The reaction of 1-fluoro-2,4-dinitrobenzene with bone of different ages

Bone from cattle of different ages was reacted with FDNB under various experimental conditions. Attempts were made to assess the difference between age groups in the size of the four categories of -amino groups of bone collagen postulated byWuthier et al. (1964). Bone from very young calves reacted with FDNB under anhydrous conditions to a much greater extent than bone from older animals. This was interpreted to mean that relatively more free -amino groups (Category I) and fewer -amino groups electrovalently associated with the mineral (Category II) are present in very young bone. Studies employing FDNB treatment simultaneous with EDTA decalcification suggested that little difference exists between age groups in the -amino groups interpreted to be more tightly bound to the bone mineral (Category III) and those interpreted to be internally bonded within collagen and inaccessible for FDNB reaction (Category IV).On the basis of FDNB reaction and measurements of bone density, ash and collagen content, it was concluded that the initial bonding between collagen and bone mineral probably occurs with the category III -amino groups. With maturation more mineral accumulates within the existing collagenous matrix. This would appear to be accompanied by an increasing electrovalent association between the mineral and free -amino groups of bone collagen.

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Zusammenfassung Knochen von Vieh wechselnden Alters wurden unter verschiedenen Versuchsbedingungen mit FDNB versetzt. Es wurde versucht, den vonWuthier et al. (1964) vorgeschlagenen Unterschied in der Verteilung der vier Kategorien von -Aminogruppen im Knochenkollagen bei verschiedenen Altersgruppen zu messen. Knochen von sehr jungen Kälbern reagierten mit FDNB unter anhydrischen Bedingungen in viel größerem Maß als Knochen von älteren Tieren. Daraus wurde geschlossen, daß relativ mehr freie -Aminogruppen (Kategorie I) und weniger -Aminogruppen elektrovalent mit dem Mineral verbunden (Kategorie II) in sehr jungem Knochen vorkommen. Studien, wobei FDNB-Behandlung gleichzeitig mit EDTA-Decalcifikation angewendet wurde, ließen vermuten, daß wenig Unterschiede zwischen den Altersgruppen besteht hinsichtlich den -Aminogruppen, welche stärker an das Knochenmineral gebunden scheinen (Kategorie III) und solchen, welche im Innern mit Kollagen gebunden vernetzt und deshalb für die Reaktion des FDNB unerreichbar sind (Kategorie IV).Auf Grund der FDNB-Reaktion und Messungen der Knochendichte, der Asche und des Kollagengehaltes kann ausgesagt werden, daß die primäre Vernetzung zwischen Kollagen und Knochenmineral wahrscheinlich in der III. Kategorie der -Aminogruppen vorkommt. Während des Wachstums lagert sich mehr Mineral innerhalb der bestehenden Kollagenmatrix ab. Dieser Vorgang scheint von einer zunehmenden elektrovalenten Verbindung zwischen Mineral und den freien -Aminogruppen des Knochenkollagens begleitet.

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Résumé Des os bovins de différents âges ont été soumis au FDNB dans diverses conditions expérimentales. Une différence dans la taille des quatre catégories de groupes -aminés du collagène osseux deWuthier et coll. (1964) a été recherchée dans les groupes d'âges différents. L'os de très jeunes veaux réagit plus fortement dans des conditions anhydres avec du FDNB que l'os d'animaux plus âgés. Ce fait semble indiquer que relativement plus de groupes -aminés libres (catégorie I) et moins de groupes -aminés associés par électrovalence avec le minéral (catégorie II), sont présents dans le très jeune os. Des études utilisant conjointement du FDNB et une décalcification par EDTA indiquent qu'il existe peu de différence en groupes -aminés en fonction de l'âge. Ces groupes semblent être intimement liés au minéral osseux (catégorie III). De même dans les groupes liés à l'intérieur du collagène et inaccessibles à la réaction au FDNB (catégorie IV), il existe peu de différence en fonction de l'âge.En tenant compte de la réaction au FDNB, de la densité osseuse et du contenu en cendres et en collagène, il semble que la liaison initiale entre le collagène et le minéral osseux se situe vraisemblablement au niveau des groupes -aminés de la catégorie III. Au cours de la croissance, plus de sels minéraux s'accumulent dans la matrice collagénique préexistante. Ce fait semble accompagné par une augmentation de l'association entre le minéral et les groupes -aminés du collagène osseux.

     

大黄总蒽醌对大鼠肾脏水通道蛋白2和水通道蛋白4表达的影响

目的 研究大黄总蒽醌对大鼠肾脏水通道蛋白（AQP）2、AQP4表达的影响及探讨大黄利尿机制。 方法 32只SD大鼠随机分为正常对照组，大黄总蒽醌低、中、高剂量组，每组8只，分别给予不同剂量大黄总蒽醌灌胃，每天1次，共灌5 d。第4天测定大鼠24 h尿量，尿液Na+水平及尿渗透浓度。5 d后处死大鼠，腹主动脉取血，进行血液生化指标检测；并留取肾脏，用免疫组化、Western印迹和RT-PCR法检测肾脏AQP2、AQP4蛋白及mRNA表达变化。 结果 与正常对照组比较，大黄总蒽醌中、高剂量组大鼠24 h尿量显著增加[(16.21±1.96) ml、(18.16±1.80) ml比（13.85±1.25）ml, P均＜ 0.05]，低剂量组大鼠尿量变化不明显；中、高剂量组大鼠肾脏AQP2蛋白及mRNA表达均显著下降（P均＜ 0.01），高剂量组大鼠肾脏AQP4蛋白及mRNA表达显著下降（P ＜ 0.01）；中剂量组大鼠AQP4蛋白表达下降（P ＜ 0.01），但mRNA表达无显著降低；低剂量组大鼠AQP2、AQP4蛋白及mRNA表达无显著变化。 结论 大黄总蒽醌能降低大鼠肾脏AQP2、AQP4蛋白及mRNA表达水平，这可能是大黄产生利尿的机制之一。     

The effect of 2,4-diamino-6-hydroxy-pyrimidine on postburn Staphylococcus aureus sepsis in rats

GTP-cyclohydrolase I (GTP-CHI) is the first and rate-limiting enzyme for the de novo biosynthesis of biopterin. The present study was to observe the effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP),an inhibtor of GTP-CHI, on the development of postburn Staphylococcus aureus sepsis. Methods: 56 male Wistar rats were randomly divided into four groups as follows: normal control group (n= 10), scald control group(n= 10),pos tburn sepsis group (n= 20) and DA HP treatment group (n= 16). In the scald control group, rats were subjected to a 20% total body surface area (TBSA) Ⅲ° scald injury, then sacrificed at 24 hrs. In the postburn sepsis group (n=20), rats were inflicted with 20% TBSA Ⅲ° scald followed by Staphylococcus aureus challenge, and they were further divided into 2 and 6 hrs groups. In the DAHP treatment group (n= 16), animals were intraperitoneally injected with a dose of 1g/kg DAHP prior to Staphylococcus aureus challenge, and then further divided into 2, 6 hrs groups. Tissue samples from liver, kidneys, lungs and heart were collected to determine GTP-CHI, inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) mRNA expression. Meanwhile, biopterin and nitric oxide (NO) levels in these tissues were also measured. Results: After the scald injury followed by Staphylococcus aureus challenge, GTP-CHI mRNA expression and biopterin levels significantly elevated in various tissues such as liver, heart, kidneys and lungs, so did the values of iNOS mRNA expression and NO formation (P＜0.01). Pretreatment with DAHP could significantly reduce GTP-CHI/biopterin induction (P＜0. 05～0. 01), and the up-regulation of iNOS/NO was also suppressed. Furthermore, DAHP administration could also inhibit the gene expression of TNF-α. 2 hrs after septic challenge, TNF-α mRNA expression in liver, kidneys and lungs in DAHP-treated group were 35.7%, 37.3% and 33.0% of those in postburn septic group, respectively. Additionally, in animals without DAHP treatment, the 6-hour mortality was 55.6% (20/36), while it was only 25.0% in DAHP-treated animals (4/16, P=0. 08). Conclusions: Early treatment with DAHP might be a potential strategy to prevent the development of postburn Staphylococcal sepsis, which appears to be associated with down-regulation of biopterin and NO formation by DAHP.     

The uncoupling agent 2,4-dinitrophenol improves mitochondrial homeostasis following striatal quinolinic acid injections

It is now generally accepted that excitotoxic cell death involves bioenergetic failure resulting from the cycling of Ca2+ and the generation of reactive oxygen species (ROS) by mitochondria. Both Ca2+ cycling and ROS formation by mitochondria are dependent on the mitochondrial membrane potential (Deltapsi(m)) that results from the proton gradient that is generated across the inner membrane. Mitochondrial uncoupling refers to a condition in which protons cross the inner membrane back into the matrix while bypassing the ATP synthase. As a consequence of this "short-circuit," there is a reduction in Deltapsi(m). We have previously demonstrated that animals treated with the classic uncoupling agent 2,4-dinitrophenol (DNP) show significant protection against brain damage following striatal injections of the NMDA agonist quinolinic acid (QA). In an effort to elucidate the mechanism of neuroprotection, we have assessed the effects of DNP on several parameters of mitochondrial function caused by QA. The results presented herein demonstrate that treatment with DNP attenuates QA-induced increases in mitochondrial Ca2+ levels and ROS formation and also improves mitochondrial respiration. Our findings indicate that DNP may confer protection against acute brain injury involving excitotoxic pathways by mechanisms that maintain mitochondrial function.     

Calculation of pH of Human Erythrocyte From the Distribution of 5,5-Dimethyl-2,4-Oxazolidinedione (DMO)

The intracellular pH of human erythrocytes in nine subjects was calculated by the DMO method and compared with direct measurements on the hemo-lysate. The standard deviation of differences between means of duplicate observations of the two methods was ± 0.111 pH unit. The amount of DMO-like substance in normal human blood was found to vary from one subject to another and must be taken into account in the calculations. It is concluded that the DMO method is of limited accuracy in estimating intracellular pH of human erythrocytes, but it may be useful in evaluating the extent of any changes.

ZUSAMMENFASSUNG

Das nach der DMO-Methode bei 9 Versuchspersonen errechnete intracelluläre pH menschlicher Erythrozyten wurde mit direkten Messungen im Haemolysat verglichen. Die Standardabweichung der Unterschiede zwischen den Mittelwerten der beiden Methoden, die jeweils aus doppelten Beobach-tungen stammten, betrug ± 0,111 pH-Einheiten. Die Menge an DMO-ähnlichen Substanzen im normalen menschlichen Blut varüerte von Fall zu Fall und musste bei den Berechnungen in Betracht gezogen werden. Die Autoren kommen zu dem Schluss, dass die DMO-Methode bei der Schätzung des intracellulären pH menschlicher Erythrozyten nur begrenzte Genauigkeit besitzt, jedoch für die Bestimmung des Ausmasses von Veränderungen nützlich sein kann.     

生物喋呤合成酶抑制剂在大鼠烫伤后金葡菌脓毒症防治中的意义

目的采用大鼠20%体表面积Ⅲ°烫伤后金黄色葡萄球菌(简称金葡菌)攻击所致脓毒症模型,探讨生物喋呤合成限速酶抑制剂-2,4-二胺-6-羟基嘧啶(DAHP)在金葡菌脓毒症防治中的意义.方法56只动物随机分为正常对照组、烫伤对照组、烫伤后金葡菌感染组和DAHP拮抗组.无菌留取动物心、肝、肺、肾组织,采用RT-RCR方法检测三磷酸鸟苷环水解酶I(GTP-CHI)、诱生型一氧化氮合酶(iNOS)及肿瘤坏死因子-α(TNF-α)基因表达,同时测定上述组织中四氢生物喋呤(BH4)和一氧化氮(NO)的水平.结果烫伤后金葡菌感染可导致组织GTP-CHI基因表达广泛上调、BH4合成显著增加.与之相对应,组织iNOS基因表达和NO水平亦明显升高,其中肝、肺改变尤为显著.给予DAHP不仅可显著抑制各组织GTP-CHI基因表达和BH4的产生,iNOS基因表达和NO的生成亦明显受抑,同时TNF-(基因表达也明显降低.此外,DAHP拮抗组动物6h死亡率明显降低(与未拮抗组相比,P=0.08,趋于统计学意义).结论早期应用DAHP进行干预可在一定程度上改善革兰阳性菌脓毒症动物的预后,其作用机理可能与DAHP抑制了体内BH4和NO的产生有关.     

Risk of tuberculosis in dialysis patients: association of tuberculin and 2,4-dinitrochlorobenzene reactivity with risk of tuberculosis

Background Dialysis patients are at increased risk of tuberculosis (TB) and anergy due to attenuated cellular immunity. Aim To define specific risks of TB in anergic and non-anergic dialysis patients. Methods A total of 272 dialysis patients were enrolled in this prospective study over a 36-month follow-up. Entering the study, participants had Mantoux and 2,4-dinitrochlorobenzene skin tests and their cell-mediated immunity (CMI)-index was estimated. Patients were classified as anergic (CMI-index ≤2 and Mantoux ≤4 mm) or non-anergic. Specific relative risks of TB were then calculated, using data from both the general population and 49 non-uremic health care workers. The independent effect of sex, age and diabetes mellitus was determined using Cox’s proportional hazard method. Results Among the 116 (42.6%) anergics 13 (11.2%), and among the 156 non-anergics 11 (7%) developed active tuberculosis. Anergics had a significantly higher risk of TB than non-anergics (Adjusted Relative Risks, 98.3, 95% CI 58.65–113.6 versus 61.6, 95% CI 13.6–72.1, P = 0.003). Tuberculin reactivity was weakly associated with the subsequent risk of TB (r = 0.51). The latter showed a steadily decreasing trend with increasing CMI-index (r = −0.99). Conclusion Anergy distorts the association of tuberculin reactivity with risk of TB. Anergic dialysis patients are at increased risk of developing active TB and chemoprophylaxis is justified in them too.     

门静脉高压症脾功能亢进脾巨噬细胞Toll样受体2、4 mRNA表达变化的研究

目的 检测门静脉高压症(PH)脾亢脾和正常脾巨噬细胞(Mφ)中Toll样受体2、4(TLR2、4) mRNA的表达差异,为进一步深入探讨Toll样受体在PH脾亢发生中的作用奠定基础.方法 选取门静脉高压症脾亢患者(均为慢性乙型肝炎患者)的手术切除脾脏(12例)为实验组,外伤性脾破裂患者的手术切除脾脏(4例)为正常对照组.贴壁培养法分离纯化脾脏组织Mφ,荧光定量PCR法对Mφ表面Toll样受体2、4 mRNA的表达进行检测,并将两组结果进行统计学分析比较.结果 与正常脾脏相比,PH脾亢脾Mφ TLR2、4 mRNA的表达水平明显增强(TLR2:2.29±0.55 vs 1.06±0.53,P <0.05;TLR4:2.32±0.41 vs 1.01±0.14,P <0.01).结论 PH脾亢脾Mφ TLR2、4的mRNA表达水平明显升高,与蛋白水平免疫组化的结果一致,进一步支持了"内毒素血症→脾脏Mφ Toll样受体活化→Mφ吞噬破坏血细胞增多"是PH脾亢发生可能机制的观点.     

新型光催化反应器降解典型有机污染物2,4-二硝基苯酚

利用ＴｉＯ２在Ｈ２Ｏ２中的溶解性，在玻璃表面形成ＴｉＯ２薄膜，制作了新型环状光催化型反应器。以２，４－二硝基苯酚为典型有机化合物，进行了降解的条件研究，并测试了反应器性能。分别考察了催化剂涂层载体、光源、搅拌以及流速对２，４－二硝基苯酚光降解的效率。结果表明：石英玻璃为最理想的光催化剂载体；水银灯对２，４－二硝基苯酚的降解速度最快，是理想的光源；搅拌能够提高反应器效率；采用适宜的流速（４５ｍｌ／ｍｉｎ）可使反应器效率达到理想值。在反应动力学研究中，２，４－二硝基苯酚的最大降解速率为Ｋｄｍａｘ＝０．３６ｍｇ／（Ｌｍｉｎ），最终的降解率达到９９．５％．     

Attenuation of bleomycin induced pulmonary fibrosis in mice using the heme oxygenase inhibitor Zn-deuteroporphyrin IX-2,4-bisethylene glycol

BACKGROUND: Pulmonary fibrosis is associated with a poor prognosis. The pathogenesis of fibrotic lung disorders remains unclear, but the extent of tissue damage due to the persistent presence of oxidants or proteases is believed to be important. The heme degrading enzyme heme oxygenase (HO) has been found to be expressed in experimental fibrosis, and generation of free iron and carbon monoxide (CO) by HO has been implicated in oxidant induced lung damage. A study was undertaken to examine the effects of the HO inhibitor Zn-deuteroporphyrin-IX-2,4-bisethylene glycol (Zndtp) on the development of pulmonary fibrosis in the bleomycin model of lung injury and repair. METHODS: Zndtp (10 micro mol/kg) was administered subcutaneously twice daily to mice 1 week following the intratracheal instillation of 0.025 U bleomycin. Animals were killed 10 or 21 days after bleomycin instillation and indices of lung damage and fibrosis were evaluated. RESULTS: Bleomycin treatment induced pulmonary cytotoxicity, increased levels of active transforming growth factor beta (TGF-beta), enhanced lung collagen accumulation, and decreased glutathione content. Zndtp administration significantly attenuated these indices. CONCLUSIONS: Administration of Zndtp in the bleomycin model resulted in appreciable alveolar cytoprotection and amelioration of pulmonary fibrosis. This molecule and its analogues may warrant further consideration in the treatment of acute lung injury and fibrotic lung disorders.     

生物喋呤合成酶抑制剂对烫伤大鼠金葡菌脓毒症的保护效应及机制

目的　探讨生物喋呤合成限速酶抑制剂 - 2 ,4 二胺 6 羟基嘧啶 (DAHP)对金黄色葡萄球菌 (简称金葡菌 )脓毒症的保护效应及机制。　方法　 5 6只Wistar大鼠随机分为正常对照组、2 0 %TBSAⅢ度烫伤对照组、烫伤后金葡菌感染组和DAHP拮抗组。无菌留取大鼠心、肝、肺、肾组织检测三磷酸鸟苷环水解酶I(GTP CHI)、诱生型一氧化氮合酶 (iNOS)及肿瘤坏死因子α (TNFα )基因表达 ,同时测定组织中四氢生物喋呤 (BH4)和一氧化氮 (NO)的水平。　结果　烫伤后金葡菌感染可导致组织GTP CHI基因表达广泛上调、BH4合成显著增加。同时 ,组织iNOSmRNA表达和NO水平亦明显升高 ,其中肝、肺改变尤为显著。给予DAHP不仅可显著抑制各组织GTP CHImRNA表达和BH4的产生 (P<0 .0 5～ 0 .0 1) ,iNOSmRNA表达和NO的生成亦明显受抑 ,同时TNFα表达也明显降低。此外 ,DAHP拮抗组动物 6h死亡率有所降低 (分别为 2 5 .0 %和 5 5 .6 % ,P =0 .0 8)。　结论　DAHP早期干预可在一定程度上改善革兰阳性菌脓毒症动物的预后 ,其机制可能与DAHP抑制体内BH4和NO的产生有关。     

二硝基苯修饰B16细胞对黑色素瘤小鼠疗效的实验研究

目的：探讨半抗原——二硝基苯（DNP）修饰的恶性黑色素瘤（恶黑）细胞激活树突状细胞（DC）后,对转移性黑色素瘤小鼠的抗肿瘤效应。方法：采用DNP修饰恶黑细胞B16（H-2^b）,在体外激活C57BL/6小鼠（H-2^b）外周血来源的DC,用于激发自体的T细胞,观察其对T细胞的增殖和特异性T细胞杀伤功能。在由B16细胞接种产生的荷瘤C57BL/6小鼠实验中,将小鼠随机分为5组,于左背部注射及于左耳背面涂抹DNP修饰B16瘤苗联合DC（DNP-B16-DC组）、B16激活DC（B16-DC组）、单纯DC（DC组）、单纯DNP（DNP组）和生理盐水（生理盐水组,每组6只）,观察各组抑瘤率和迟发型超敏反应。结果：经DNP修饰的B16细胞激活的DC,诱发的T细胞增殖能力和对B16细胞的特异性杀伤效应均明显高于未其修饰的B16细胞组和DC组。荷瘤小鼠实验显示,DNP-B16-DC组和单纯DNP组小鼠耳肿胀明显增加,其迟发型超敏反应明显强于其他各组。实验中DNP组有1只小鼠用药后20d死亡,其余小鼠DNP-B16-DC组抑瘤率（96.16%）明显高于B16-DC组（50.11%）、DC组（22.12%）和DNP组（57.79%,P〈0.01）,显示DNP-B16-DC具有更强的抑瘤作用。结论：DNP修饰B16所激活的DC可以诱导更强的特异性抗肿瘤作用。     

除草剂2甲4氯钠致肾病综合征合并急性肾功能衰竭一例

2甲4氯钠(MCPA-Na)属于苯氧乙酸类除草剂,广泛应用于水稻、小麦等农作物控制阔叶类杂草生长.对哺乳动物毒性较低,皮肤接触和呼吸道吸入导致严重后果的少有报道.     

Toll样受体2、4与全身性感染研究进展

全身性感染是由感染所致的临床综合征,是一种高发率和高病死率疾病,目前仍然是一项严重的临床问题。尽管在全身性感染治疗方面,人们已经取得了长足的进步,但是迄今为止,仍然没有有效的病因学治疗方法应用在全身性感染患者身上。这很可能是由于全身性感染的免疫病理学发病机制没有得到完全阐明。近来,TLR在全身性感染免疫机制中所起到的作,用在多种动物体内得到验证,TLR、TLR4在其中的作用尤为关键。TLR导致的体内信号传导,使炎症反应级联放大。此外,TLR还能诱发机体免疫功能紊乱．致使机体对病原体清除效率降低。这些原因都可以导致全身性感染的病情的进一步进展。所以阻断TLR传导道路可能会抑制全身性感染发生和发展的进程。这也为临床医生最终攻克全身性感染提供了崭新的思路和临床治疗靶点。     

Characterization of the exfoliative antispermatogenic agent 1-(2,4-dichlorobenzyl)-H-indazole-3-carboxylic acid in the rhesus monkey.

The effects of oral doses of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (DICA) on spermatogenesis in the rhesus monkey (Macaca mulatta) was studied. Four animals given five daily 50 mg/kg doses or three or five daily 500 mg/kg doses showed that DICA was an exfoliating antispermatogenic compound. The inhibition of spermatogenesis was only partially reversible following 500 mg/kg doses of DICA. Weekly and monthly 50 mg/kg doses of DICA only partially inhibiting spermatogenesis as measured by electro-ejaculated sperm counts. Response in individual monkeys ranged from azoospermia to no effect. Testicular biopsies confirmed this finding. DICA did not affect serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), or testosterone concentrations. The blood absorption or urinary excretion rates of uniformly tritiated DICA in the animals that responded well did not differ from those monkeys that responded poorly. DICA metabolites were not detected in monkey urine. Serum testosterone concentrations appeared to vary with the season of the year, but FSH concentrations and ejaculated sperm count did not.     

1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (DICA), an exfoliative antispermatogenic agent in the rat.

The oral administration of 50 mg DICA/kg at nine weekly or four monthly intervals produced partially reversible infertility in male rats as judged by the results of serial mating and testicular histology. Oral 500 mg DICA/kg doses administered at the same intervals produced permanent sterility. Single oral doses of 50 or 500 mg DICA/kg elevated mean FSH concentrations on days 2, 3, and 7 but did not affect LH or testosterone. Mean plasma concentration peaked at 74 micrograms/ml 4 hr after a 50 mg/kg dose of uniformly tritiated DICA; 24 hr later, it had declined rapidly to 5.5 micrograms/ml. The drug did not have a strong affinity for any tissue studied including the testis. DICA-induced exfoliation of immature germ cells was first observed 4 hr after administration and led to significantly reduced testis weights by day 2. Neither single doses of 10--250 mg DICA/kg nor five daily doses of 10--100 mg DICA/kg reduced seminal vesicle, ventral prostate, or body weights of male rats. Chronic weekly DICA administration did reduce mean seminal vesicle weight. These studies have shown that DICA is an effective, partially reversible antifertility agent that directly affects the rat testis.     

The mitochondrial uncoupling agent 2,4-dinitrophenol improves mitochondrial function, attenuates oxidative damage, and increases white matter sparing in the contused spinal cord

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.     

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method in canine pancreas preservation--the effect of 2,4 dinitrophenol on graft viability and adenosine triphosphate tissue concentration.

Tissue concentrations of adenosine triphosphate have been previously associated with successful pancreas preservation using the two-layer cold storage method in a canine autotransplantation model. To clarify the role of ATP vs. oxygenation per se, we used 2,4 dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. DNP caused no toxicity in pancreas grafts preserved for 24 hr in Euro-Collins' solution or 48 hr in University of Wisconsin solution. Tissue concentration of ATP and viability of pancreas grafts, defined as maintenance of normoglycemia for 5 days following transplantation, were compared among six groups after a preservation interval of 24 or 48 hr. After 24 hr all grafts were viable, whether preserved using simple cold storage in EC (group 1a), two-layer (EC/perfluorochemical [PFC]) method (group 2a), or two-layer (EC+DNP/PFC) method (group 3a); respective graft survival was 4/4 (100%), 5/5 (100%), and 4/5 (80%); one of five dogs in group 3a died of a cause unrelated to the pancreas. ATP levels were higher in group 2a compared with group 1a (7.47 +/- 0.47 vs. 1.41 +/- 0.53 mumol/g dry weight, P less than 0.01) and lower in group 3a compared with group 2a (1.25 +/- 0.37 vs. 7.47 +/- 0.47, P less than 0.01). After 24 hr, we observed no difference in viability despite ATP concentration differences. However after 48 hr preservation, graft viability varied among the groups: 0/4 (0%), 4/4 (100%), and 0/3 (0%) in groups 1b, 2b, and 3b, respectively. ATP tissue concentration was again higher in group 2b after two-layer (EC/PFC) method preservation (7.91 +/- 1.21 vs. 1.21 +/- 0.31 mumol/g dry weight, P less than 0.01) compared with EC preservation (group 1b). DNP again caused a significant decrease in tissue ATP in group 3b (0.61 +/- 0.07 vs. 7.91 +/- 1.21, P less than 0.01). The two-layer (EC/PFC) method clearly protected pancreas viability, and inhibition of ATP production using DNP caused loss of viability in this model. We conclude that oxygenation of the pancreas during preservation by the two-layer method allows continued ATP production within the graft. Metabolic processes vital to cellular integrity can be maintained, which produces an extended period of preserved pancreatic viability.