Occurrence of immunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary

The localization of adipocyte-type fatty acid binding protein (A-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Solitary round cells showing the distinct immunoreactivity for A-FABP were detected in 1–6 antral follicles. In sets of two consecutive sections in a mirror alignment on slide glasses which were treated for immunoreactivity for A-FABP and TUNEL reaction separately, cells immunoreactive for A-FABP appeared in the same antral follicles as containing cells exhibiting TUNEL-reaction. In immunoelectron microscopy, A-FABP-immunopositive cells were found to contain highly electron-dense nuclei of round, irregular or crescent shapes together with cytoplasmic remnants without any features of macrophages or cells of extrinsic origin. Therefore the cells were identified as apoptotic granulosa cells. The apoptotic cells immunoreactive for A-FABP were often seen to be enclosed/engulfed in adjacent cells exhibiting normal ultrastructures without containing numerous lysosomes. The present findings suggest that A-FABP is involved in the apoptosis of ovarian granulosa cells, probably through its interaction with peroxisome proliferator activated receptors.     

Persistent reovirus infection of CHO cells resulting in virus resistance.

We obtained a persistently infected line of Chinese hamster ovary cells by selection for resistance to reovirus infection. The cells were persistently infected by a population of viruses that were (i) cytopathic for parental chinese hamster ovary cells and (ii) similar to wild-type reovirus in molecular characteristics. The growth rate, plating efficiency, and morphology of the cells were altered. A large majority of the cells in the population were infected. There was no detectable interferon present in the medium. The cells were relatively resistant to a wide range of viruses.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Rapid establishment of highly migratory cells from cancer cells for investigating cellular functions

Abstract

Cell migration is closely involved in cancer cell invasion into surrounding tissue and metastasis to the distant organs. It is crucial for understanding the molecular mechanisms that regulate cell migration in cancer cells. The aim of this study is to establish a rapid induction method of highly migratory cells from cancer cells. Osteosarcoma MG-63 and colon cancer DLD1 cells were seeded at 1?×?10⁵ cells in 6-well plates. After 10?min, unattached cells were washed off three times with PBS. The cells which remained attached on the bottom of plates were cultured in DMEM containing 10% FBS. When the cells reached approximately 80% confluence, cells were harvested using trypsin/EDTA. The harvested cells were seeded in other 6-well plates and incubated for 10?min. The unattached cells were washed off and attached cells were further cultured. By repeating this procedure 11–12 times for 2?months, highly migratory MG63-A12 and DLD-A11 cells were obtained from MG-63 and DLD1 cells, respectively. In cell motility assay, the cell motile activities of MG63-A12 and DLD-A11 cells was 10.3 and 13.7 times higher than those of the parental cells, respectively. This procedure is useful to generate highly migratory cells for investigating cellular functions during tumor progression in cancer cells.     

Steroid production from 17alpha-hydroxypregnenolone and dehydroepiandrosterone by human granulosa cells in vitro.

Granulosa cells were aspirated 3--4 h before the expected time of ovulation from 10 follicles of 4 patients treated with gonadotrophins: 4 of the follicles were immediately preovulatory. The granulosa cells were cultured for 10 h with 17alpha-hydroxypregnenolone or dehydroepiandrosterone and samples of medium removed at 3 and 10 h were assayed for 6 steroids. Granulosa cells were unable to synthesize androgens from endogenous substrate or undertake conversions via the delta5 pathway, but cells from all follicles were capable of aromatizing exogenous androgens to oestrogens although this capability was reduced in cells from follicles beginning to luteinize. Granulosa cells from preovulatory follicles synthesized more progesterone from endogenous substrate than cells from follicles which had not begun to luteinize. The results provide further support for the two-cell theory of oestrogen biosynthesis whereby granulosa cells aromatize androgens which are synthesized by the thecal cells in vivo.     

Variations in the Size of Mitotic Cells in the Root Meristem

Variations in the length of mitotic and interphase cells were analyzed in various tissues of wheat roots and in the cortex of maize roots. Reliable differences were shown in the length of mitotic cells in individual file clones of cells of the same tissue. The mean lengths of dividing cells in different roots differed to a lesser extent than those of different files in the same tissue of one root. Within the file, the length of the sister simultaneously dividing cells differed the least, while the difference of lengths of the neighbor simultaneously dividing nonsister cells was bigger. The mean length of interphase cells in any file was always less than that of mitotic cells by a factor of 1.45. This ratio was almost invariable for files and tissues in both the plants we studied and corresponded to that of an exponentially growing cell population. In addition, a very small number of cells were found (less than 1%) in meristems, which are longer than the mitotic cells. The length of these cells exceeded those of mitotic cells by less than twice. The origin of such cells is discussed. The length of mitotic cells near the quiescent center is more variable than in the middle of the meristem in the cortex of both plants. In the meristem basal part, the mitotic cells were no longer than those in the middle of the meristem but there were no small dividing cells. In the wheat epidermis, the cells are differentiated into trichoblasts and atrichoblasts and, therefore, the length of the dividing cells is highly variable. The cell length is essential for their transition to mitosis for all studied proliferating meristem cells.     

Studies on the capacity of B cells as well as T cells to serve as accessory cells for the activation of herpes simplex virus-specific cytolytic T cells

Experiments were conducted in an effort to determine the ability of B and T lymphocytes to serve as APC for the activation of HSV-primed splenic T cells to become class I-restricted, HSV-specific CTL. The results showed that both freshly isolated splenic B cells as well as LPS and dextran sulfate (L/D)-activated B cells were effective at stimulating the generation of CTL during a 5-day in vitro culture. There was no requirement for the addition of exogenous IL-2 to the culture and, since murine B cells do not appear to express either membrane or secreted IL-1, this lymphokine appears to either not be required for the activation of virus-specific CTL or to be provided by the T cells themselves. When normal B cells were separated into fractions enriched for resting vs activated cells and then tested for their ability to stimulate the generation of HSV-specific CTL, it was found that while the activated B cells were quite effective at stimulating the generation of CTL, resting B cells were ineffective at carrying out this function. In contrast to normal B cells, normal T cells were unable to act as APC. However, Con A-activated T lymphoblasts were equivalent to L/D B cells in their ability to mediate the generation of CTL activity. L/D B cells that had been pulsed with HSV and then incubated at 37 degrees C for greater than 1 h could be fixed with paraformaldehyde and were still able to function as APC. The finding that L/D B cells, that had been fixed at 1 h or less after exposure to HSV, were unable to function as APC suggested that either active Ag "processing" steps may be required for the presentation of Ag in the context of class I molecules or that there is a requirement for the synthesis of viral protein Ag before presentation.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems: II. Subpopulations which mediate selective and nonselective lysis of normal and colon carcinoma target cells in vitro

Spleen cells from WF rats immunized to allogeneic lymphoid cells or to syngeneic colon carcinomas and from unimmunized controls were separated by countercurrent distribution in aqueous two-phase systems. The cells were assayed for cytotoxicity to allogeneic fibroblasts or syngeneic colon carcinoma cells and to syngeneic fibroblasts in a 24-hr ⁵¹Cr-release assay. Cells from immunized rats which selectively lysed the specific target cells were repeatedly found in one area of the distribution separate from the majority of cells, which nonselectively lysed syngeneic fibroblasts as well. A similar subpopulation which nonselectively lysed all target cells assayed was recovered from the spleens of unimmunized rats. The cells were also assayed for the ability to lyse antibody-coated thymocytes in a 4-hr ⁵¹Cr-release assay. The peak of K cells was found to overlap partially that of cells with nonselective cytotoxicity.     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Cell-surface changes during in vitro differentiation of pluripotent embryonal carcinoma cells

When aggregates of HM-1 embryonal carcinoma (EC) cells were exposed to 10(-6) M retinoic acid for 2 days and cultured in medium lacking retinoic acid, they differentiated to nerve cells, endoderm cells, and myoblasts. Cells 2 days after initial exposure to retinoic acid were not significantly different from the parental EC cells, as judged by cell-surface architecture and by reactivity to lectins. On the fourth day, the surface of the aggregates was covered with two kinds of cells distinguishable from the parental cells. The round cells with short villi seemed to be precursors to endoderm cells. Receptors for Dolichos biflorus agglutinin (DBA) newly appeared and receptors for peanut agglutinin (PNA) were still expressed on their surfaces. The other cells, which were round cells with a few processes, might be precursors to nerve cells. PNA receptors had disappeared from their surfaces, and DBA receptors were not expressed. On the sixth day of differentiation, possible precursors to myoblasts were detected; they were flat cells with smooth surfaces. These cells lacked cell-surface receptors for the two lectins, while the precursor cells and the myoblasts excreted intercellular fibers reacting with PNA. HM-1 cells synthesized much embryoglycan, the structure of which was similar to that of the glycan isolated from quasinullipotent F9 cells. The only difference was that the glycan from HM-1 cells lacked DBA binding sites. Synthesis of fucosylated embryoglycan mainly decreased between the second and fourth day of differentiation. As above, cell-surface changes occurred mainly between the second and fourth day. The period seems to be important in determining the fate of the cells, since endoderm cells were scarcely seen among differentiated cells which had been continuously exposed to 10(-6) M retinoic acid during the period.     

小鼠骨髓高增殖潜能内皮祖细胞的培养与纯化（简报）

1997年Asahara等在人外周血中发现内皮祖细胞（endothelial progenitorcell．EPC）,随后有文献报道,从骨髓中分离出EPC．异体骨髓移植研究显示成体外周血EPC来源于骨髓。许多作者报告EPC参与生理性和病理性血管新生．具有潜在的临床应用前景。目前关于EPC的不少基本生物学问题尚不完全清楚,其生理和病理意义也有争议。     

Cellular composition of a cerebral hemisphere primary culture

In this overview attention is given to available markers and methods for characterizing cell elements in a culture system. Primary cultures from newborn rat cerebral hemispheres were grown for 14 days. The population of cells was dominated by astrocytic glial cells (60–70%), but cells with properties of macrophages, endothelial-like cells, mesenchymal-like cells, ependymal-like cells, and oligoblasts were also found. Neither mature neurons nor oligodendroglial cells were observed. The enrichment in astroglial-like cells makes the cultures a satisfactory astroglial-cell model, at least for some purposes.     

The requirement for surface Ig signaling as a prerequisite for T cell:B cell interactions. A possible role for desialylation

Models for T cell:B cell collaboration suggest that activated B cells process and present Ag to Th cells which subsequently induce B cell proliferation and differentiation. In contrast to activated B cells, resting B cells have generally been shown to be less efficient APC. If this model of T:B collaboration is physiologically correct, then resting B cells must undergo a phenotypic change that permits effective interaction with T cells. In this report, the requirement for rapid signaling through surface Ig on resting B cells for the induction of T:B interaction was investigated with an in vitro clustering assay. Resting splenic B cells were unable to form specific conjugates with T cell clones, unless the B cells were first treated with neuraminidase to remove sialic acid. In contrast, LPS-activated B cells were able to form conjugates without prior treatment. The ability of antibody against LFA-1 or L3T4 to inhibit cluster formation depended on the state of B cell activation in that anti-LFA-1 and anti-L3T4 mAb inhibited cluster formation by neuraminidase-treated resting B cells, but not by LPS-activated B cells. In addition, Ag-specific B cells which were isolated by their capacity to bind specific Ag were able to form clusters without any additional treatment. Moreover, treatment of resting splenic B cells with anti-mu-antibody induced clustering potential in B cells in as little as 10 min, suggesting that signaling through surface Ig was sufficient to induce this phenotypic change in B cells. Furthermore, activation of protein kinase C and Ca2+ mobilization were shown to be involved in that PMA and ionomycin treatment were also able to induce clustering potential in resting B cells. The rapid induction of clustering potential in resting B cells after signaling through surface Ig may represent a fundamental change in B cell physiology which occurs after recognition of specific Ag and may be required for effective cognate recognition between resting hapten-specific B cells and carrier-specific T cells. The potential role of desialylation for the induction of T:B interaction is discussed.     

Linear density gradient separation of human lymphocyte subsets. II. Characterization of cells responding in secondary MLC and CML.

Lymphocytes were separated on linear density gradients (LDG) after they had been sensitized in vitro against allogeneic cells and had reverted to small cells. Cells from individual density fractions were restimulated with autologous, specific, or third-party cells and assayed 48 hr later for their response in secondary mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML). Memory cells capable of responding in secondary MLC were broadly distributed and found in both heavy and light fractions. The various density classes of memory cells differed with respect to the degree of their specificity for the restimulating cells. In secondary MLC the greatest specificity for the originally sensitizing cells and the least cross-reactivity for third-party cells were primarily features of light- and medium-density cells. Memory killer cells for CML were fairly homogeneously grouped. Following restimulation, killers were enriched in light to medium fractions also, as was previously seen at the peak of the response on Day 6.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.     

体外培养牛白细胞中环形泰勒焦虫(Theileria annulata)的增殖、释放与感染

在体外培养的牛外周血白细胞中,环形泰勒焦虫裂殖子与裂殖体寄生于宿主细胞的细胞质中,并且随着宿主细胞的分裂而分到两个子细胞中。焦虫染色质粒的分裂方式为二分裂,随着焦虫颗粒的不断增殖,逐渐发育为成熟的裂殖体。体外培养感染焦虫的牛白细胞可通过伪足与细胞裂解两种途径向培养液中释放焦虫颗粒。释放到培养液中的焦虫颗粒对体外培养的健康牛外周血白细胞具有感染能力,感染细胞能在体外连续传代培养。     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.     

Comparison of dark- and light-adapted carp retinas with NADPH diaphorase staining

The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,