Cerebroside synthesis as a measure of the rate of remyelination following cuprizone-induced demyelination in brain

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12-week time-course, control animals showed slight increases in the content of oligodendroglial-specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of (3)H(2)O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down-regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl-CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin-related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings

One bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar and residual dipolar couplings have been measured at sites in an ¹⁵N, ¹³C, 50% ²H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that ¹H-¹³C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the H_methylC_methylC angle are found to be small, with average differences on the order of 0.3 ± 0.1° and 0.4 ± 0.2° between CH₃, CH₂D and CH₃, CHD₂ isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.     

Converging catabolism of 2,4,6-trinitrophenol (picric acid) and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A

Initial F₄₂₀-dependent hydrogenation of 2,4,6-trinitrophenol(picric acid) generated the hydride -complex of picrate and finally the dihydride complex.With 2,4-dinitrophenol the hydride -complex of 2,4-dinitrophenolis generated. The hydride transferring enzyme system showed activity against several substituted2,4-dinitrophenols but not with mononitrophenols. A K_m-value of0.06 mM of the hydride transfer for picrate as substrate was found. The pH optimaof the NADPH-dependent F₄₂₀ reductase and for the hydride transferase were 5.5and 7.5, respectively. An enzymatic activity has been identified catalyzing the releaseof stoichometric amounts of 1 mol nitrite from 1 mol of the dihydride -complexof picrate. This complex was synthesized by chemical reduction of picrate and characterizedby ¹H and ¹³C NMR spectroscopy. The hydride -complex of 2,4-dinitrophenolhas been identified as the denitration product. The nitrite-eliminating activitywas enriched and clearly separated from the hydride transferring enzyme system byFPLC. 2,4-Dinitrophenol has been disproven as a metabolite of picrate (Ebert et al. 1999)and a convergent catabolic pathway for picrate and 2,4-dinitrophenol with thehydride -complex of 2,4-dinitrophenol as the common intermediate has been demonstrated.     

类胡萝卜素对亚油酸甲酯氧化的抑制作用

研究溶液中类胡萝卜素对2 ,2’ -偶氮二 (2 ,4短杠二甲基戊腈 ) (AMVN)引发的亚油酸甲酯氧化的抑制作用及色素的消耗变化。表明 β-胡萝卜素 ,叶黄素及胭脂树橙都按依赖于剂量的方式抑制亚油酸甲酯氢过氧化物的形成。41.7×10 -6mol/L的叶黄素及玉米黄质的抑制活性相近 ,高于β-胡萝卜素与胭脂树橙的活性 ,后两者活性近似。在83.3×10 -6mol/L浓度下鸡油菌黄质活性高于叶黄素 ,后者又高于 β-胡萝卜素与胭脂树橙 ,最后两者仍近似。结果表明下列五种类胡萝卜素对亚油酸甲酯氧化的抑制能力为 :鸡油菌黄质>玉米黄质≈叶黄素> β-胡萝卜素≈胭脂树橙。类胡萝卜素在抑制脂质氧化过程中 ,本身逐渐损失消耗 ,4h后在试验所用浓度下最高剩余量不超过20 %。其中鸡油菌黄质与胭脂树橙的消耗速度慢于 β-胡萝卜素与玉米黄质。     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

A simplified procedure for the purification of human N-acetylglucosaminyltransferase-III from human hepatocellular carcinoma tissues

Human N-acetylglucosaminyltransferase-III (GnT-III) has not been hitherto purified from either tissue or cell line. Although rat GnT-III has been purified from rat kidney tissue, the procedure involves laborious and complex steps. We have developed a simplified procedure based on a QAE (diethyl[2-hydroxypropyl]aminoethyl)-Sepharose and an immunoaffinity chromatography technique. The immunoaffinity chromatography utilized a monoclonal antibody to human aglycosyl recombinant N-acetylglucosaminyltransferase-III. With the new procedure, human N-acetylglucosaminyltransferase-III was purified from hepatocellular carcinoma tissues with an increase in the specific enzyme activity of 378-fold.     

Prebiotic Methylation and the Evolution of Methyl Transfer Reactions in Living Cells

An hypothesis is presented for theprebiotic origin of methyl groups and the evolution ofmethyl transfer reactions in living cells. This hypothesis,described in terms of prebiotic and early biotic chemicalevolution, is based on experimental observations in our laband in those of others, and on the mechanisms of enzymaticmethyl transfer reactions that occur in living cells. Ofparticular interest is our demonstration of the reductivemethylation of ethanolamine and glycine in aqueous solutionby excess formaldehyde. These reactions, involving prebioticcompounds and conditions, are mechanistically analogous tothe de novo origin of methyl groups in modern cellsby reduction of methylene tetrahydrofolate. Furthermore,modern cellular methyl transfers from S-adenosylmethionineto amine nitrogen may involve formaldehyde as anintermediate and subsequent reductive methylation, analogousto the prebiotic chemistry observed herein.     

光氧化胁迫下几种植物叶片的超氧自由基产生速率和光合特性

以甲基紫精 (MV) 0～ 1mmol/L在 15 0 0 μmolm-2s-1光下处理C3 植物花生、水稻和C4 植物玉米、甘蔗的叶圆片 30min ,O- ·2 产生速率随MV浓度提高而加快。MV浓度超过 10 μmol/L ,光合放氧出现负值并持续增大。光氧化作用降低叶绿素荧光参数Fv/Fm ,ΦPSⅡ和qP,而qN 则或提高 (C3 植物 ,MV 10 μmol/L )或几乎不变甚至有所降低 (C4 植物或C3 植物在高浓度MV下 )。热耗散系数KD 的变化与qN 相似。与C4 植物相比 ,C3 植物的O- ·2 产生速率和光下吸氧的速率较高 ,SOD活性较低 ,Fv/Fm ,ΦPSⅡ和qP 的降低幅度比C4植物大。对田间玉米叶片的MV涂抹试验看到 ,MV降低光合CO2 同化速率的同时 ,也降低气孔导度、PEP羧化酶和SOD活性 ,Ci增大。结果表明MV光氧化作用刺激PSⅠ的O2 光还原作用 ,引起PSⅡ部份失活 ,气孔部份关闭和关键酶类氧化失活 ,从而抑制了光合作用     

茉莉酸甲酯诱导烟草幼苗抗炭疽病与PAL活性及细胞壁物质的关系

探讨了茉莉酸甲酯(methyl jasmonate, Me-Ja)诱导烟草幼苗抗炭疽病与苯丙氨酸解氨酶(PAL)活性、木质素和富含羟脯氨酸蛋白(HRGP)含量的关系。 Me-Ja处理烟草幼苗不仅可以提高幼苗抗炭疽病的能力,而且明显提高幼苗的PAL活性、木质素和HRGP含量; 3个不同抗性品种的烟草幼苗的PAL酶活性、木质素和HRGP含量三者与感病程度之间的负相关都达到显著水平,表明PAL、木质素和富含羟脯氨酸蛋白(HRGP)在茉莉酸甲酯诱导抗病中起关键作用。