Various coated vesicles are implicated in the intracellular transport between different compartments. In vitro reconstitution is a powerful experimental system to study molecular mechanisms involved in assembly of coat proteins from cytosol onto membranes as well as formation of coated vesicles. Liposomes have been recently utilized in the cell-free systems. In this review, we summarize studies on reconstitutions of coated vesicles or coated structures on liposomes. A novel method using dynamic light scattering (DLS) to quantify vesicle formation from liposomes also is described. Our recent study on the role of phospholipids in vesicle formation, where the DSL assay is used in combination with lipid analysis, also is introduced.

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Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33°C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33°C, but did not at nonpermissive temperature (39°C). High expression level of large T-antigen in the nuclei was observed at 33°C, whereas the expression level was gradually decreased in a time-dependent manner at 39°C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33°C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E₂. Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E₂ were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.

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We have reported that an inhibitor of interleukin-3 (NIL-3) is produced from murine bone marrow cells in response to excess stimulation of interleukin-3. In this report, we attempted the purification of the NIL-3 activity from bone marrow culture supernatant in the presence of interleukin-3. The purified NIL-3 activity was a protein with relative molecular weight of 54.5 kDa (SDS-PAGE), which inhibited the growth of IL-3 dependent DA-1 cell growth in a dose dependent manner. The N-terminal amino acid sequence of purified NIL-3 activity was determined to be homologous to beta-2 glycoprotein I (apolipoprotein H: APO-H). The gene expression of APO-H was detected by nested-PCR in STIL-3 C5-CM stimulated total bone marrow cells and STIL-3 C5-CM stimulated bone marrow fraction 2 (Fr. 2) which has been reported as a hematopoietic stem cell rich fraction. These observations indicate the possibility that the APO-H is the NIL-3 which was produced from bone marrow cells in response to excess IL-3 stimuli.

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The involvement of actin filaments from the host cell on the process of invasion of trypomastigote forms of Trypanosma cruzi was analyzed in seven different cell lines. Prior incubation of all cell lines with cytochalasin D, under conditions which interfere with actin filaments, markedly inhibited parasite internalization and increased parasite attachment. Attached parasites were readily ingested following washing of the drug-treated cells. Cytochalasin treatment interfered with the distribution of actin filaments of the host cell as evaluated by visualization of the filaments using confocal laser scanning microscopy of cells incubated in the presence of FITC-phalloidin. Concentration of actin filaments could be observed in most, but not all, parasites in the process of internalization. We also treated LLCMK 2 and macrophage cells with Jasplakinolide, a drug that stabilizes actin filaments, before interaction with the trypomastigote forms. This drug partially inhibits parasite invasion into the cells. Prior incubation of the host cells in the presence of colchicine, which interfere with microtubules, also inhibited parasite internalization into the cells.

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Apoptosis plays an important role during embryonic development. Apoptotic cell death is executed by caspases and can be regulated by the Bcl-2 family of genes. Ribonuclease protection assay was used to investigate the expression of selected apoptosis-related genes of the Bcl-2 family, pro-apoptotic Bax, Bad and anti-apoptotic Bcl-2, during differentiation of murine embryonic stem cells (ES) mediated by all-trans-retinoic acid. The mRNA expression of caspase 3, caspase 6 and certain pro-inflammatory cytokines was also investigated simultaneously. ES cells exposed to 1 μM all-trans-retinoic acid on day 8, 9 and 10 of differentiation revealed increased expression of Bax and Bad compared to the vehicle-treated cells. No effect on Bcl-2 mRNA was noted after all-trans-retinoic acid treatment. Increased mRNA expression of caspase 3 and caspase 6 in all-trans-retinoic acid-exposed ES cells suggested that caspases play an important role in retinoic acid-mediated apoptosis during ES differentiation. Increase in the expression of TNFα and macrophage migration inhibitory factor (MIF) was noted in retinoic acid-treated cells on day 14. Significant increase observed in interferon γ inducing factor (IGIF/IL-18) mRNA expression in all-trans-retinoic acid-treated cells on day 14 and 17 did not translate to increased INFγ expression. No change in the expression of other pro-inflammatory cytokines was noted with all-trans-retinoic acid treatment. The function of TNFα, IGIF/IL-18 and MIF in all-trans-retinoic acid-treated cells during ES differentiation and apoptosis is still speculatory. Results suggested that RA-mediated apoptosis during neural differentiation of ES cells involves up-regulation of caspase 3, caspase 6, Bad, and Bax.

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A heparin-binding growth factor, midkine, is the product of a retinoic acid-responsive gene. Since retinol plays critical roles in lung development and treatment of bronchopulmonary dysplasia, and midkine has been implicated in the maturation of lung explants and in cytoprotection, we herein examined midkine expression during postnatal development of the lungs and hyperoxic lung injury. Midkine protein transiently increased to a maximum level at around 4 days postnatal. Immunohistochemistry revealed that the amounts of midkine increased in resident alveolar cells, but not in smooth muscle cells or the large airway epithelium. If neonatal mice were exposed to >95% oxygen, lung development was impaired and midkine expression was suppressed. In contrast, when adult mouse lungs as well as in vitro cultured lung adenocarcinoma cells were exposed to hyperoxia, midkine expression was not affected. Furthermore, a pronounced induction of midkine by retinoic acid was observed in neonatal lungs. The results indicate that midkine expression is associated with postnatal lung development, but not necessarily with hyperoxic cell damage.

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In order to elucidate the mechanism controlling the biogenesis of the Golgi complex, we have studied whether the expression of a resident membrane protein p138 of the Golgi complex is dependent upon the cell cycle. The protein level of p138 in human KB cells was increased during thymidine block to synchronize the cells in the early-S phase, but changed little from S to G2 after release from the block. On the other hand, the mRNA level of the p138 gene was constant during the block. The change in mRNA level in the cells was small with a low peak at S to G2. Both p138 protein and mRNA levels decreased after cell division and then rose rapidly to the same level as those of log-phase cells in the next G1 to S. Thus, translation of p138 protein was upregulated in the cells at the early-S phase. However, we found also that the p138 protein level increased during an arrest at G2/M caused by etoposide. The kinetics of centrosome duplication apparently differ from those of p138 protein production. The duplication occurred mainly at S to G2 after the release from thymidine block, while the ratio of cells containing duplicated centrosomes increased gradually during the block. Taken together, these results show that both the translation and transcription of p138 protein are regulated independent of the cell cycle and dissociated from the duplication of the centrosome. Rather, the expression of p138 protein seems to be coupled with a change in cell size since both thymidine block and etoposide inhibition resulted in an apparent increase in cell size.

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