脑缺血大鼠海马超微结构的形态定量研究

对大鼠双侧颈总动脉夹闭2小时,经透射电镜观察海马,用Weibel氏形态定量法分别测量了海马1区和3区的线粒体和突触的表面积密度(Svi)、表面积—体积比(Si/Vi),并与对照组进行了比较。结果表明:缺血时海马1区受损较严重,表现为线粒体体积缩小,数量减少,Svi下降;突触数量有增多的趋势,但突触的Svi与Si/Vi变化不明显。缺血组海马3区受损较轻,线粒体和突触的结构基本正常,其Svi、Si/Vi与对照组差别不显著,提示了3区对缺血有较强的耐受性。     

‘Distensibility’ of the papaverine-relaxed vascular bed in human subcutaneous tissue

The effect of an increase in vascular transmural pressure upon the blood flow in two subcutaneous vascular beds, maximally dilated by papaverine was studied in 6 healthy humans. Blood flow was measured on the dorsum of the hand and at the lateral malleolus by the local ¹³³Xe washout technique. Increase in vascular transmural pressure was induced by lowering the labelled area various distances below heart level. Lowering the area caused an increase in blood flow. The increase was less pronounced in the legs than in the hand. As arterial perfusion pressure head remained constant during lowering, this indicates that the relative decrease in vascular resistance was smaller in the leg than in the hand. Experimental edema did not influence the relative decrease in vascular resistance. The results suggest that ‘distensibility’ of the resistance vessels is smaller in the leg than in the hand. This might be due to a structural adaptation of the vascular wall in vessels often subjected to increased hydrostatic pressure.     

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats following UVA-irradiation

Ultrastructual characteristics of the dorsal skin responses to a single irradiation of UVA (1100 kJ/m2) were examined in Wistar-derived hypotichotic WBN/ILA-Ht rats (HtRs). In the epidermis, mitochondrial swelling of some keratinocytes and dissociation of keratinocytes due to intercellular edema developed at 3 hours (h) after irradiation and continued to 48 h. At 6 h, in addition to these changes, necrosis of keratinocytes accompanied with infiltration of neutrophils was also observed in some portions, and epidermal hyperplasia with many keratinocytes showing nucleolar hypertrophy and some mitotic keratinocytes was observed at 48 h. In the dermis, mitochondrial swelling and/or partial cytoplasmic destruction in capillary endothelial cells and edema with inflammatory cell infiltration were observed at and after 3 h, and extravasation of erythrocytes was found in some capillaries at 48 h. Mitochondrial swelling was also frequently found in pericytes and fibroblasts. Inflammatory cells were mainly composed of neutrophils throughout the experimental period. Mild degranulation of mast cells which also showed mitochondrial swelling was observed at and after 3 h, and a close special relationship between mast cells and fibroblasts or neutrophils was sometimes observed. In conclusion, the most prominent change in the dorsal skin of HtRs exposed to UVA was degeneration of capillary endothelial cells, resulting in edema and inflammatory cell infiltration, and the most characteristic cytopathic effect of UVA was mitochondrial swelling, and it was common to keratinocytes, capillary endothelial cells, pericytes, mast cells, and fibroblasts.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.     

Effect of beta-amyloid peptide fragment 25-35 on nonselective permeability of mitochondria

Beta-Amyloid peptide fragment 25-35 potentiated phosphate- and calcium-induced opening of mitochondrial channels and caused swelling of mitochondria (even without exogenous calcium and phosphate). These changes were accompanied by accumulation of lipid peroxidation products in mitochondria. Specific inhibitors of mitochondrial channels ADP and cyclosporine A prevented beta-amyloid peptide-induced swelling of mitochondria. Our findings suggest that potentiation of mitochondrial channel opening is an important component of the neurodegenerative effect of beta-amyloid.     

Apoptosis and apoptosis-associated perturbations of peripheral blood lymphocytes during HIV infection: comparison between AIDS patients and asymptomatic long-term non-progressors

This study was designed to compare the degree of lymphocyte apoptosis and Fas-Fas ligand (FasL) expression in AIDS patients and long-term non-progressors (LTNPs) and correlate these parameters with apoptosis-associated perturbations in lymphocyte function. LTNPs had a lower frequency of apoptotic CD4+ and CD8+ T cells compared with subjects with AIDS. This correlated with a lower frequency of cells expressing Fas and FasL. The frequency of selected lymphocyte populations exhibiting a disrupted mitochondrial transmembrane potential (DeltaPsim) and increased superoxide generation was lower in LTNPs than in patients with AIDS; these abnormalities were associated with lower levels of caspase-1 activation in LTNPs. The results indicate a significantly reduced level of apoptosis and apoptosis-associated parameters in LTNPs than in patients developing AIDS. Based on these findings, a crucial role for mitochondria can be predicted in the process of lymphocyte apoptosis during the evolution of AIDS.     

Purification and enzymatic activity of an NADH-fumarate reductase and other mitochondrial activities of Leishmania parasites

A 65 kD membrane-associated NADH-fumarate reductase subunit, which has a molecular weight similar to that of one of the enzyme subunits from bacteria, was purified from Leishmania donovani promastigotes. NADH-fumarate reductase and other mitochondrial enzymatic activities of L. major and L. donovani promastigotes and amastigotes were investigated. The presence of NADH-fumarate reductase was demonstrated in digitonin-permeabilized L. major promastigotes and mitochondria of L. major and L. donovani promastigotes and amastigotes. The activity of solubilized NADH-fumarate reductase was measured in L. major and L. donovani promastigotes. Succinate exhibited a clear concentration-dependent inhibitory effect on fumarate reductase, whereas fumarate also exhibited a clear concentration-dependent inhibitory effect on succinate dehydrogenase. The data indicate that fumarate reductase is an obligatory component of the respiratory chain of the parasite. Since the enzyme is an important component in the intermediate metabolism in the Leishmania parasite and is absent in mammalian cells, it could be a potential target for antileishmanial drugs.     

The role of Bcl-2 family members in the progression of cutaneous melanoma

The overwhelming problem of cutaneous melanoma is chemoresistance. Subversion of the biochemical changes that lead to chemoresistance intersects the apoptosis pathways. The mitochondrion has been a focal point of this intersection for the development of therapeutic strategies aimed at reducing the progression of melanoma. The Bcl-2 family of apoptotic regulators is arguably the most pivotal component to this mitochondrial response. The shear number of studies conducted on the relationship between melanoma and Bcl-2 members prompted us to evaluate the literature available and discern some rational utility of the data. We have found that there are striking inconsistencies for the expression of Bcl-2 family proteins with melanoma progression, particularly for Bcl-2. Roughly one-third of the data suggests an increase in Bcl-2 expression with advancing melanoma, while another third suggests a decrease. Furthermore, the remaining third found on the whole, a detectable level of Bcl-2 in all tissues of melanocytic origin. These discrepancies are difficult to rectify in light of the apparent success of recent clinical trials utilizing Bcl-2 antisense strategies. The general consensus in the literature is that pro-apoptotic Bax is decreased with melanoma progression while anti-apoptotic Bcl-x_L and Mcl-1 appear to increase with progression. We suggest that the biochemical techniques being used for analysis present too great of a heterogeneity, which could be mitigated with more standard procedures and reagents. Finally the utility of ‘multi-specific’ antisense tactics could be a more effective way of targeting advanced melanoma disease. This revised version was published online in July 2006 with corrections to the Cover Date.