沙鼠松果腺細胞之超微結構、鋅碘鈉染色與嗜鉻反應性之研究

沙鼠松果腺細胞之一般超微結構與其他嘴齒類動物相似：細胞外形不規則，且具有許 多胞質突起，核呈圓形或卵圓形有明顯之核仁；線粒體呈長形且有扁平型脊或管狀脊 和緻密之基質，高爾基氏器發達，平滑內質網及粗糙內質網亦多，胞質內有許多微小 管與胞質絲。有些松果腺細胞具有較大之胞質突起，胞質內含有許多包被小泡（ coated vesicle , 30-50 nm ) ；另外，在血管附近與松果腺實質內可見到串珠狀之 神經斷面（ varicosities ) ，其內含有許多澄清小泡（clea : vesicles ）與密心 小泡（ dens-cored vesicles ）。經鋅碘餓染色後，鋅碘鐵可染色部分之松果腺細 胞，但不是全部之松果腺細胞皆可染色；胞質突起內之包被小泡及神經斷面內之澄清 小泡亦均可用鋅碘餓染色。經嗜鉻反應後，松果腺細胞並無嗜鉻反應，但松果腺之神 經斷面內之密心小泡之數目增多且其密心之電子密度增高；顯示神經斷面內之密心小 泡均可用嗜鉻反應染色。由以上之結果吾人推論， ( 1 ）沙鼠松果腺之一些細胞之 胞器如內質網等，及其胞質突起內之包被小泡可能含有 cystine / cystein ，這些 胞器可能與鈣離子之收集與松果腺 cconcretion 之生成有關； ( 2 ）沙鼠松果腺之 神經斷面內之突觸小泡（ synaptic vesicles ) I 勾可能含有神經傳導物質 monoamines 。 The ultrastructure and cytochemistry of the gerbil pineal gland were studied by the conventional electron microscopy, zinc iodide- osmium tetroxide (ZIO) staining and chromaffin reaction. Conventional electron microscopy revealed that the ultrastructure of gerbil pinealocytes are similar to other rodents, i.e. irregular cell contour with numerous cytoplasmic processes, round or oval nucleus and prominent nucleoli, elongated mitochondna with flattened and tubular cristac and dense matrix, well-developed Golgi apparatus and its associated structures , abundant elements of endoplasmic reticulum -- both smooth and rough varieties, and bundles of microfilament and microtubule in the cytoplasm. Some pinealocyte processes contain numerous small clear and “slightly coated” vesicles. Numerous profiles of varicosities containing small dense-cored and clear vesicles were frequently encountered. After ZIO treatment, ZIO staining was preferentially localized in the cytoplasm of some, but not all, of the gerbil pinealocytes. Numerous small clear vesicles (30-50 nm in diameter) in the process of the pinealocytes or in the varicosities of the nerve fibers showed strong ZIO-philia. After chromaffin reaction treatment, the number and electron density of small clear and dense-cored vesicles in the profiles of nerve varicosities increased and this indicates that some of the small clear and dense-cored vesicles in the varicosities are reactive. It is thus concluded that (1) the vesicles in the pinealocytes may be rich in cystine and/or cysteine and possibly the organelle is involved in the sequestering calcium ion during the calcification of the pineal concretions, and (2) the small dense- cored and clear vesicles in the nerve fibers in the gerbil pineal parenchyma may contain both serotonin and primary biogenic amines

Chromaffinity, uranaffinity and argentaffinity of small granule-containing (SGC) cells in rat superior cervical ganglia

A systemic examination on the small granule-containing (SGC) cells in rat superior cervical ganglia was conducted by conventional and cytochemical electron microscopy including chromaffin, argentaffin and uranaffin reactions. According to the fine structure of dense cored vesicles (DCVs) in the cytoplasm, three types of small granule-containing (SGC) cells were revealed - Type 1: 90 - 160 nm vesicles with cores of moderate or low electron density; Type 11: 130 - 330 nm vesicles, polymorphic with highly electron dense cores; Type 111: elongated vesicles (170 nm x 60 nm) with cores of moderate to low electron density. The majority of SGC cells were the Type 1 cells (78%) and Type 11 and 111 cells made up 13% and 9% of SGC cell population, respectively. Cytochemical results demonstrated that only the Type 11 cells displayed a positive chromaffin reaction and al1 three types of SGC cells showed argentaffinity and uranaffinity. The present study is the first to demonstrate the argentaffin reaction at ultrastructural level in SGC cells of sympathetic ganglia. Based on the results of the present study we also concluded that (1) the DCVs of Type 11 SGC cells contained noradrenaline and (2) biogenic amines and nucleotides (ATPs) coexisted in the DCVs of al1 three types of SGC cells

Comparison of ultrastructure and lectin histochemistry on the anterior medial gland of nasal septum in rat and gerbil

The anterior medial gland (AMG), located in the submucosa of rodent nasal septum, is suggested to provide fluid for humidification of inspired air. Tremendous variation of the environmental air humidity, on which rats and gerbils depend to live, leads us to expect a multiplicity on ultrastructure and various subcellular glycoconjugate distribution within the AMG acinar cells between these two species. Electron microscopy revealed that: (1) The nucleus of AMG acinar cells in rat was irregular-shaped, but that in gerbil was round or elliptical; (2) Secretory granules in rat AMG acinar cells contained homogenous content with various electron density. However, two types of secretory granules in gerbil AMG acinar cells were found: one with lamellated-structure and high electron density, while the others had particulate materials; (3) Myoepithelial cells were present in the acinus of medial and posterior regions in rat AMG, but absent in gerbil; and (4) Nerve terminals were present only in the medial and posterior rat AMG, but in all three regions of the gerbil AMG. Lectin histochemistry demonstrated that: (1) Rat and gerbil AMG acinar cells expressed strong affinity toward Con A and WGA, but neither showed any reactivity toward UEA and PNA; and (2) Varying degrees of reactivity toward different lectins, including DBA, PNA, SBA and EBL, were recognized in rat and gerbil AMG acinar cells. We confirm the species variation on the ultrastructure and lectin histochemistry of AMG in rats and gerbils, and speculate that these variations may be due to the different living environment

The permeability of capillaries among the small granule-containing cells in rat superior cervical ganglia, an ultrastructural lanthanum tracer study

The permeability of blood capillaries associated with small granule-containing (SGC) cells in rat superior cervical ganglia was investigated at ultrastructural level by employing ionic lanthanum as an electron dense tracer. In rat superior cervical ganglia, the majority of blood capillaries were nonfenestrated. Both fenestrated and nonfenestrated capillaries were observed in the area associated with SGC cells. Lanthanum tracer was observed in the lumina1 surface, the interendothelial cleft and the subendothelial perivascular spaces of both fenestrated and nonfenestrated capillaries associated with SGC cells. The external lamina of the Schwann cell which surrounded the neurons, nerve fibres and SGC cells were clearly delineated by the lanthanum tracer. Furthermore, the perineuronal space, the periaxonal space, and the pericellular space of the SGC cells were readily accessible to the lanthanum ion. The results demonstrated an absence of blood-nerve barrier, bloodganglionic and blood-SGC cell barrier to the lanthanum ion in the parenchyma1 area of the SGC cells in rat superior cervical ganglia. It is proposed that lanthanum may pass through the endothelial cells via 1) the fenestrae of fenestrated capillaries, 2) the intercellular junctions of both fenestrated and nonfenestrated capillaries, i.e., a paracellular pathway; and 3) the process of endocytosislexocytosis, i.e., a transcellular pathway. to reach the subendothelial space and be distributed in the parenchyma of SGC cells in rat superior cervical ganglia

Analysis of NQO1 polymorphisms and p53 protein expression in patients with hepatocellular carcinoma

NAD(P)H: quinone oxidoreductase 1 (NQO1), a cytosolic enzyme which catalyzes the twoelectron reduction of quinone compounds, has been suggested to prevent the generation of semiquinone free radicals and reactive oxygen species, thus protecting cells from oxidative damage. However, the enzymatic activity of NQO1 strongly depends on the individual genetic polymorphism of the NQO1 gene. A common NQO1 polymorphism is a C to T transition at position 609, which results in an inactive enzyme. Recent studies showed that NQO1 is an important enzyme for stabilizing p53 protein, which is involved in antitumorigenesis. Thus, the lack of enzymatic activity in the homozygous C609T NQO1 polymorphism may play a pivotal role in tumor development. This study aimed to investigate the relationship between C609T NQO1 polymorphism and p53 expression in human hepatocellular carcinoma (HCC). Genotyping of NQO1 was performed on 100 HCC specimens by PCR-RFLP analysis. In addition, NQO1 and p53 protein expression in HCC samples at different TNM stages was determined by immunohistochemistry. Our data showed that (1) the frequency of C609T NQO1 was significantly increased in TNM stage III HCC patients; (2) no significant association was found between p53 expression and C609T polymorphism of NQO1 gene; and (3) a tumor/non-tumor (T/N) ratio > 1.27 of NQO1 expression revealed by real-time qPCR analyses was positively correlated with poorer survival in patients with tumors >5 cm, suggesting that an increase of NQO1 expression may be an indicator of advanced tumor progression. This study provides important information about NQO1 genotypes and its expression to HCC tumor development and progression

ß-Lapachone accelerates the recovery of burn-wound skin

ß-lapachone is a quinone of lapachol extracted from the bark of lapacho tree. Recent findings demonstrated that punched skin wounds of mice healed faster with ß-lapachone treatment. The present study investigates the effects of ß-lapachone on burn-wound skin of C57BL/6 mice injured by a 100°C iron stick. Our results indicated that wounds treated with ß-lapachone recovered faster than those treated with control ointment containing no ß-lapachone. On the third day after burning, the area of ß-lapachone treated-wound was 30% smaller than wound treated with control ointment. H&E and immunohistochemistry staining showed that burn-wound skin treated with ointment containing ß-lapachone healed faster in its epidermis, dermis, and underlying connective tissues with more macrophages appeared than those treated with control ointment alone. RAW264.7 cell, a macrophage-like cell line derived from BALB/C mice, was used as a model for scrutinizing the effect of ß-lapachone on macrophages. We found that the proliferation and the secretion of EGF and VEGF by macrophages were higher in cultures treated with ß-lapachone and that ß-lapachone can also increase the release of EGF with TNF-α pretreatment. We conclude that ß-lapachone plays an important role in accelerating burn wound healing, and that ß-lapachone not only can raise the proliferation of macrophages but also increase the release of VEGF from macrophages

Involvement of endoplasmic reticulum stress and activation of MAP kinases in B-lapachone-induced human prostate cancer cell apoptosis

ß-Lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the ß-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of ß-lapachone. ß- Lapachone-induced DU145 cell apoptosis was dosedependent and accompanied by cleavage of procaspase- 12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent ß-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of ßlapachone- treated cells. These findings show that ßlapachone- induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of ßlapachone

Sulindac compounds facilitate the cytotoxicity of β-lapachone by up-regulation of NAD(P)H quinone oxidoreductase in human lung cancer cells.

β-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of β-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, β-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to β-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in β-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of β-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers