beta-carotene) the MMC-action increases about 1.8-fold. In aerated media vitamin B1 causes an about 4-times increase of the MMC-efficiency, but by adding vitamin B1 and C the MMC-activity decreases by a factor of two, whereas in the presence of B1, C, E-ac., and beta-carotene it rises again to 2.6-fold. In environment saturated with N2O (conversion of e(-)aq into OH radicals) a different picture is observed. The presence of vitamin B1 or vitamin B1 + C causes a strong decrease of the MMC-efficiency, but the addition of all vitamins (B1, C, E-ac., and beta-car.) leads to a small increase of the cytostatic action. The results demonstrate the influence of vitamin B1 used individually or in combination with other antioxidants on the MMC-efficiency and the strong effect of the environment. The results are of interest for the application of MMC in radiotherapy.     

Effects of various vitamins and coenzymes Q on reactions involving alpha-hydroxyl-containing radicals

Effects of vitamins B, C, E, K and P, as well as coenzymes Q, on formation of final products of radiation-induced free-radical transformations of ethanol, ethylene glycol, alpha-methylglycoside and glucose in aqueous solutions were studied. Based on the obtained results, it can be concluded that there are substances among vitamins and coenzymes that effectively interact with alpha-hydroxyl-containing radicals. In the presence of these substances, recombination reactions of alpha-hydroxyalkyl radicals and fragmentation of alpha-hydroxy-beta-substituted organic radicals are suppressed. It has been established that the observed effects are due to the ability of the vitamins and coenzymes under study to either oxidize alpha-hydroxyl-containing radicals yielding the respective carbonyl compounds or reduce them into the initial molecules.     

The role of natural antioxidants in preserving the biological activity of endothelium-derived nitric oxide

Endothelium-derived nitric oxide (EDNO) is a pivotal molecule in the regulation of vascular tone via the stimulation of vascular smooth muscle cell relaxation and concomitant vasodilation. In addition, EDNO exerts a number of other potent antiatherogenic effects, including inhibition of leukocyte-endothelial interactions, smooth muscle cell proliferation, and platelet aggregation. Endothelial vasodilator dysfunction has been observed in patients with CAD or coronary risk factors such as hypercholesterolemia, hyperhomocysteinemia, essential hypertension, diabetes mellitus, smoking, and aging. Most of these conditions are associated with increased oxidative stress, particularly increased production of superoxide radicals and elevated levels of oxidized LDL, both of which can attenuate the biological activity of EDNO. The levels of superoxide and oxidized LDL can be decreased by administering the small molecule antioxidants vitamins E and C. Vitamin C also spares intracellular thiols, which in turn can stabilize EDNO through the formation of biologically active S-nitrosothiols. Here we review the role that vitamins E and C and thiol compounds play in endothelium-dependent vasodilation. Understanding the mechanisms of the reversal of endothelial dysfunction by natural antioxidants will lead to successful therapeutic interventions of CAD and its clinical sequelae.     

Interaction of vitamin B1 with bovine serum albumin investigation using vitamin B1-selective electrode: potentiometric and molecular modeling study

Vitamin B₁ or thiamin is one of the B vitamins. All B vitamins help the body to convert food (carbohydrates) into fuel (glucose), which produces energy. The B vitamins are necessary for healthy skin, eyes, hair, and liver. It also could help the nervous system function properly, and is necessary for brain functions. Drug interactions with protein can affect the distribution of the drug and eliminate the drug in living systems. In this study, the binding of thiamine hydrochloride (vitamin B₁) to bovine serum albumin (BSA) was evaluated using a new proposed vitamin B₁ (thiamine)-selective membrane electrode under various experimental conditions, such as pH, ionic strength, and protein concentration; in addition molecular modeling was applied as well. The binding isotherms plotted based on potentiometric data and analyzed using the Wyman binding potential concept. The apparent binding constant was determined and used for the calculation of intrinsic Gibbs free energy of binding. According to the electrochemical and molecular docking results, it can be concluded that the hydrophobic interactions and hydrogen binding are major interactions between BSA and vitamin B₁.