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Biomedical subjects

A Okuda

Publications and source records attributed to A Okuda.

At least 91 records · Page 5Linked to original sources
PubMed1989

Mechanism of selective killing by dilinoleoylglycerol of cells transformed by the E1A gene of adenovirus type 12.

Rat 3Y1 fibroblasts transformed by the E1A gene of adenovirus type 12 (E1A-3Y1 cells) are highly sensitive to the cell-killing effect of 1,3-dilinoleoylglycerol (DLG) administered in a culture medium, whereas the parental 3Y1 cells are less sensitive (H. Shimura et al., Cancer Res., 48: 578-583, 1988). The selective cytotoxicity of DLG to E1A-3Y1 cells was markedly reduced by the simultaneous administration of nonspecific antioxidants such as vitamin E, butylated hydroxytoluene, and ascorbic acid. Specific scavengers for oxygen radicals had no effect. Lipoxygenase inhibitors (nordihydroguaiaretic acid, esculetin, and baicalein) reduced the DLG-mediated selective cytotoxicity, whereas cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) showed no effect. The intracellular and extracellular contents of the products from lipid peroxidation as measured by the thiobarbituric acid test were significantly greater in E1A-3Y1 cells than in the parental 3Y1 cells. In comparison with DLG, linoleic acid and monolinoleoylglycerol were equally toxic to E1A-3Y1 and parental 3Y1, and trilinoleoylglycerol was weakly toxic to both types of cells. Scanning electron microscopy revealed that numerous holes about 0.2 micron in diameter were scattered all over the surface of the E1A-3Y1 cells after treating the cultures with DLG. These results suggest that; (a) the DLG-mediated cytotoxicity to the E1A-transformed cells is attributable to lipid peroxidation; (b) the structural property of DLG is essential to the E1A specificity of cytotoxicity; and finally (c) the destruction of the cell membrane is the basis of cytotoxicity of DLG.

Adenovirus Early Proteins↗
PubMed1989

Structural and functional analysis of an enhancer GPEI having a phorbol 12-O-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene.

We have recently identified a typical enhancer, termed GPEI, located about 2.5 kilobases upstream from the transcription initiation site of the rat glutathione transferase P gene. Analyses of 5' and 3' deletion mutants revealed that the cis-acting sequence of GPEI contained the phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)-like sequence in it. For the maximal activity, however, GPEI required an adjacent upstream sequence of about 19 base pairs in addition to the TRE-like sequence. With the DNA binding gel-shift assay, we could detect protein(s) that specifically binds to the TRE-like sequence of GPEI fragment, which was possibly c-jun.c-fos complex or a similar protein complex. The sequence immediately upstream of the TRE-like sequence did not have any activity by itself, but augmented the latter activity by about 5-fold.

Animals↗
PubMed1989

Transient increase in the c-fos mRNA level after change of culture condition from serum absence to serum presence and after cycloheximide addition in rat 3Y1 fibroblasts.

When 3Y1 cells resting at a saturation density were mitotically stimulated with serum, the c-fos mRNA level markedly increased in a short period of time and then decreased rapidly to an undetectable level. Subsequent serum deprivation followed by serum re-addition or subsequent cycloheximide addition caused a transient re-increase in the c-fos mRNA level. These results can be explained by assuming that the continuous expression of the c-fos gene at a minimum level is necessary for the eventual initiation of S phase, and that the over-expression of the c-fos gene occurs when the control of the gene expression is transiently disturbed by the change of the culture condition.

Animals↗
PubMed1989

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals.

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.

Acid Phosphatase↗
PubMed1989

Increase in c-fos and c-myc mRNA levels in untransformed and SV40-transformed 3Y1 fibroblasts after addition of serum: its relationship to the control of initiation of S phase.

When rat 3Y1 fibroblasts were exposed to serum after 7.5 h of S, G2, and M phases in the absence of serum, the c-fos and c-myc mRNA levels markedly increased. This marked increase was also observed when density-arrested cells were stimulated with fresh serum to initiate proliferation. Increase in the c-fos and c-myc mRNA levels was not observed in cells that had traversed 7.5 h in these phases in the presence of serum. Cells passing through S, G2, and M phases in the absence of serum delayed entry into the next S phase approximately 8 h compared to control cells incubated in the presence of serum. Also, when density-arrested cells were stimulated with serum for 5 h, then deprived of serum for 8 h, and then incubated in serum again, the c-fos and c-myc mRNA levels increased. In this last case, the total excess time of serum exposure required to enter S phase was only 2 h, indicating that cells had not returned to the initial density-arrested state during the serum deprivation period. The increase in c-fos and c-myc mRNA levels following addition of serum after incubation in the absence of serum was also observed in SV40-transformed 3Y1 cells. The entry of SV40-transformed cells into S phase was not markedly affected by the absence of serum. These results can be explained by assuming that there is a process leading to the initiation of S phase that is operating or accumulating continuously in all cell cycle phases. In 3Y1 cells the expression of the c-fos and c-myc genes is required at any cell cycle phase, and the increase in c-fos and c-myc mRNA levels in response to changes in serum concentration simply reflects the possible overexpression due to the delay of a hypothesized negative feedback regulation. In SV40-transformed 3Y1 cells, the process leading to the initiation of S phase operates normally in response to growth factors, and the SV40 large T antigen supplements or enhances the process in the absence of the growth factors.

Animals↗
PubMed1989

Prostaglandin E2 initially inhibits and then stimulates bone resorption in isolated rabbit osteoclast cultures.

Osteoclasts were isolated from the long bones of neonatal rabbits and cultured on devitalized bovine bone slices for 8, 24, 48 and 72 h with and without prostaglandin E2 (PGE2) (10(-6) M). The number of osteoclasts present at the end of the culture periods was counted after staining the cells for tartrate resistant acid phosphatase (TRAP). After removal of the cells, the resorption lacunae excavated by the osteoclasts were observed by scanning electron microscopy (SEM) and their size and depth calculated by computer-assisted morphometric and stereomorphometric techniques. PGE2 had no effect on the number of TRAP positive multinucleated osteoclasts, but decreased the number of TRAP positive mononuclear cells. The total area of the excavated pits and the area excavated per osteoclast in PGE2-treated cultures were decreased by 62 and 58% respectively after 8 h in culture. After 24 h in culture, the total excavated area and the excavated area per osteoclast were still 44 and 38% lower in the PGE2-treated cultures than in the corresponding control cultures. However, after 48 h of culture, resorptive activity in PGE2-treated cultures was consistently greater than in control cultures. In the course of a 48 h culture period, the PGE2 concentration decreased from 1.0 x 10(-6) to 0.3 x 10(-6) M. Thus, despite the continuous presence of PGE2, the resorptive activity of osteoclasts not only recovered from the transient inhibitory effect of PGE2, but was actually greater than in the control cultures. This confirms that the effects of PGE2 in isolated osteoclast preparations are inhibitory in short term cultures, but shows that the effects of PGE2 in such preparations are stimulatory in longer term cultures. Proliferating stromal cells with osteoblast-like characteristics comprised approximately 45% of the 'osteoclast' cultures at the start of the cultures, but their number increased to 93% of the total cell population at 48 h and to 98% at 72 h. Our results suggest that the PGE2-induced stimulation of osteoclastic activity represents an indirect effect mediated by stromal cells derived from bone marrow. Our results also indicate that the increased resorptive activity in PGE2 treated cultures can be accounted for by an increase in the size of the resorption lacunae and is not caused by an increase in osteoclast number.

Acid Phosphatase↗
PubMed1989

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature.

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.

Animals↗
PubMed1989

Frequency of cell transformation by the small DNA tumor viruses: infection of proliferating cells and quiescent cells.

Small DNA-containing tumor viruses (simian virus 40, mouse polyoma-virus, and adenoviruses) malignantly transform fibroblasts of the susceptible rodents. Fibroblasts can exist, in vitro and in vivo, in either of the two states: the proliferating state or the quiescent state. In the present study, we examined whether the state of fibroblasts at the time of exposure to these DNA viruses affects the frequency of transformation. Dense-focus formation in monolayer culture of rat 3Y1 fibroblasts was used to quantitate transformation. Results show that the frequency of transformation by simian virus 40 and mouse polyomavirus was reduced when cells were in the proliferating state at the time of virus inoculation as compared to cells in the quiescent state, whereas that by adenovirus type 12 was similar in the two cellular states. The reduction of the frequency of transformation in proliferating cells infected with simian virus 40 was also observed in BALB/c 3T3 mouse cells. Mechanisms underlying the difference between the two cellular states and the difference between the papovavirus and adenovirus in this aspect of transformation remain to be investigated.

Adenoviridae↗
PubMed1989

Selective killing of transformed fibroblasts by combined treatment with cycloheximide and aphidicolin.

The possibility of selective killing of transformed cells in a mixed population of untransformed and transformed cells was examined using a cell culture system of rat 3Y1 fibroblasts (parental 3Y1 cells, 3Y1 cells transformed with either SV40, polyoma virus, Rous avian sarcoma virus, E1A gene of adenovirus type 12, or H-v-ras oncogene). The principle of the selective killing is as follows. Under suboptimal culture conditions, untransformed cells are inhibited from progressing through G1 phase and retain viability, while transformed cells are not arrested. When DNA synthesis is inhibited for a long period, both types of cells in S phase die. Therefore, if we administer inhibitors of G1 progression and of DNA synthesis simultaneously to a cell population consisting of untransformed and transformed cells, most untransformed cells are arrested in G1 phase, retaining viability, while transformed cells leak from the G1 phase, cease DNA synthesis, and gradually die The present study shows that all types of transformants in stationary-phase cultures (consisting of cells mainly with a G1 DNA content) were killed to higher extents compared with untransformed cells, during incubation at lower cell densities with a combination of cycloheximide (G1 inhibitor) and aphidicolin (DNA-synthesis inhibitor). However, cycloheximide reduced the killing effect of aphidicolin by changing the irreversible DNA-synthesis inhibition to a reversible inhibition. The availability of G1 inhibitors that do not interfere with the irreversibility of inhibition of DNA synthesis is required for the treatment of cancer based on this idea.

Animals↗
PubMed1989

Proliferative behaviour of fibroblasts in plasma-rich culture medium.

We examined the proliferative behaviour in tertiary culture cells of human skin fibroblasts (HSF) as well as cells of the rat 3Y1 diploid fibroblast line placed on a plastic substratum in a nutrient-rich medium containing high concentrations of platelet-poor plasma (PPP). Autochthonous human PPP was used for the HSF cells and heat-treated (at 56 degrees C for 30 min) bovine PPP was used for the 3Y1 cells. In both types of cells, the saturation cell density rose with increasing PPP concentration and reached a plateau at 30-90% PPP. When the cells were cultivated in serum, the saturation densities were the same as those with the same concentrations of corresponding PPP in both the HSF and 3Y1 cells. When the cells were arrested at a saturation cell density in a medium containing 10% PPP and then were refed with a fresh medium containing 90% PPP, DNA synthesis and cell division occurred in both types of fibroblasts. This effect was either reduced in the HSF cells or enhanced in the 3Y1 cells by the addition of platelet lysate. TGF-beta 1 added to the fresh medium containing 90% PPP also inhibited the induction of DNA synthesis in the HSF cells but not in the 3Y1 cells. The inhibitory effect of the platelet lysate was neutralized by anti-TGF-beta 1 IgG. On the other hand, PDGF added to the fresh medium had no effect on either type of cells. These results suggest that cultured fibroblasts are capable of proliferating on a plastic substratum under fluid conditions that essentially reflect the fluid environment of the body, as long as sufficient nutrients are supplied. Platelet lysate represses the proliferation of HSF cells, possibly through the inhibitory effect of TGF-beta 1, and promotes the proliferation of 3Y1 cells by growth factor(s) other than PDGF and TGF-beta 1.

Animals↗
PubMed1988

Segmental autotransplantation of the pancreas after total pancreatectomy for advanced periampullary carcinoma--a case report.

A case is reported here in which segmental autotransplantation of the pancreas was performed after total pancreatectomy for advanced periampullary carcinoma in an attempt to preserve pancreatic endocrine function. The postoperative course was uneventful. The requirement of insulin decreased after the operation and the daily profile examination, done 5 months following surgery, showed a permissive fluctuation of blood sugar levels without insulin injection. Thus, segmental autotransplantation of the pancreas offers a method of preserving pancreatic endocrine function after total pancreatectomy for periampullary carcinoma in selected patients.

Carcinoma↗
PubMed1988

Gas exchange and development of chicken embryos with widely altered shell conductance from the beginning of incubation.

The O2 uptake of chicken embryos confined in the eggshell (MO2) is governed by a shell diffusive conductance (GO2) and PO2 difference between ambience and air space, suggesting that a relation between GO2 and air space PO2 (PAO2) is hyperbolic at constant MO2. Upon wide alteration of GO2 from the beginning of incubation, the MO2 was measured on day 16 of incubation and the relation between GO2 and PAO2 examined. The MO2 increased hyperbolically with increasing GO2, reached maximum at control conductances and decreased with further increase in GO2. From these changes in MO2 with GO2, an equation was derived predicting PAO2 as a quadratic function of inverse gO2 (mass-specific conductance, i.e., GO2 standardized by fresh egg mass), and the relation between air space PO2 and shell conductance was no longer hyperbolic. The arterialized blood PO2 (PaO2) of the allantoic vein measured individually was also expressed by a quadratic equation of inverse gO2. While for widely altered conductance the MO2 was little related to PaO2, the mass (embryo)-specific O2 uptake increased with PaO2. The excess water loss associated with increased conductance was involved in changes in these variables.

Animals↗
PubMed1988

Short-term effects of altered shell conductance on oxygen uptake and hematological variables of late chicken embryos.

The preceding report on the O2 uptake (MO2) of chicken embryos whose shell conductance (GO2) was altered from the beginning of incubation showed that the MO2 was decreased despite increased GO2 [Okuda, A. and H. Tazawa (1988) Respir. Physiol. 74: 187-198]. This was attributed to an excess water loss which reduced the growth of the embryos. The present study was designed to investigate the short-term effects of altered GO2, obviating the effect of excess water loss, on the MO2 and simultaneously on the hematological variables of embryos on days 16-17 and days 18-19 of incubation. The MO2 measured 5 h after increasing the GO2 was neither decreased nor increased significantly. The diffusing capacity of the chorio-allantoic membrane, which was estimated using the Bohr integration procedure, decreased as the GO2 was increased. When the GO2 was decreased, on the other hand, the decrease in MO2 was not so large as expected from the decrease in GO2, for both 16- and 18-day-old embryos. The effect of reduced GO2 on MO2 was more prominent in 18-day-old embryos than 16-day-old embryos. One-day-long hypoxia due to decreased GO2 induced erythropoiesis in 18-19-day embryos, but did not do so in 16-17-day embryos. The increase in hematocrit value of the latter group of embryos was attributed to an increase in cell volume due to concurrent hypercapnia.

Allantois↗
PubMed1988

Multiple regulatory elements and phorbol 12-O-tetradecanoate 13-acetate responsiveness of the rat placental glutathione transferase gene.

We have analyzed the cis-acting regulatory DNA elements of the placental rat glutathione S-alkyltransferase (GST-P) gene. Various regions of the 5' flanking sequence were fused with a bacterial chloramphenicol acetyltransferase gene. The transcriptional activity of each construct was determined by the transient expression assay after introduction into a hepatoma cell line. Multiple regulatory elements were identified. Two enhancing elements were located 2.5 and 2.2 kilobases upstream from the transcription start site and designated GST-P enhancers I and II (GPEI and GPEII, respectively). A consensus sequence of the phorbol 12-O-tetradecanoate 13-acetate responsive elements was present in the GPEI and at position -61. GPEII contained two of the simian virus 40 and one of the polyoma enhancer core-like sequences. A silencing element was also found 400 base pairs upstream from the cap site. In accordance with the above observation, endogenous GST-P gene was found to be stimulated when the rat fibroblast line 3Y1 was treated with phorbol 12-O-tetradecanoate 13-acetate. Phorbol 12-O-tetradecanoate 13-acetate enhanced the expression of the transfected GST-P gene to a much higher degree in HeLa cells than in the hepatoma cells, which constitutively expressed the endogenous GST-P. The results are discussed in terms of the specific derepression of GST-P gene during hepatocarcinogenesis in the rat.

Animals↗
PubMed1988

Commitment to ploidy conversion of 3Y1 cells during metaphase arrest by colcemid.

Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.

Animals↗
PubMed1988

Elongation of G1 phase by transient exposure of rat 3Y1 fibroblasts to caffeine during the previous and present generations.

When density-arrested rat 3Y1 fibroblasts were stimulated to enter S phase by seeding sparsely in fresh medium, caffeine inhibited this stimulation. When compared at the doses that gave the same levels of the inhibition of entry into S phase, caffeine inhibited protein synthesis to a far lesser extent than cycloheximide. This indicates that caffeine affects some event(s) specific to entry into S phase rather than general protein synthesis. When cells synchronized at early S phase were exposed to caffeine, progression of S and G2 phases was prolonged by only 1 h (from 6 h to 7 h). However, after removal of caffeine at mitosis, the G1 phase was prolonged for 5 h (from 11 h to 16 h). These results are consistent with our model that the initiation of S phase is regulated throughout the period between the adjacent S phases. When cells were incubated with normal medium containing serum during the S and G2 periods, a subsequent 6-h pulse exposure to caffeine caused prolongation of G1 phase for 7 h (from 11 h to 18 h). On the other hand, when cells were incubated in the absence of serum during these periods, the prolongation was only 2 h (from 16 h to 18 h). Similarly, entry into S phase was prolonged only 2 h, when a 6-h pulse exposure to caffeine was given immediately after release from density arrest or serum-deprivation arrest. These results indicate the involvement of the relaxation process, which is not affected by caffeine, when serum-deprived cells or density-arrested cells restore the process prerequisite for entry into S phase.

Animals↗