High-throughput immuno-electron microscopy is required to capture the protein–protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell’s plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Children with either fetal alcohol spectrum disorder (FASD) or attention-deficit/hyperactivity disorder (ADHD) display deficits in attention and executive function (EF) and differential diagnosis of these two clinical groups may be difficult, especially when information about prenatal alcohol exposure is unavailable. The current study compared EF performance of three groups: children with heavy prenatal alcohol exposure (ALC); nonexposed children with attention-deficit/hyperactivity disorder (ADHD); and typically developing controls (CON). Both clinical groups met diagnostic criteria for ADHD. The EF tasks used were the Wisconsin Card Sorting Test (WCST), the Controlled Oral Word Association Test (COWAT), and the Trail Making Test (TMT). Results indicated different patterns of deficit; both clinical groups displayed deficits on the WCST and a relative weakness on letter versus category fluency. Only the ALC group displayed overall deficits on letter fluency and a relative weakness on TMT-B versus TMT-A. In addition, WCST performance was significantly lower than expected based on IQ in the ADHD group and significantly higher than expected in the ALC group. These results, which indicate that, although EF deficits occurred in both clinical groups, the degree and pattern of deficit differed between the ALC and ADHD groups, may improve differential diagnosis. (JINS, 2008, 14, 119–129.)

Fas ligand (FasL) induces programmed cell death, or ‘apoptosis’, in cells expressing its cognate receptor, Fas (CD95/APO-1). There is evidence that FasL precludes inflammatory reactions from sites of ‘immune privilege’ by triggering Fas-mediated apoptosis of infiltrating pro-inflammatory cells. The ability of FasL to impair immune responses is being pursued as a possible means of protecting tissue transplants from immunological rejection, and therapeutic promise has been reported in some experiments. However, FasL is becoming an enigmatic molecule, exhibiting pro-inflammatory activity independently of its ability to mediate immune downregulation. FasL can recruit and activate neutrophils and macrophages in some experimental situations. Triggering of Fas in some cell types has been shown to upregulate expression of certain pro-inflammatory cytokines and chemokines, providing an unexpected link between apoptosis and inflammation. FasL appears to contribute to the destruction of Fas-sensitive end-organ cells during inflammation. This appears to occur in two ways: (1) direct killing by cytotoxic immune effector cells expressing FasL; or (2) autocrine cell suicide of end-organ cells that upregulate their own FasL in the inflammatory context. Depending on the condition, or the site of inflammation, either or both mechanisms may occur. Prevention of Fas-mediated end-organ apoptosis and enhancement of Fas-mediated apoptosis of inflammatory cells are emerging as potential anti-inflammatory therapeutic goals.

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.

The molecular mechanisms leading to ovarian follicular atresia in the typical pathways of programmed cell death remain to be clarified. Here we have demonstrated that the apoptotic signalling pathway in MRL-+/+ (MRL/+) murine oocytes is through the Fas receptor followed by the activation of caspase-3. In contrast, we found that the aberrant expression and dysfunction of the mutant Fas receptor in MRL-lpr/lpr (MRL/lpr) murine oocytes caused by insertion of the early transposable element (ETn) into the Fas gene were associated with an inability to activate the caspase cascade (especially caspase-3) and to induce nuclear DNA fragmentation. These findings indicate that the induction of apoptosis in MRL/lpr murine oocytes did not occur in the presence of a defective Fas receptor lacking the death domain to trigger the caspase cascade, suggesting a failure to induce ovarian follicular atresia.

Interferon-gamma (IFN-γ) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-γ affects the parasite-infected host cells. The effect of IFN-γ on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-γ, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-γ. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-γ-treated cells following N. caninum infection. Moreover, IFN-γ treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-γ induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

In lpr mice the insertion of an early transposable element (ETn) into intron 2 of the Fas gene, which mediates apoptosis, causes the development of massive lymphadenopathy, splenomegaly and autoimmune disease. In the present study we investigated the influence of this mutation on ovarian development of lpr mice. By means of in situ hybridisation, the expression of Fas mRNA was detected at the same levels in the ovarian cells of MRL/MpJ-lpr/lpr (MRL/lpr) mice as in those of MRL/MpJ- +/+ (MRL/+) mice. However, indirect immunofluorescence (IIF) staining with anti-Fas monoclonal antibody (mAb) on the membrane of follicle and egg of MRL/lpr mice was significantly weaker than that of MRL/+ mice. Furthermore, the expression level of Fas protein at the 45 kDa band from ovarian cell lysates of MRL/lpr mice was much lower than that of MRL/+ mice. The co-incubation of Spodoptera frugiperda (Sf9)-Fas ligand (L) cells with eggs of MRL/+ mice resulted in apoptosis of eggs, as detected by the terminal deoxynucleotide transferase mediated dUPT-nick end labelled (TUNEL) method. In contrast the co-incubation of Sf9-FasL cells with eggs of MRL/lpr mice did not generate apoptosis in eggs. Following intraperitoneal administration of anti-Fas mAb into both types of mice, most oocytes, a proportion of granulosa cells in the ovary and hepatocytes in liver of MRL/+ mice were positively stained by the TUNEL method, corresponding to the appearance of DNA fragmented ladders by DNA fragmentation assay, while negative signals were obtained in those cells of MRL/lpr mice. As the mice aged, the ovarian size of MRL/lpr mice was found to be much larger than that of MRL/+ mice due to the increased number of ovarian follicles. Therefore, the ovarian adenopathy in MRL/lpr mice was strongly suggested to be caused by the dysfunction of Fas antigen in the ovary.