Serial section analysis of mouse hepatic peroxisomes

Summary The ultrastructure and organization of mouse hepatic peroxisomes were investigated using serial thin sections and the alkaline diaminobenzidine technique for visualization of the peroxidatic activity of catalase. Mouse periportal hepatocytes exhibit three classes of peroxisomes which display morphological and cytochemical heterogeneity: 1) large, circular to ovoid organelles containing a crystalline nucleoid, 2) small, circular to elongate, anucleoid particles, and 3) tail-like extensions which are devoid of both catalase activity (only traces of reaction deposits) and a crystaline core.Serial section analysis reveals that these profiles correspond to three diverse interconnecting peroxisomal segments which constitute a highly complex organelle. In particular, the large nucleoid-containing peroxisomal segment exhibits an intimate relationship to the endoplasmic reticulum. However, direct membrane continuities between the two compartments are never observed.With respect to the complex structure of the organelle the following conclusions can be drawn concerning biochemical studies on liver peroxisomes: 1) During homogenization and subcellular fractionation procedures, fragmentation of peroxisomes into particles of different size classes should be expected. 2) These peroxisomal fragments are inhomogeneous with respect to their matrix contents and possess at least one rupture site on their membrane surface. 3) Soluble matrix and, to a lesser degree, membrane components of peroxisomes contribute to the soluble fraction. 4) Crude microsomal fractions are regularly contaminated by peroxisomal membrane fragments.This study was supported by a grant of the Deutsche Forschungsgemeinschaft, Fa 146/1-2     

Pathology of hepatic peroxisomes and mitochondria in patients with peroxisomal disorders

Summary The morphology of hepatic peroxisomes in five patients with metabolic disorders believed to be due to inherited defects of peroxisomal function or biogenesis is described. Electron microscopy and cytochemical staining for catalase were used to identify peroxisomes in two boys with infantile Refsum's disease (IRD), a girl with autopsy confirmed neonatal adrenoleukodystrophy (NALD), and two boys with pseudo-Zellweger syndrome (PZS). In the patients with IRD and NALD hepatic peroxisomes were significantly reduced in size and number and contained electron dense centres. In the liver of the patients with PZS the peroxisomes were enlarged. Morphologically abnormal peroxisomes were also detected in autopsy tissue from one boy with PZS using electron microscopy. Lamellar-lipid inclusions and mitochondria with crystalline inclusions and/or abnormal cristae are also described in two patients, one with IRD, the other with NALD.     

Adiponectin reduces connective tissue growth factor in human hepatocytes which is already induced in non-fibrotic non-alcoholic steatohepatitis

Connective tissue growth factor (CTGF) is induced in liver fibrosis and enhances the activity of transforming growth factor β (TGFβ). Recently we have shown that the hepatoprotective adipokine adiponectin downregulates CTGF in primary human hepatocytes (PHH). In the current study, the mechanisms mediating suppression of CTGF by adiponectin and the well described downstream effector of adiponectin receptor 2 (AdipoR2), peroxisome proliferator activated receptor α (PPARα), were analyzed in more detail. Adiponectin downregulated CTGF mRNA and protein in primary human hepatocytes (PHH) and suppression was blocked by a PPARα antagonist indicating that AdipoR2 is involved. The PPARα agonists fenofibrate and WY14643 also reduced CTGF protein in these cells. Adiponectin further impaired TGFβ-mediated upregulation of CTGF. Phosphorylation of the TGFβ downstream effectors SMAD2 and –3 was reduced in PHH incubated with adiponectin or PPARα agonists suggesting that early steps in TGFβ signal transduction are impaired. CTGF and TGFβ mRNA levels were increased in human non-fibrotic non-alcoholic steatohepatitis (NASH), and here AdipoR2 expression was significantly reduced. Current data show that CTGF and TGFβ are already induced in non-fibrotic NASH and this may be partly explained by low adiponectin bioactivity which interferes with TGFβ signaling by reducing phosphorylation of SMAD2/3 and by downregulating CTGF.     

Intracellular changes in rat hepatocytes after intratracheal administration of highly dispersed silicon dioxide and uridine effects on these changes

Rat hepatocytes were examined under electron microscope at early terms after intratracheal administration of highly dispersed silicon dioxide powder against the background of uridine treatment. Penetration of powder particles into hepatocyte cytoplasm, nuclei, mitochondria, and peroxisomes and development of bacteria in these cells were observed. Uridine reduced the destructive effect of powder on the organelles, increased glycogen content in hepatocytes, and inhibited the formation of capsulated bacterial forms in these cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 596–600, May, 2006     

罗格列酮、辛伐他汀合用对兔动脉粥样硬化形成的影响

目的探讨罗格列酮、辛伐他汀合用对兔动脉粥样硬化（AS）形成的影响及可能的作用机制。方法40只日本大耳白兔随机分为五组,每组8只。正常对照组：普通颗粒饲料喂饲11周。高脂模型组：高脂饲料（1％胆固醇＋8％猪油＋普通颗粒饲料）喂饲11周。罗格列酮组：高脂饲料喂饲同时口服罗格列酮0．5mg·kg^-1·d^-1。辛伐他汀组：高脂饲料喂饲同时口服辛伐他汀2．5mg·kg^-1·d^-1。联合用药组：高脂饲料喂饲同时口服罗格列酮0．5mg·kg^-1·d^-1和辛伐他汀2．5mg·kg^-1·d^-1.12周末实验结束后获取血浆和主动脉全长标本进行生化和病理分析。结果各用药组与高脂模型组比较C-反应蛋白（CRP）、内皮素（ET）浓度明显降低,以联合用药组降低最明显（P〈0．01）;一氧化氮（NO）水平升高,以联合用药组升高最显著（P〈0．01）。罗格列酮组、联合用药组与高脂模型组比较,血浆非对称二甲基精氨酸（ADMA）水平下降（P〈0．01）。辛伐他汀组、联合用药组与高脂模型组比较,血浆总胆固醇（TC）、甘油三脂（TG）、低密度脂蛋白胆固醇（LDL—C）明显降低,高密度脂蛋白胆固醇（HDL-C）水平明显升高（P〈0．01）。高脂模型组、各用药组主动脉内膜有不同程度的脂质斑块形成,内膜增厚程度不同,高脂模型组脂质斑块最多,内膜／中膜厚度比值90．17±23．03用药组斑块明显减少,以联合用药组脂质斑块最少,内膜／中膜厚度比值最小13．56±0．72（P〈0．01）。结论罗格列酮、辛伐他汀通过抑制炎症反应,改善内皮功能、调脂等各自不同途径抑制动脉硬化的形成,联合应用具有协同作用。     

Effect of peripheral axotomy on the fine structure and histochemistry of the Rolando substance: Degenerative atrophy of central processes of pseudounipolar cells

Summary Disappearence of fluorid-resistant acid phosphatase activity from the ipsilateral Rolando substance after transection of the peripheral nerve, is shown to be due to the cessation of enzyme supply from dorsal root ganglion cells to their central terminals. This is accompanied by (or ensues in consequence of) a fine structural derangement of these terminals (degenerative atrophy). Fine structural alterations of axon terminals undergoing degenerative atrophy, though similar to some extent to those seen during early phases of a Wallerian degeneration, are markedly different. Also myelinated nerve fibers, both in the dorsal horn and in dorsal columns, are affected by degenerative atrophy. This important, new trophical feature of sensory ganglion cells suggests a delicate metabolic balance between peripheral and central axonal branches of bipolar (pseudounipolar) cells. Degenerative atrophy raises serious implications in evaluating hodological experiments based upon Wallerian degeneration and offers new perspectives for theoretical and clinical neurology.     

Investigation of children for mitochondriopathy confirms need for strict patient selection, improved morphological criteria, and better laboratory methods

We studied muscle biopsies of 103 pediatric patients in whom clinical suspicion for disorder of energy metabolism was highest in 13 patients, intermediate in 8 patients, and lowest in 82 patients. Electron transport complex (ETC) enzyme activity measurements were available in 96 of 103 patients. Most children with unclassified encephalopathy before biopsy had negative or equivocal morphological and biochemical evaluation for disorder of energy metabolism (72/85). The incidence of ETC abnormality and morphological abnormality in muscle from 39 patients with clinical encephalomyopathy (groups I, II, and III) was 20% and 38%, respectively. In 21 children with high or intermediate clinical suspicion of mitochondriopathy, light microscopy was confirmative in 12, ultrastructure was confirmative in 15, and major ETC abnormality was present in only 4 (29%) of 14. In 82 children with lower clinical suspicion of mitochondriopathy, morphological criteria at both the light and electron microscopic level were absent, and major abnormality of ETC activity was uncommon, in 9 (11%) of 82. Partial reductions of ETC activity occurred in 15 (18%) of 82, but are of uncertain significance. Ragged blue fibers were more prevalent in infants with mitochondriopathy than ragged red fibers. Increase of large, but not small, subsarcolemmal mitochondrial aggregates based on succinate dehydrogenase histochemistry is a useful indicator for mitochondriopathy. Thus, a distinction should be made between small aggregates (normal) and large aggregates. Using strict criteria to define pathological mitochondria, we concluded that electron microscopy is a powerful tool in the diagnosis of mitochondriopathy mainly when clinical suspicion is high. We found no consistent difference in the frequency of mitochondrial "proliferation" as currently defined or in citrate synthase activity in any group. Better patient selection in infants and children and better methods for investigation of mitochondriopathy are needed.     

Inspissation of pancreatic zymogen material in cystic fibrosis

Cystic fibrosis is characterized by the elaboration of abnormal, thick, tenacious mucus resulting in obstructive disease in sites such as the lung and pancreas. In the pancreas, acinar plugs of mucus have been reported as the earliest recognizable morphologic lesion in cystic fibrosis. Since mucus is not normally elaborated within the pancreatic lobular tissue, the mechanism of accumulation of mucus in acini is enigmatic. To investigate this phenomenon, well-preserved autopsy pancreatic tissue was studied ultrastructurally. This study demonstrated very prominent mucous metaplasia in these diseased organs. Acinar plugs, though, developed before mucous metaplasia. Subsequent histochemical study was performed, which demonstrated that the early acinar plugs exhibited the same staining properties as zymogen granules and were distinct from the staining pattern of mucus in pancreatic tissue of cystic fibrosis patients. These findings, then, indicate that zymogen material, not mucus, becomes inspissated in the acini of the pancreas in early cystic fibrosis, and that subsequent mucous metaplasia occurs as the obstruction and exocrine atrophy progress.     

Niemann-Pick disease

Summary The results of a complex analysis of liver tissue are presented (four biopsy and two autopsy samples) obtained from six patients with Niemann-Pick disease (NPD) with a gross deficiency of sphingomyelinase (SMase) accompanied by a typical increase in sphingomyelin (SM). There were five cases of NPD type A (four of them with an atypical, prolonged course) and one case of type B. By means of lipid histochemistry it was possible to demonstrate SM storage both in hepatocytes and in the reticuloendothelial system (RES) of the liver (Kupffer cells and portal macrophages) and to show in two siblings with NPD type A a so-far undescribed centrilobular storage pattern. Enzyme histochemistry revealed a secondary deficit of nonspecific esterase activity and acid -galactosidase in liver storage macrophages and varying degrees of suppression of hepatocytic enzyme activities as a reaction to lipid storage of sudden onset. Ultrastructurally, it was possible to demonstrate cholesterol in lysosomes by using digitonin fixation, the involvement of Ito cells in lipid storage, the aggregation of storage lysosomes with certain other organelles and their occasional connections with the endoplasmic reticulum. The problems of possible lipid extraction during processing were considered as a cause of pronounced lysosomal electron-lucidity and of the ultrastructural identification of the participating lipopigment. The significance of the findings is discussed in relation to the existing classification and, particularly, to the stored lipid dilemma of cases of NPD type C.     

肝门阻断和再开放对兔胰腺功能的影响

目的：观察肝门阻断（HVO）及再开放（HVR）后胰腺内外分泌功能的改变。方法：选择健康日本大耳白兔25只，体重2.3-3.0 kg，分别于HVO前、HVO 10、20 min及HVR后（10、30、60、120 min）不同时点取血，并在HVO前、HVO 20 min、HVR后120 min 3个时点取胰腺组织，电镜下观察其超微结构的变化。结果：HVO时，血浆葡萄糖、胰岛素水平及一氧化氮代谢产物（NO₂^-/NO₃^-）含量均明显低于阻断前，胰高血糖素/胰岛素及丙二醛（MDA）浓度显著高于阻断前（P<0.05或P<0.01），并随阻断时间延长而加重；血浆淀粉酶、脂肪酶、游离脂肪酸水平则无明显差异（P>0.05）；胰岛细胞的线粒体肿胀、粗面内质网扩张，细胞核形态、结构基本正常，而胰腺腺泡细胞变化则不明显。HVR后上述差异逐渐不明显，至120min才接近阻断前水平。结论：急性肝门阻断和再开放对胰腺内分泌功能有较大的影响。     

Influences of endurance training on the ultrastructural composition of the different muscle fiber types in humans

To investigate changes in the ultrastructure of the different muscle fiber types induced by endurance training ten sedentary subjects (five women and five men) were exercised on bicycle ergometers 5 times a week for 30 min. After 6 weeks of training there were significant changes in (+14%), in the percentage of type I (+12%) and type IIB fibers (–24%) as well as in the volume densities of mitochondria. The latter increased 35% in type I, 55% in type IIA and 35% in type IIB fibers. The relative increase in subsarcolemmal mitochondria was larger than in interfibrillar mitochondria in all fiber types. There was also a significant increase in the volume density of intracellular lipid in type II fibres. It is concluded that high intensity endurance training leads to an enhancement of the oxidative capacity in all muscle fiber types.     

Peroxisomes in sebaceous glands

Summary The ultrastructure of peroxisomes in partially differentiated cells of the mouse preputial gland was investigated using serial thin sections and three-dimensional reconstruction as well as the alkaline diaminobenzidine technique for visualization of the peroxidatic activity of catalase.An analysis of serial sections indicates that the different types of intensely stained peroxisomal profiles, classified according to their shape, represent random planes through highly complex peroxisomes. These organelles exceed 4 m in length and exhibit a focal heterogeneity with respect to their size, shape and enzyme distribution. The graphical three-dimensional reconstruction demonstrates that the most intricate peroxisomes are characterized by tortuous, elongate, and branched tubular segments of varying diameter equipped with enlarged terminal hollow-spherical structures which engulf areas of cytoplasm.A close spatial relationship is established between adjacent peroxisomes and peroxisomes and mitochondria, the latter two of which synchronously develop into highly complex structures. A close association is also observed between peroxisomes and the endoplasmic reticulum, whereby membrane continuities between the two compartments cannot be demonstrated.These observations are inconsistent with traditional concepts concerning peroxisomal shape and size, the number per cell, as well as their biogenesis from the endoplasmic reticulum.The functional significance of individual highly complex peroxisomes and their assemblage forming an extensive netlike membraneous system throughout the cell is discussed with respect to intracellular energy transport and trans-membrane electron exchange.This paper is dedicated to Prof. Dr. O. Pflugfelder on the occasion of his 80th birthdayThis study was supported by a grant of the Deutsche Forschungsgemeinschaft, Fa 146/1-2     

Cell biology, clinicopathological profile, and classification of gastro-enteropancreatic endocrine tumors

 Recent developments in the field of endocrine cell biology and pathology at both morphological and molecular levels are briefly outlined and discussed as a basis for endocrine tumor characterization. The main tools available for identifying the endocrine nature of the tumors, their pathogenetic interpretation, and experimental reproduction with special emphasis on tumor antecedents are reported. Based on this, classifications of endocrine tumors of the pancreas and gastrointestinal tract are developed, covering most clinical (hyperfunctional syndromes included), pathological, and biological patterns, with special emphasis on tumor prognosis. Received: 20 June 1997 / Accepted: 15 September 1997     

Ultrastructural study of magnesium dependent adenosine triphosphatase (Mg-ATPase) activity in rat liver peroxisomes after administration of aspirin and clofibrate

A total of 36 male F344 rats was fed a diet containing 1% aspirin or 0.25% clofibrate for 1, 3 and 4 weeks. Hepatomegaly was more evident with aspirin than with clofibrate. The activity of magnesium dependent adenosine triphosphatase (Mg-ATPase) was localized on the limiting membrane of aspirin-induced peroxisomes, but it was much weaker than that in control and clofibrate-induced peroxisomes. Alteration in the ultrastructure of peroxisomes and mitochondria became more evident in Week 4 of the experiment than in Week 1. These ultrastructural changes also basically returned to normal conditions after one week withdrawal of the chemicals. This study was presented in part at the 24th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Okayama, September 17–19, 1992.     

Vascular tumors of the mammary gland

Summary A total of five haemangiosarcomata and two benign haemangiomas arising in the mammary gland have been studied electron microscopically and by histochemical techniques. Malignant tumors were mainly composed of endothelial cells reactive to alkaline phosphatase and adenosine triphosphatase, and of pericytes and undifferentiated mesenchymal elements. A juvenile haemangioma showed a more structured wall with an increase of endoplasmic reticulum and filaments, and a diminution of membrane modulations and rod-like tubular bodies. A cavernous haemangioma showed an ultrastructure very similar to normal vessels.The ultrastructural and histochemical data suggest a blood vessel origin of mammary angiosarcomas and show that vascular neoplasms of the breast, benign or malignant, are composed of a combined proliferation of the different cell types present in the vessel wall, as described in other organs.Juvenile haemangioma and cavernous haemangioma show a more complex structure in their walls with stratification of the different types of cells and a continuous wall. The features of endothelium in juvenile haemangioma suggest a low functional activity rather than morphological de-differentiation.     

Target-targetoid phenomenon of the human muscle fibers

Summary Target and targetoid fibers in a muscle biopsy from a patient with paralysis of the deltoid and supraspinatus muscles were studied by light and electron microscopy. The probable cause of the neuropathy was tumor compression.Target and targetoid change was exclusively confined to hypertrophic or normal-sized fibers. Morphometric evaluation of the target and targetoid fibers showed no significant difference between them. With the electron microscope, up to 4 structural zones were seen in the typical target fiber but many were devoid of either zone 2 (halo) or zone 3, or both.It was conceivable that focal irregularity and streaming of Z-bands were the primary alterations in the process of target-targetoid fiber formation, and that this phenomenon was induced both by partial residual innervation as well as re-innervation.     

过氧化体增殖物激活型受体对血管内皮细胞PAI-1转录的调节

目的:探讨血管内皮细胞中过氧化体增殖物激活型受体(PPARs)的表达及其对纤溶酶原激活物抑制剂-1(PAI-1)转录的调节作用。方法:培养人脐静脉内皮细胞(HUVECs)并提取总RNA,通过逆转录聚合酶链式反应(RT-PCR)扩增PPARα、δ和γ;以PAI-1启动子控制表达氯霉素转移乙酰酶(CAT)报告基因的重组质粒PAI-pCAT转染人血管内皮细胞株ECV304、并同时分别共转染250、500、1000ng的PPARα或PPARγ表达载体,酶联免疫吸附方法(ELISA)测定CAT表达量-启动子片段转录活性。结果:HUVECs中可检测到PPARα、δ和γ的mRNA水平表达,并且表达量PPARγ明显小于PPARα(P＜0.01)和PPARδ(P＜0.01);增加PPARα表达可剂量依赖地提高ECV304细胞PAI-1转录,而增加PPARγ表达对ECV304细胞PAI-1的转录无影响。结论:血管内皮细胞存在3种PPARs的表达,其中PPARα涉及对PAI-1基因转录的诱导调节。     

Selective hepatic vagotomy blocks pancreatic glucagon's satiety effect

To test the hypothesis that hepatic vagal afferents mediate the satiety effect of pancreatic glucagon, we tested the effects of selective surgical and pharmacological lesions of the abdominal vagus on glucagon's potency to inhibit feeding. Surgical disconnection of only the hepatic branch of the abdominal vagus blocked glucagon's satiety effect as well as total abdominal vagotomy. However, abdominal vagotomy that spared the hepatic branch did not change glucagon's satiety effect. Glucagon also inhibited feeding after pharmacological blockade of peripheral postganglionic muscarinic receptors with atropine methylnitrate. All these results are consistent with the hypothesis that hepatic vagal afferents mediate the satiety effect of pancreatic glucagon.