Biomedical subjects

A Tsukamoto

Publications and source records attributed to A Tsukamoto.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structural characteristics of cationic liposomes with potent enhancing effect on retroviral transduction into human hepatoma cells.

To improve the efficiency of retroviral transduction into human hepatoma cells, new liposomes were prepared using different cationic and neutral lipids. Their effect on the retroviral transduction was evaluated using PLC/PRF/5 hepatoma cells and Moloney murine leukemia virus-derived retrovirus carrying herpes simplex thymidine kinase gene. Those liposomes consisted of cationic lipids with a long spacer were highly efficient in enhancing retroviral transduction and also less cytotoxic. On the other hand, the length of hydrophobic domains of neutral lipids was not correlated with the efficiency in enhancing the retroviral transduction. These results suggest that liposomes which effectively enhance retrovirus transduction can be developed by using cationic lipids with new spacers.

Carcinoma, Hepatocellular↗

PubMed1996

Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities.

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy-1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy-1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Cationic liposomes enhance retrovirus-mediated multinucleated cell formation and retroviral transduction.

A retroviral vector carrying herpes simplex virus thymidine kinase (HSV-TK) gene was introduced into psi 2 packaging cells (psi 2tkn) and XC tumor cells (XCtkn). psi 2tkn, XCtkn and XC cells co-cultured with TK-carrying cells were killed by ganciclovir (GCV) treatment. The growth of XC cells transplanted into nude mice was also suppressed by intratumoral injection of psi 2tkn or XCtkn cells and subsequent GCV administration. In addition, the XC cells cultured with either psi 2tkn cells or cell-free retrovirus suspension formed giant multinucleated cells. The multinucleated cell formation was specific to the combination of XC cells and the retrovirus produced by the psi 2tkn cells. Cationic liposomes enhanced the retrovirus-induced multinucleated cell formation and retroviral transduction. The correlation between the two actions of liposomes suggests that liposomes which enhance multinucleated cell formation are potent enhancers of retroviral transduction.

Animals↗

PubMed1996

Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.

Animals↗

PubMed1996

A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells.

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker-negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.

Adult↗

PubMed1996

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.

Animals↗

PubMed1996

CD34+Thy-1+Lin- stem cells from mobilized peripheral blood.

Over the last ten years there has been increasing use of mobilized peripheral blood (MPB) progenitor cells as grafts for autologous transplantation. Among the cells comprising these MPB autografts is a subpopulation of CD34+Thy-1+Lineage (Lin)- cells, which is enriched for hematopoietic stem cell (HSC) activity. The percentage of CD34+ cells which express Thy-1 is higher in some samples of MPB than in bone marrow (BM). Using myeloid and erythroid cell depletion prior to high speed cell sorting, it is possible to purify sufficient numbers of CD34+Thy-1+Lin-HSCs from a MPB leukapheresis sample for use as an autograft. CD34+Thy-1+Lin-cells will potentially provide a tumor-depleted autograft for cancer patients. This HSC population is also being studied as a potential target for gene transfer for the treatment of patients with HIV, cancer and a variety of genetic disorders.

Animals↗

PubMed1996

Detection of a primitive megakaryocyte progenitor cell in human fetal bone marrow.

Ontogeny-related changes in megakaryocyte (MK) progenitor cells were a analyzed to further define the cellular hierarchy occurring during human MK development. CD34+ cells were selected from human low-density adult bone marrow (ABM) or unfractionated fetal bone marrow (FBM) and assayed for MK colony formation in a serum-depleted fibrin clot assay system. At days 3, 7, 12, 16, 21, and 28 of incubation, MK colonies were analyzed for colony number, size and number of foci of development. Unifocal CFU-MK-derived colonies cloned from FBM formed after fewer days of in vitro culture and were 2.6-fold larger than those colonies cloned from ABM, However, the frequency of CFU-MK derived colonies cloned from ABM was significantly greater. the MK colonies cloned from FBM morphologically consisted of both pure MK colonies and mixed MK-containing colonies, in which a core of CD41- cells were surrounded by CD41+MKs. Large colonies resembling the primitive burst-forming unit-MK (BFU-MK) also were assayable from both FBM and ABM. These BFU-MK-derived colonies appeared after fewer days of incubation when FBM was assayed compared to ABM, but at a significantly lower frequency. In addition, large unifocal MK colonies consisting of >300 cells (300-1000) appeared from cells cloned from FBM but not ABM. This type of colony, which appears to represent a unique type of MK progenitor cell, was termed a high proliferative potential cell-MK (HPPC-MK). such colonies represent a relatively rare MK progenitor. We conclude that there are a significant ontologic changes in human MK progenitor cell development. The kinetics of MK colony formation from ABM differs significantly from that of FBM. Furthermore, the proliferative capacity of fetal MK progenitor cells appear to be far greater than that of ABM. In addition, a more primitive lineage-restricted MK progenitor cell with extensive proliferative capacity, the HPPC-MK, was assayable exclusively from FBM.

Adult↗

PubMed1996

Fetal bone marrow CD34+CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.

We have investigated the expression of CD41a (gpIIbIIIa) on a subpopulation of human fetal bone marrow (FBM) CD34+ progenitor cells. Human FBM CD34+Lin- cells were subfractionated into CD41a+ and CD41a- subpopulations by flow cytometry. All the megakaryocyte colony-forming cells (CFU-MK) and almost all the burst-forming units-megakaryocyte (BFU-MK) were found within the CD41a+ subpopulation. In addition, a 14-fold greater number of granulocyte-macrophage colony-forming units (CFU-GM) and a five-fold greater number of mixed lineage progenitor cells (CFU-mix) were observed within the CD34+Lin-CD41a+ subpopulation compared to the CD34+Lin-CD41a- subpopulation. The high proliferative potential of CD34+Lin-CD41a+ cells was demonstrated by their capacity to expand in in vitro culture containing human plasma and recombinant Mpl ligand (thrombopoietin [Tpo]) with production of over 80% CD41b+ (gpIIb+) MKs. However, in long-term bone marrow cultures, the CD34+Lin-CD41a- population contained a significantly higher frequency of cobblestone area-forming cells (CAFC) than the CD34+Lin-CD41a+ population, indicating the presence of a primitive hematopoietic stem cell (HSC) population within the CD34+Lin-CD41a- subset. These data suggest that fetal CD34+Lin-CD41a+ cells are enriched for MK progenitor cells (CFU-MK and BFU-MK), myeloid progenitors, and CFU-mix but do not contain the more primitive CAFC.

Antigens, CD34↗

PubMed1995

Gene therapy of hepatoma: bystander effects and non-apoptotic cell death induced by thymidine kinase and ganciclovir.

A retroviral vector carrying herpes simplex virus thymidine kinase gene was constructed, and transfected into the psi 2 packaging cells. The replication-defective retrovirus produced by this cell line (psi 2 tkn cells) was transduced into XC rat hepatoma cells, from which a cell line (XCtkn2) highly sensitive to ganciclovir was cloned. Ganciclovir suppressed the growth of XCtkn2 hepatoma and psi 2tkn cells. Both of these HSV-tk-carrying cells treated with ganciclovir showed potent 'bystander effect' on co-culturing with genetically unmodified XC hepatoma cells. In addition, intratumoral injection of XCtkn2 and psi 2tkn cells into the XC hepatomas transplanted in nude mice and subsequent ganciclovir administration suppressed in vivo growth of the hepatomas. Flow cytometry disclosed that the ganciclovir-treatment increased the relative number of XCtkn2 hepatoma and psi 2tkn cells at the G2 phase of the cell cycle. However, the nuclear fragmentation and internucleosomal DNA cleavage were not observed, indicating that the death of XCtkn2 hepatoma and psi 2tkn cells treated with ganciclovir was not apoptotic.

Animals↗

PubMed1995

Apoptosis of human hematopoietic progenitor cells induced by crosslinking of surface CD43, the major sialoglycoprotein of leukocytes.

Interactions of hematopoietic progenitor cells (HPC) with bone marrow stroma, mediated by adhesion molecules, are assumed to be critically important to the regulation of hematopoiesis. However, the specific roles of individual adhesion molecules involved in these interactions are poorly understood. Here, a monoclonal antibody, MEM-59, recognizing CD43, an adhesion molecule highly expressed on HPC, is shown to induce apoptosis in this cell population. This process operates at the single-cell level, and its initiation requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated cells originating from this primitive cell population, as well as peripheral lymphocytes, do not undergo apoptosis in response to the CD43-mediated stimulation. Thus, CD43 may function as a negative regulator of early hematopoietic events, delivering a signal for apoptosis of HPC.

Antibodies, Monoclonal↗

PubMed1995

Apoptosis and nuclear levels of p53 protein and proliferating cell nuclear antigen in human hepatoma cells cultured with tumor promoters.

Anticancer drugs etoposide and mitomycin C increased nuclear p53 protein and decreased proliferating cell nuclear antigen (PCNA) of PLC/PRF/5 human hepatoma cells. These changes were followed by DNA fragmentation and apoptosis. Teleocidin antagonized both apoptosis and alterations of nuclear p53 protein and PCNA induced by these anticancer drugs. In contrast, thapsigargin antagonized only drug-induced nuclear accumulation of p53 protein. Therefore, the inhibition of apoptosis appears not to be the common mechanism of tumor promotion. Both tumor promoters suppressed the increase in nuclear p53 protein, suggesting that an inadequate DNA repair due to the reduced nuclear accumulation of p53 protein might be playing important role in enhancing carcinogenesis.

Apoptosis↗

PubMed1995

Lack of the expression of EBNA-2 and LMP-1 in T-cell neoplasms possessing Epstein-Barr virus.

We investigated 34 cases of T-cell neoplasm [15 cases of T-cell granular lymphocytic leukemia (T-GLL), 10 cases of T-cell non-Hodgkin's lymphoma (T-NHL), six cases of T-cell chronic lymphocytic leukemia (T-CLL), and three cases of cutaneous T-cell lymphoma] to study their association with Epstein-Barr virus (EBV). In 4 (three T-NHL and one T-GLL) of 34 cases, EBV genome was detected in a single episomal form, while polyclonal EBV-DNA was detected in one (T-NHL) of the remaining cases. All three cases of T-NHL having monoclonal EBV episome showed histologically diffuse large-cell lymphoma and developed leukemic conversion. Phenotypic analysis showed that two of these four cases were CD4+, CD8-, and the remaining two cases were CD4-, CD8+. The cells from all four cases were confirmed to be in T-cell lineage by detecting the rearrangement of T-cell receptor (TCR) beta or gamma chain gene. By reverse transcription-polymerase chain reaction (RT-PCR), EBNA-1 was detected at low levels, and neither EBNA-2 nor LMP-1 were found in any of the three cases examined. Lack of the expression of EBNA-2 and LMP-1 was also confirmed by immunocytochemical staining. The cells of these four cases did not show rearrangement or overexpression of c-myc and bcl-2 genes by Southern and Northern blots, and the mutation of p53 gene was detected in only one patient. These results suggest that other latent gene products of EBV or other cellular oncogenes are involved in the development of Japanese T-cell neoplasm after EBV infection.

Antigens, Viral↗

PubMed1994

Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34.

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Animals↗

PubMed1994

Thapsigargin-induced persistent intracellular calcium pool depletion and apoptosis in human hepatoma cells.

We found that thapsigargin (Tg), a non-phorbol ester type tumor promoter that specifically inhibits endoplasmic reticulum Ca(2+)-ATPase, transiently increased the level of cytosolic free calcium ([Ca2+]i) and subsequently induced chromatin condensation, nuclear fragmentation, and internucleosomal DNA cleavage in cultured PLC/PRF/5 human hepatoma cells. These alterations were followed by the loss of plasma membrane integrity and by cell death. Epidermal growth factor (EGF) and vasopressin similarly elevated [Ca2+]i without causing DNA fragmentation which is characteristic of apoptosis. Consequently, the elevation of [Ca2+]i itself was not sufficient for causing Tg-induced cell death. On the other hand, preculturing the cells with Tg completely suppressed Ca2+ mobilization induced by EGF and vasopressin; a result that strongly suggests that Tg depleted the endoplasmic reticulum Ca2+ pool. Such depletion is hypothesized to induce apoptotic cell death in this hepatoma cell line by changing the nuclear Ca2+ levels which probably produce a structural change in chromatin.

Apoptosis↗

PubMed1994

Analysis of human hematopoietic stem cell populations.

Purification of human hematopoietic stem cells (HSC) may be useful clinically for preparation of tumor-free grafts to be used for autologous transplantation and as targets for gene therapy. To analyze the phenotype of the human HSC, assays were used that measure the unique properties of stem cells, i.e., their long-term repopulating ability and their multilineage potential. These assays include: (1) an in vitro long-term hematopoietic culture system, using the murine bone marrow stromal cell line SyS1, which supports both B lymphopoiesis and myelopoiesis; (2) fetal human bone grafts implanted in SCID-hu mice, in which maintenance of CD34+ cells and B and myeloid differentiative capacity of candidate stem cell populations may be measured; (3) fetal human thymus grafts in SCID-hu mice, which allow the analysis of in vivo T-cell potential of a candidate stem cell population. Stem cells in adult bone marrow (ABM) or cytokine-mobilized peripheral blood (MPB) are thought to express CD34 but lack expression of markers indicating lineage commitment. This CD34+ Lineage- (Lin-) subpopulation has been isolated by fluorescence-activated cell sorting and tested for activity in the assays described here. CD34+ Lin- cells from both ABM and MPB demonstrated long-term engraftment in the SCID-hu bone model. This CD34+ Lin- population can be subfractionated further using an antibody to Thy-1. The Thy-1+ subset of CD34+Lin- cells is enriched for both long-term culture-initiating cells (LTC-1C) and has the ability to engraft in vivo.

Animals↗

PubMed1993

Involvement of down-regulation-resistant protein kinase C in teleocidin inhibition of cell proliferation and calcium mobilization induced by epidermal growth factor and vasopressin in human hepatoma cells.

Teleocidin, a phorbol ester-type tumor promoter, inhibits cell proliferation and calcium mobilization induced by epidermal growth factor and vasopressin in PLC/PRF/5 hepatoma cells. These inhibitory effects of teleocidin were observed even after a prolonged exposure of the hepatoma cells to this promoter, suggesting the presence of down-regulation-resistant protein kinase C in this hepatoma cell line. Column chromatography of cytosolic fractions showed three separate peaks of protein kinase C activity, two being down-regulation-sensitive while one was down-regulation-resistant. This down-regulation-resistant PKC is suggested to be responsible for the inhibitory effect of teleocidin on cell proliferation and calcium mobilization induced by epidermal growth factor and vasopressin.

Calcium↗

PubMed1993

Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, enhances c-fos expression but antagonizes vacuole formation of human hepatoma cells induced by teleocidin.

Teleocidin, a phorbol ester-type tumor promoter, enhanced actin redistribution, vacuole formation and c-fos expression of PLC/PRF/5 hepatoma cells. This tumor promoter also inhibited calcium mobilization induced by epidermal growth factor (EGF). Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, elevated cytosolic calcium, enhanced c-fos expression and antagonized the vacuole formation induced by teleocidin without interfering with actin redistribution and Lucifer yellow uptake. On the other hand, a calcium ionophore ionomycin elevated both cytosolic Ca2+ and c-fos mRNA but could not antagonize the vacuole formation induced by teleocidin. From these results it was speculated that the Ca2+ leak from the endoplasmic reticulum rather than the elevation of cytosolic Ca2+ appeared to be responsible for the specific inhibition of vacuole formation by thapsigargin.

Actins↗