Lysosomes and pancreatic islet function

Summary The relation between qualitative and quantitative glucose-dependent alterations of lysosomes in pancreatic islets and the function of the islets was studied. Isolated islets of the mouse were maintained in tissue culture for one week in either 28, 5.5 or 3.3 mmol/l glucose. Insulin biosynthesis, insulin secretion and insulin content of the cultured islets were determined. After culture, the islets were subjected to acid phosphatase cytochemistry and examined by electron microscopy and ultrastructural morphometry. Islets cultured in 28 mmol/l glucose both produced and secreted insulin rapidly. Such islets seemed, however, unable to maintain more than small amounts of granule-stored insulin. Islets cultured at the lower concentrations of glucose displayed a reduced insulin secretion, which apparently resulted in considerable amounts of intracellularly stored insulin. In all cultured islets different types of lysosomes, identified by their acid phosphatase reactivity, could be seen. Dense bodies, i.e., lysosomes characterized by a homogeneous, very fine, particulate content of high density, seemed to predominate at all concentrations of glucose. It is suggested that, in the islets, the dense bodies correspond morphologically to primary lysosomes. Other types of lysosomes with inclusions of various kinds, which were frequent at the two lower concentrations of glucose, may correspond to secondary lysosomes. Morphometry revealed differences between the size distributions of lysosomes in the three experimental groups. Thus, the average lysosomal size was inversely proportional to the concentration of glucose in the culture medium. However, the numerical density of lysosomes was greatest at the highest glucose concentration. The observation of secondary lysosomes, containing material resembling secretory granules, suggests that the increased size and lowered number of lysosomes in islets cultured at low glucose concentrations may depend on a crinophagic process. Such a process, together with insulin biosynthesis and insulin secretion, may be of physiological importance for control of the secretory granule content within the pancreatic B-cell.     

Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium

Summary The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.     

Ultrastructural,cytochemical and functional studies on the eosinophilic granulocytes of larval lampreys

Summary Blood from larval lampreys (ammocoetes) contains a small number of eosinophilic granulocytes which are formed in the protospleen and kidney. Both immature and mature forms of this cell type are present in the blood and these are easily identified from other cell types due to the prominent eosinophilic granules that fill the cytoplasm. Ultrastructurally, these granules are electron-dense, largely unstructured, Golgi-derived and contain acid phosphatase but not peroxidase. Eosinophilic granulocytes ingest bacteria but fail to internalise colloidal carbon. The functional and phylogenetic significance of these cells is discussed.     

Endocytosis by platelets during cationized ferritin-induced aggregation

Summary Localization of cationized ferritin (CF) particles in the process of CF-induced aggregation of rabbit platelets was investigated by electron microscopy. CF particles attached to the surface membrane of discoidal platelets immediately after the addition of CF. Some platelets were connected to each other through the CF particles located on their surfaces. At 30 s after the addition of CF, aggregation of platelets in round form was observed. During the time course of aggregation, CF particles moved to the interplatelet spaces. Also CF particles were found in the open canalicular system, the membrane component of which was stained with ruthenium red. On the other hand, CF particles were also found in ruthenium-red-negative vesicles in platelets. At 180 s after, CF particles containing vacuoles, which showed acid phosphatase activity, were observed in the aggregates. These results suggest that part of CF particles may be incorporated into the cytoplasma by endocytosis.     

Growth with hydrogen,and further physiological characteristics of Desulfotomaculum species

Growth of Desulfotomaculum orientis, D. ruminis, D. nigrificans and the Desulfotomaculum strains TEP, TWC and TWP, that were newly isolated with sulfate and fatty acids, was studied using defined mineral media. Four of these strains grew with hydrogen plus sulfate as the only energy source. Under these conditions the growth yield of D. orientis in batch culture was 7.5 g cell dry mass per mol sulfate reduced. Growth on methanol with growth yields of about 6 g cell dry mass per mol sulfate was obtained with D. orientis and strain TEP. All strains tested grew slowly with formate as electron donor. Fatty acids from propionate to palmitate were utilized by the strains TEP, TWC and TWP. D. orientis and the strains TEP and TWC were able to utilize the methoxyl groups of trimethoxybenzoate for growth. D. orientis was found to grow chemoautotrophically with hydrogen, carbon dioxide and sulfate; during growth with C₁-compounds no additional organic carbon source was required. Furthermore, D. orientis was able to grow slowly in sulfate-free medium with formate, methanol, ethanol lactate, pyruvate or trimethoxybenzoate. Under these conditions acetate was excreted, indicating the function of carbon dioxide as electron acceptor in a homoacetogenic process. A growth-promoting effect of pyrophosphate added to the medium of Desulfotomaculum species was not observed. The results show a high catabolic and anabolic versatility among Desulfotomaculum species, and indicate that electron transport to sulfate can be the sole energy conserving process in this genus.     

Widespread tissue distribution and synthetic pathway of polyunsaturated C24:2 sphingolipids in mammals

Sphingolipids are multifunctional lipids and a major constituent of the cell membranes of eukaryotes. Although the fatty acid (FA) moiety of sphingolipids is usually a saturated or monounsaturated FA, polyunsaturated FA (PUFA)-containing species also exist in mammalian tissues. In the present study, we showed that C24:2 PUFA-containing ceramide is one of the seven major ceramide species in a wide range of tissues. C24:2 ceramide levels were especially high in spleen and small intestine; in the former, it was the fourth most abundant ceramide species. However, both the synthetic pathway and the physiological function of C24:2 ceramide had yet to be identified. Tracer analysis using deuterium-labeled linoleic acid (C18:2) revealed that C24:2 ceramide is produced via elongation of linoleic acid. We also found that the FA elongase ELOVL1 and the ceramide synthase CERS2 were involved in C24:2 ceramide production. Sphingolipids are known to form lipid microdomains in membranes; however, in a detergent-resistant membrane (DRM) assay, we observed a lower proportion of C24:2 sphingomyelin in the DRM fraction than of saturated sphingomyelins, suggesting that C24:2 sphingolipids may act to negatively regulate lipid microdomain formation. Our findings expand our knowledge of sphingolipid diversity, and provide insight into how different sphingolipid molecular species play different functions in biological membranes.     

n−3 polyunsaturated N-acylethanolamines are CB2 cannabinoid receptor-preferring endocannabinoids

Anandamide, the first identified endogenous cannabinoid and TRPV1 agonist, is one of a series of endogenous N-acylethanolamines, NAEs. We have generated novel assays to quantify the levels of multiple NAEs in biological tissues and their rates of hydrolysis through fatty acid amide hydrolase. This range of NAEs was also tested in rapid response assays of CB₁, CB₂ cannabinoid and TRPV1 receptors. The data indicate that PEA, SEA and OEA are not endocannabinoids or endovanilloids, and that the higher endogenous levels of these metabolites compared to polyunsaturated analogues are a correlate of their slow rates of hydrolysis. The n?6 NAEs (AEA, docosatetraenoyl and docosapentaenoyl derivatives) activated both CB₁ and CB₂ receptors, as well as TRPV1 channels, suggesting them to be ‘genuine’ endocannabinoids and ‘endovanilloids’. The n?3 NAEs (eicosapentaenoyl, docosapentaenoyl and docosahexaenoyl derivatives) activated CB₂ receptors and some n?3 NAEs (docosapentaenoyl and docosahexaenoyl derivatives) also activated TRPV1 channels, but failed to activate the CB₁ receptor. We hypothesise that the preferential activation of CB₂ receptors by n?3 PUFA NAEs contributes, at least in some part, to their broad anti-inflammatory profile.     

亚麻酸对肝癌细胞HepG2节律的调控研究

目的:探讨多不饱和脂肪酸亚麻酸对肝癌细胞HepG2节律钟的影响。方法:通过50%的马血清刺激诱导HepG2细胞同步化,利用亚麻酸处理同步化后的HepG2细胞。进一步利用荧光定量PCR和western-blot检测节律钟关键基因的变化。结果:HepG2细胞经过亚麻酸处理后节律钟关键基因芳烃受体核转位蛋白3(brain and muscle Arnt-like protein-1,BMAL1)和蓝光受体蛋白1(Cryptochrome 1,CRY1)在转录水平的表达水平有所降低。与此一致的是,在蛋白质水平CRY1和BMAL1的表达同样受到亚麻酸的抑制。同时发现,CRY1的转录水平的节律周期有明显的缩短。进一步研究发现,亚麻酸对HepG2细胞的脂肪酸合成关键基因脂肪酸合酶(Fatty acid synthase,FASN)和硬脂酰辅酶A脱氢酶(Stearoyl-CoA desaturase 1,SCD1),以及免疫促炎因子白细胞介素-6(Interleukelin-6,IL-6)和白细胞介素-8(Interleukelin-8,IL-8)的mRNA的表达具有明显的抑制效应。结论:亚麻酸影响了肝癌HepG2细胞水平的节律基因的表达水平以及缩短了节律基因的周期,并且对于HepG2细胞的脂肪酸合成以及免疫促炎因子有明显的抑制效应。