Transcriptional regulation of the gene for the myeloid calcium binding protein, MRP14, was investigated in human monocytic leukemia cell lines. The MRP14 gene was not expressed in monoblastic ML-1 cells, promonocytic U-937 cells, or promyelocytic HL-60 cells. On the other hand, the gene was expressed in monocytic THP-1 cells and in the HL-60 cells treated with 1, 25-dihydroxyvitamin D₃ (VD₃). The level of MRP14 in VD₃-treated HL-60 cells was two-fold higher than that in THP-1 cells. Among several known transcription factor binding motifs, nuclear protein(s) of VD₃-treated HL-60 cells and THP-1 cells bound to the CCAAT/enhancer binding protein (C/EBP)-binding motif that was located in the upstream region of the MRP14 gene (-81), as evidenced by the competitive gel mobility-shift assay. An antibody for C/EBP α supershifted the nucleoprotein complex in THP-1 cells but not in the VD₃-treated HL-60 cells, whereas an antibody for C/EBP β blocked the formation of the complex with the nuclear factor of the HL-60 cells but not with that of THP-1 cells. An anti-C/EBP δ antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBP α and -β were able to bind to the C/EBP motif, and that C/EBP α bound to the motif in THP-1 cells and C/EBP β bound to that in the VD₃-treated HL-60 cells. Furthermore, to examine the transcriptional activity of the C/EBP motif, we transfected several constructed luciferase reporter DNAs into HL-60 cells and THP-1 cells. The luciferase activity of the C/EBP motif in HL-60 cells was increased by VD₃ treatment. The C/EBP motif in the MRP14 gene was confirmed to function as a regulatory region in VD3-treated HL-60 cells and THP-1 cells by the assay. Since C/EBP β was also detected in VD₃-untreated HL-60 cells by immunoblotting, VD₃ activated C/EBP β to bind to the motif, probably through post-translational modification.

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Murine tracheal epithelial cell lines, TM01 and TM02-3, were established from a primary culture of tracheal cells of adult transgenic mice bearing a temperature-sensitive simian virus (SV40) large T-antigen gene. Both TM01 and TM02-3 cells, which grew until confluent monolayers were formed, maintained tight contact with neighboring cells, and retained the characteristics of epithelial cells with microvilli on the surface. These cells grew at a permissive temperature (33°C), but did not at a nonpermissive temperature (39°C), indicating that TM01 and TM02-3 cells undergo temperature-sensitive growth. Large T-antigen was expressed only in the nuclei at 33°C. Sepharose CL-4B column chromatography using a ¹⁴C-glucosamine hydrochloride, indicating that both cells produced high molecular weight glycoconjugates, and suggesting that these cells may originate from mucus-producing cells. TM01 cells expressed intercellular adhesion molecular-1 (ICAM-1) in both unstimulated and stimulated (1, 000 U/ml tumor necrosis factor-a and 500 U/ml interferon-γ) conditions, whereas TM02-3 cells expressed ICAM-1 only under stimulated conditions. We conclude that these cell lines may serve as a useful model to study the tracheal cell functions under defined in vitro conditions.

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The spatial characteristics of inositol 1, 4, 5-trisphosphate (IP₃)-induced quantal Ca²⁺ release were examined by imaging Ca²⁺ concentrations within Ca²⁺ stores ([Ca²⁺]_L) in permeabilized HSY cells. The image of mag-fura-2 fluorescent ratio with dual excitation (344 nm/360 nm) demonstrated that a sequential application of different concentrations of IP₃ (0.1, 0.3, 10 μM) resulted in a stepwise decrease in the ratio at all regions of the cytoplasm. This change in the ratio suggests that the stepwise decrease in [Ca²⁺]_L is associated with the quantal Ca²⁺ release. To monitor the change in [Ca²⁺]_L within a single organelle, IP₃-dependent changes in the mag-fura-red fluorescence of permeabilized cells were studied by confocal microscopy. Applications of increasing concentrations of IP₃ caused a stepwise increase in fluorescence within ER-like reticulum structures of the cytoplasm. This finding suggests that the [Ca²⁺]_L in a single Ca²⁺ store was not depleted by submaximal concentrations of IP₃, and supports the steady-state model of quantal Ca²⁺ release.

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Methylation of cytosine in the genomic DNA plays an important role in mammalian embryogenesis. DNA methyltransferase activity, which contributes mainly to the maintenance of the methylation pattern during proliferation, is under the control of the cell cycle, its activity being higher in the S phase than in the other phases (Adams, R.L.P., 1990, Biochem. J. 265, 309-320). In the present study, we examined how DNA methyltransferase is regulated in the cells arrested at S phase by aphidicolin treatment. The activity and protein levels of DNA methyltransferase in the nuclei were kept constant in proliferating mouse erythroleukemia cells, and increased about twofold after 6 h incubation in the presence of aphidicolin. This increase of DNA methyltransferase levels by aphidicolin treatment was positively correlated with the cell population at S phase. De novo synthesis of DNA methyltransferase protein was increased by the treatment. In addition, the relative half life of pulse labeled DNA methyltransferase was prolonged by aphidicolin treatment. Both increase in synthesis and prolongation of half life of DNA methyltransferase in S phase contributed to the increase of the activity and the protein levels by aphidicolin treatment. Prolongation of half life was abolished by cycloheximide, suggesting that newly synthesized protein(s) with a short half life participated in the degradation of DNA methyltransferase.

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To elucidate the mechanism by which red blood cells (RBC) participate in thrombus formation, we investigated the mechanism of adhesion between human RBC. Our study showed that the morphology of RBC was changed by various cationic reagents, inducing adhesion between RBC. When RBC suspended in PBS buffer containing sodium phosphate (PBS(Na)) or potassium phosphate (PBS(K)) were treated with cationic reagents, stronger adhesion occurred between RBC treated with the latter. When concentrations of the reagents were low, adhesion was released and the RBC resumed its original morphology after washing. However, when the concentrations of reagents were high, the morphology did not normalize, although the adhesion was released. When fresh RBC were treated with cationized ferritin (CF), CF bound to the periphery of RBC membranes and induced adhesion. However, when RBC were induced to adhere strongly by a cationic reagent, no binding of CF to the membrane was not observed. When RBC were treated with CF, bindings between substances outside the membranes and bindings between the membranes and substances outside the membranes were observed. When RBC treated with neuraminidase to remove 85-90% of sialic acid were treated with the cationic reagents, both adhesion between RBC and morphological change were reduced. When RBC were pretreated with polyclonal antibody against human RBC membrane band 3 protein, treatment with the cationic reagents did not induce adhesion and morphological change of RBC. Further, when RBC induced to adhere by the cationic reagents were treated with the polyclonal antibody against band 3, in the case of weak adhesion, the adhesion was released and the RBC resumed its original morphology. However, in the case of strong adhesion, the morphology did not return to normal although the adhesion was released.
These results suggest that the adhesion between RBC induced by cationic reagents was due to changes in the charge on the membrane surface, involving polysaccharide chains and membrane surface proteins.

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It has been argued that 70 kDa heat shock cognate (hsc73)-like molecules may be expressed on the surface of certain cells, but direct evidence of this has yet to be found. To clarify whether this molecule belongs to hsc73 itself, the membrane protein fraction of Daudi cells was isolated by Triton X-114 phase separation and the reactivity of this membrane protein fraction was assessed with monoclonal antibodies (mAbs) which react with 70 kDa heat shock protein (hsp) family, i.e., NT22, A15 and 3A3. In western blotting analysis, mAb NT22-deflned protein (pNT22) was clearly detected as a membrane protein of Daudi cells with an approximate molecular size of 70 kDa, whereas pNT22 was not recognized by anti-cytoplasmic hsc73/hsp72 mAbs A15 or 3A3. By using deleted recombinant hsc73 proteins, it was determined that mAb NT22 recognizes the N-terminal 350-372 ami no acid stretches of the hsc73 protein. mAb NT22 also reacted with the cell surface protein of Daudi cells in FACS analysis. Taken together, our present data strongly suggest that pNT22 may be a novel hsc73-like protein that is localized in the plasma membrane.

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The initial association of tachyzoites of Toxoplasma gondii with a host cell induces an endocytic process which leads to the formation of a vacuole known as the parasitophorous vacuole (PV). We analyzed the parasite-host cell interaction process using either parasites or host cells whose membrane was previously labeled with probes specific for proteins, sialoglycoconjugates and lipids, and then allowed to interact for periods varying from 5 minutes to 24 hours. The fate of the fluorscents probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, FITC-Thiosemicarbazide or DTAF, which label membrane proteins, siloglycoconjugates and lipids, respectively, a uniform labeling of the cell surface was observed before interaction. When allowed to interact with T. gondii, labeling for PKH26 and DTAF, but not for FITC-Thiosemicarbazide, was observed initially at the region of contact between the two cells and subsequently on the membrane lining the PV and the intravacuolar parasites. These observations show that some, but not all, membrane components contribute to the formation of the PV membrane. Previously labeled parasites attach to the host cell surface but lose the fluorescent probes during the invasion process so that no labeled parasites were seen within the PV. These observations point to the existence of a dynamic process of membraneassociated components of the parasite and host cell during the interaction process.

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We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1, 106 and 1, 107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1, 339-1, 395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.

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The effects of cytochalasin B (CB), which acts on microfilaments, on sperm penetration into eggs of a teleost fish were investigated ultrastructurally. Eggs from the rose bitterling, Rhodeus ocellatus ocellatus, were pre-treated in physiological saline containing CB and inseminated in water also containing CB. Microvilli at the sperm entry site (SES) under the micropyle disappeared or shortened in length following CB treatment. However, a spermatozoon attached to and fused with the SES of CB-treated eggs. The spermatozoon present in a swollen mass (SM) remained at the egg surface even after membrane fusion and did not enter the cortex. It was unclear whether or not sperm movement from the cortex to the inner cytoplasm is CB-sensitive. The SM formed and plugged the micropyle. CB did not inhibit cortical alveolus breakdown. Based on the present experiments with fish eggs, it is concluded that CB inhibits sperm movement from the egg surface to the cortex, but not sperm attachment (binding), membrane fusion and SM formation during the process of sperm penetration.

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