Reductive dechlorination and degradation of mirex and kepone with Vitamin B12.

Vitamin B12s effects the reductive dechlorination of mirex (dechlorane) in protic solvent systems, under both catalytic and stoichiometric conditions, mainly to yield compounds of composition C10Cl12-nHn, with n = 1-8, in which the basic dihomocubane cage structure is retained; the formation of cage-opened, reductively dehalogenated derivatives of 4,7-methanoindene occurs only to a very minor extent. The corresponding reactions of kepone (chlordecone), in contrast, occur with predominant formation of indene derivatives. Under certain mild conditions, vitamin B12s induces a fragmentation of kepone leading to the destruction of the dihomocubane moiety and the formation of an isolable organocobalamin having a C3Cl3H2 residue attached to the cobalt atom. In strongly alkaline media, the reaction of kepone with vitamin B12s may in addition yield high-molecular-weight condensation products of unknown constitution. Reactions of this type are of interest as prototypes of soil-decontamination processes.     

Changes in lipid composition of mucosa secretion from chick glandular stomach intermediate zone in A-vitaminosis

The lipid composition of secretion and the vitamin A content in it were studied. Secretion of normal chickens is shown to contain 96 +/- 3.1 mg of lipids, 82.5 +/- 3.4 of substances extracted by the solvent hexane-diethyl ester, 30 +/- 2.1 of unsaponifiable lipids and 2.43 +/- 0.285 micrograms of vitamin A per 1 g of dry residue. With A-avitaminosis the secretion amount is twice as large with vitamin A absent; the lipid content is almost twice as low, the total weight of substances extracted by the solvent hexane-diethyl ester is four times as low and that of the lipid unsaponifiable fraction--2.5 times as low. Quantitative and qualitative changes in lipid zones were detected by means of thin-layer chromatography and UV-spectroscopy, and structural changes in the secretion--by electron microscopy. Vitamin A is suggested to participate in formation of macromolecular lipid systems of the secretion.     

The content of lipid peroxidation products and the activity of antioxidant enzymes in the myocardium and liver of rats with various vitamin E allowances

After 2 month of feeding vitamin E-supplemented diet (100.6 and 0 mg/kg; group I-control, II and III, respectively) the concentration of lipid peroxidation products (diene conjugates, malondialdehyde, Schiff's bases) and activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase) was estimated in rat heart and liver. Although the content of alpha-tocopherol in organs of group II was significantly decreased, the concentration of peroxidation products and enzyme activities was unchanged. Moreover, these parameters were constant in rat liver of group III. The heart was more sensitive because in group III to vitamin E deficiency (the alpha-tocopherol level was dropped fourfold) the concentration of diene conjugates and malondialdehyde was increased and superoxide dismutase activity was decreased. Thus insufficiency of vitamin E may result in selective alterations of myocardial functions. In addition, vitamin E may be useful instrument for correction of free radical oxidation and antioxidant system activity in the heart.     

Effect of excess and deficiency of vitamin A on the utilization of FFA by liver and skeletal muscle.

Fatty acid metabolism in liver and skeletal muscle has been studied in rats treated with high doses of vitamin A and in those made vitamin A-deficient. Ingestion of 30,000 IU of vitamin A for two days resulted in increased incorporation of palmitate-1-14C into triglycerides but not into phospholipids. Accumulation of hepatic triglycerides was observed in vitamin A-fed rats. Deficiency of vitamin A did not cause any change in the triglyceride or phospholipid content of the liver. The rate of hepatic fatty acid oxidation and ketogenesis was markedly increased in vitamin A-fed rats. The experimental evidence indicated that vitamin A may have a stimulatory effect on these processes apart from that exerted by the high plasma FFA level in vitamin A-fed rats. Oxidation of palmitate-1-14C into C32 by skeletal muscle (latissimus dorsi) was also increased as a result of vitamin A administration. Vitamin A deficiency did not cause any change in fatty acid oxidation by liver and skeletal muscle. Hepatic palmitoyl-CoA synthetase activity was decreased in vitamin A-deficient rats. The results presented suggest that vitamin A may be required for the uptake and utilization of fatty acids by liver and akeletal muscle.     

Anomalous antioxidant effects in selenium- and vitamin E-deficient liver mitochondria

In this study, we investigate the mechanisms of two anomalous protective effects of exogenous vitamin E that had previously been postulated to involve either a specific antioxidant effect or a non-antioxidant function of the vitamin. These atypical vitamin E effects were observed during the prevention of NAD-induced respiratory decline occurring in homogenates and mitochondria prepared from vitamin E- and selenium-deficient rat liver. The study showed neither hypothesis to be true; rather, the two effects, one in homogenates and the other in isolated mitochondria, were explained by other mechanisms. The protective effect against respiratory decline in homogenates was found to result from interference in the thiobarbituric acid assay for lipid peroxidation by ethanol (the conventional solvent for vitamin E addition). With other non-interfering solvents, inhibition of lipid peroxidation by vitamin E, in contrast to previous studies, correlated perfectly with prevention of respiratory decline. The atypical vitamin E effect occurring in isolated mitochondria—and consisting of a requirement for cytosol proteins for the prevention of respiratory decline by exogenous vitamin E—was found to be caused by the prevention of adverse glass effects and not by the action of vitamin E-specific binding proteins. Frequent failures in the combined protective effect of vitamin E and cytosol, which had been a major complication of respiratory decline studies, were found to be caused by phospholipase activity generated during isolation procedures. Irreversible deactivation of respiratory enzymes by lipid peroxidation was found not to be involved in the respiratory decline mechanism. In memoriam: Klaus Schwarz, MD, 1914–1978.     

维生素K2对四氯化碳诱导的急性大鼠肝损伤的保护作用

探讨枯草芽胞杆菌产物维生素K2(MK-7)对四氯化碳诱导的急性肝损伤的保护作用及机制。

选取SPF级实验用6~8周龄SD雄性大鼠30只, 体质量200~220 g, 随机分为5组(每组6只): 空白组、肝损伤模型组、模型+溶剂组、模型+水飞蓟素组和模型+维生素K2组。空白组不做额外处理, 其余各组均予四氯化碳染毒, 溶剂组给予与维生素K2组相同体积的大豆油, 水飞蓟素组添加水飞蓟素100 mg/100 g体质量, 维生素K2组用维生素K2每日灌胃(给药剂量为2 μg/100 g体质量)。1周后观察大鼠体征和肝脏外观、肝脏炎症及微肉芽肿、肝细胞空泡化结构, 检测血液AST、ALT、MDA、SOD、TNF-α和IL-6含量。

与肝损伤模型组大鼠比, 模型+维生素K2组肝脏指数下降(t=3.250 0, P=0.031 4)。HE染色结果显示, 与肝损伤模型组大鼠比, 模型+维生素K2组空泡化程度明显降低, 损伤较为弥散。与肝损伤模型组大鼠比, 模型+维生素K2组肝功、氧化应激指标及相关炎症因子水平降低[AST(t=4.283 0, P=0.012 8)、ALT(t=2.582 0, P=0.041 6)、MDA(t=7.028 0, P=0.005 9)、SOD(t=3.384 0, P=0.011 7)、TNF-α(t=3.459 0, P=0.013 5)、IL-6(t=2.422 0, P=0.041 8)]。

维生素K2可减轻大鼠急性肝损伤程度, 其作用可通过改善抗氧化酶体系、抑制氧化应激反应及降低炎性因子水平而实现。

     

Subcellular distribution of free and esterified forms of vitamin a in the pigment epithelium of the retina and in liver

• 1.1. The distribution of vitamin A was examined in various subcellular fractions of rat liver and bovine retinal pigment epithelium. In rat liver, the major portion of the vitamin is in the cytosol, whereas in pigment epithelium, it is concentrated mainly in the microsomes. The microsomal vitamin A of pigment epithelium is tightly bound to membranes, as shown by the inability to release it except by organic solvent extraction or incubation with Triton X-100.
• 2.2. In both tissues, two different forms of cytosol vitamin A could be distinguished by ultracentrifugation. The major portion in liver is in the flotating lipid phase and consists mainly of retinyl ester. The remainder (less than 10% of the total) is in the underlying infranatant; about 90% of the vitamin A in this fraction is esterified. By contrast, two-thirds of the vitamin A of pigment epithelial cell cytosol is in the infranatant; it consists of both esterified and unesterified retinol. The floating layer in the pigment epithelial cytosol consists entirely of retinyl ester.
• 3.3. These two forms of cytosol vitamin A in the pigment epithelium could also be separated by gel filtration on Sephadex G-100 which yielded two distinct fluorescent peaks. The first, which appeared in the void volume and corresponded in all probability to the floating layer obtained by ultracentrifugation, consisted only of retinyl ester. The second peak, which was eluted in approximately the same position as myoglobin, contained only unesterified retinol. It was abolished completely by preincubation with pronase. These findings support the view that the second peak represents the endogenous retinol-retinol binding protein complex of pigment epithelial cytosol. The fluorescent enhancement of the retinol bound to protein in this peak was about 4–5-fold compared to retinol in organic solvents.

     

Crystal structure of a secondary vitamin D3 binding site of milk beta-lactoglobulin

Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).     

Evidence of p53 mutation in an early stage of liver cancer in European flounder, Platichthys flesus (L.)

A number of flounders dwelling in highly contaminated coastal areas of Northern Europe develop liver tumours. In order to increase our understanding of the molecular pathogenesis of these sporadic tumours, we examined p53 mutations in eleven hyperplasia and six adenoma. p53 introns 4 to 8 were first sequenced to allow individual amplification of exons 5 to 8. DNA extracted from formalin-fixed livers was amplified and PCR products were directly sequenced. Two major results were obtained. (i) Flounders from different geographical areas displayed a high rate of sequence variation. Base substitutions were identified in both tumour and normal tissues and thus may be considered as polymorphic variations in individuals. (ii) One mutation was detected in two hyperplastic foci from the same flounder. This mutation was a T:A to A:T transversion at codon 147, resulting in the replacement of valine for glutamic acid. This residue took place in the L2 loop of the DNA binding surface. Its substitution by an hydrophilic and charged residue could thus impair p53 (protein) biological activity.     

Identification of vitamin K1 chromenol--a novel metabolite of vitamin K1 formed in vitro by a component in blood

A newly recognized metabolite of vitamin K1, vitamin K1 chromenol, is produced when the vitamin is added to the plasma or serum of a number of species. The metabolite was identified by comparison of its uv and mass spectra and high-performance liquid chromatographic retention times with those of the synthetic vitamin K1 chromenol. In aqueous solution vitamin K chromenol decomposed to a variety of products and reacted with nucleophilic substances. Optimal conditions for its formation and evidence that chromenol formation may be an enzyme catalyzed reaction are presented.     

Activity of radiation degradation products of vitamins A and E to haemolyse erythrocyte

Exposure of vitamin A acetate in freely dissolved state to γ-radiationin vitro caused a dose dependent degradation accompanied by the formation of new products. The radiation degradation products were separated by chromatography using step gradient elution. The parent molecule, vitamin A acetate, induced negligible haemolysis of erythrocytes. In contrast, the polar products formed by irradiation were found to be potent haemolysing agents. A highly polar product, eluted with methanol revealed maximum haemolytic activity. Acetylation of these products resulted in loss of their haemolytic properties. Similarly, vitamin E acetate, a known stabilizer of the biomembranes, after irradiation yielded products which caused haemolysis of erythrocytes. It was demonstrated that irradiation introduces hydroxyl groups which impart haemolytic properties to the radiation degradation products of vitamin A     

Quantitative Characterization of Local Protein Solvation To Predict Solvent Effects on Protein Structure

Characterization of solvent preferences of proteins is essential to the understanding of solvent effects on protein structure and stability. Although it is generally believed that solvent preferences at distinct loci of a protein surface may differ, quantitative characterization of local protein solvation has remained elusive. In this study, we show that local solvation preferences can be quantified over the entire protein surface from extended molecular dynamics simulations. By subjecting microsecond trajectories of two proteins (lysozyme and antibody fragment D1.3) in 4 M glycerol to rigorous statistical analyses, solvent preferences of individual protein residues are quantified by local preferential interaction coefficients. Local solvent preferences for glycerol vary widely from residue to residue and may change as a result of protein side-chain motions that are slower than the longest intrinsic solvation timescale of ～10 ns. Differences of local solvent preferences between distinct protein side-chain conformations predict solvent effects on local protein structure in good agreement with experiment. This study extends the application scope of preferential interaction theory and enables molecular understanding of solvent effects on protein structure through comprehensive characterization of local protein solvation.     

Critical evaluation of the existence of so-called tissue-bound lithocholate in human liver tissue by selected ion monitoring

Monohydroxy bile acids in liver tissue may be of importance because of their hepatotoxicity and strong cholestatic effects. Recently, the existence of lithocholate in liver tissue in two forms was suggested by Nair et al. (Lipids. 1977. 12: 922-929) i.e., either in free form or as so-called tissue-bound lithocholate released exclusively by cholylglycine hydrolase treatment. The presence of the latter aroused much interest in relation to its hepatotoxicity and possible role in tumor induction. In the present investigation lithocholyl-epsilon-L-lysine, proposed as the predominant tissue-bound bile acid, was synthesized and its metabolic behavior was tested. Lithocholyl-epsilon-lysine was not deconjugated by cholylglycine hydrolase treatment but only by alkaline hydrolysis. Bile acids in seven cirrhotic and three noncirrhotic liver samples were extracted with 95% ethanol-0.1% ammonium hydroxide. The bile acids in the extract and residue were quantified by glass capillary gas-liquid chromatography using selected ion monitoring. The presence of so-called tissue-bound lithocholate could not be substantiated in either cirrhotic or noncirrhotic liver tissues. Nearly complete extraction of lithocholate was achieved by the use of organic solvent alone. Therefore, tissue-bound lithocholate, if it exists at all, may be attached to tissue by a physical linkage which can be disrupted by the use of conventional organic solvent.     

Content of dolichol and retinol in isolated rat non-parenchymal liver cells

The liver sinusoids, that are considered as a functional unit, harbour four types of sinusoidal cells (Ito, Kupffer, endothelial and pit cells). Dolichol content has been determined in many tissues and subcellular compartments, alteration has been reported in many types of liver injury, but until now no data are available on its content in every type of sinusoidal non-parenchymal liver cells. Dolichol and retinol metabolism might intersect in their traffic in biological membranes. Intercellular as well as intracellular exchange of retinoids is an essential element of important processes occurring in liver cells. It has been suggested that the role of dolichol, besides being a carrier of oligosaccharides in the biosynthesis of N-linked glycoproteins, may be to modify membrane fluidity and permeability, and facilitate fusion of membranes. Dolichol in the membrane is intercalated between the two halves of the phospholipid bilayer, but its exact disposition is not known and the movement and distribution of retinoid in membranes may vary with the geometry of the membranes. Therefore the aim of this study is to obtain a global understanding of the sinusoidal system regarding dolichol and retinol content in each type of isolated rat liver sinusoidal cell, in normal conditions and after vitamin A administration. The information that can be drawn from the present results is that with normal vitamin A status of the animal, the dolichol content is almost uniform in all liver cells. After vitamin A supplementation, a great increase of dolichol, together with the known increase of retinol, can be measured only in a subpopulation of the Ito cells, the Ito-1 subfraction. Therefore in the cells that are present in the hepatic sinusoid, different pools of dolichol may have separate functions. Because retinol traffic among cells, membranes and plasma still remains to be fully understood, roles of dolichol in the exchange of vitamin A among sinusoidal liver cells are discussed. © 1998 John Wiley & Sons, Ltd.     

Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells

Vitamin E is the primary lipophilic antioxidant in mammals. Lack of vitamin E may lead to an increase of cytotoxic phospholipid-peroxidation products (PL-Ox). However, we could previously show that alimentary vitamin E-depletion in rats did not change the concentrations of dienes, hydroperoxides, and platelet-activating factor-related oxidation products in alveolar type II cells (TII cells). We hypothesized that vitamin E deficiency increases the activity of enzymes involved in the degradation of PL-Ox. Degradation of PL-Ox may be catalyzed by phospholipase A2, PAF-acetylhydrolase, or peroxiredoxins (Prx's). Alimentary vitamin E deficiency in rats increased the expression of Prx-1 at the mRNA and protein levels and the formation of Prx-SO3, but it did not change the expression of Prx-6 or the activity of phospholipase A2 and PAF-acetylhydrolase in TII cells. H2O2-induced oxidative stress in isolated TII cells activated protein kinase Calpha (PKCalpha) and increased the expression of Prx-1 and Prx-6. Inhibition of PKCalpha in isolated TII cells by long-time incubation with PMA inhibited PKCalpha and Prx-1 but not Prx-6. We concluded that the expression of Prx-1 and -6 is selectively regulated in TII cells; PKCalpha regulates the expression of Prx-1 but not Prx-6. Prx-6 expression may be closely linked to lipid peroxidation.     

Apoptosis-inducing activity of a driselase digest fraction of green tea residue

We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70% ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.     

Effect of ligand binding on the conformation of human plasma vitamin D binding protein (group-specific component).

Several techniques have been used to demonstrate that the binding of specific ligands to human plasma vitamin D binding protein induces a change in protein conformation. Apoprotein and holoprotein show circular dichroism spectra of similar form in the peptide region with double minima at 207 and 218 nm. The minimum mean residue ellipticity of apoprotein (20.6 X 10(3) degrees.cm2.dmol-1) is decreased by about 8% after vitamin D3 binding, suggesting a small change in the backbone conformation. Spectrofluorimetric studies showed that 25-hydroxycholecalciferol causes a saturable enhancement of intrinsic fluorescence of human vitamin D binding protein and alters the pH profile of protein fluorescence, suggesting that there are alterations in the local environment of tryptophan residue(s) after ligand binding. Furthermore, in the presence of 25-hydroxycholecalciferol, the rate of chemical modification of the amino groups in human vitamin D binding protein is decreased and the susceptibility of intact vitamin D binding protein to proteolytic degradation is reduced, suggesting that some surface sites in the vitamin D binding protein molecule are less accessible to external agents. In addition, although the absorbance of vitamin made if difficult to interpret the ultraviolet spectra of holoprotein and apoprotein, the presence of vitamin D binding protein appears to stabilize the vitamin in an aqueous environment, a phenomenon that may be of physiological importance.     

固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

RCNPRED: prediction of the residue co-ordination numbers in proteins

The RCNPRED server implements a neural network-based method to predict the co-ordination numbers of residues starting from the protein sequence. Using evolutionary information as input, RCNPRED predicts the residue states of the proteins in the database with 69% accuracy and scores 12 percentage points higher than a simple statistical method. Moreover the server implements a neural network to predict the relative solvent accessibility of each residue. A protein sequence can be directly submitted to RCNPRED: residue co-ordination numbers and solvent accessibility for each chain are returned via e-mail. AVAILABILITY: Freely available to non-commercial users at http://prion.biocomp.unibo.it/rcnpred.html.