Histamine-induced cortisol secretion from bovine adrenocortical cells co-cultured with bovine adrenomedullary cells

Inflammation Research -     

Demonstration of a Na+-dicarboxylate cotransporter in bovine adrenocortical cells

Our study found the uptake of [14C]succinate into bovine adrenocortical cells to be sodium-dependent, inhibited by lithium, and to have an apparent K(m) of 146 mumol/l. Succinate uptake was inhibited by glutarate, fumarate, alpha-ketoglutarate, and maleate but not by 2,3-dimethylsuccinate or cis-aconitate, specific inhibitors of the basolateral Na(+)-dicarboxylate transporter of renal proximal tubule cells. Succinate uptake was highest at pH 6.0 and decreased with increasing pH. Transport of succinate was not significantly inhibited by citrate at pH 7.4 whereas at pH 6.0 inhibition of succinate uptake by citrate was small but significant. The affinity of the adrenal dicarboxylate transporter towards succinate ranges in between the low affinity of the renal luminal dicarboxylate transporter and the high affinity of the respective basolateral transporter. The pH dependency of succinate uptake and the missing inhibition by citrate at pH 7.4 differ from both the luminal and from the basolateral dicarboxylate transporters in kidney, liver, intestine, and placenta. These functional characteristics provide evidence for the existence of a Na(+)-dicarboxylate cotransporter in adrenocortical cells which may supply cholesterol metabolism with reducing substrates.     

Demethylation of repetitive DNA sequences in neuroblastoma

Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer-based approach for the analysis of two-dimensional separations of human genomic restriction fragments. Through the use of methylation-sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two-dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor. Genes Chromosom Cancer 17:234–244 (1996). © 1996 Wiley-Liss, Inc.     

Long-term culture of bovine trophoblastic cells

Summary Procedures for the isolation and cultivation of bovine cotyledonary trophoblastic cells from early second trimester placentas are described. Collagenase digestion yielded a single cell suspension of both uninucleate and binucleate cells with minimal contamination with other cell types. Optimal growth conditions were obtained through supplementation of medium with epidermal growth factor, insulin, transferrin, and selenium. Trophoblastic cells survived multiple passages, cryopreservation, and serum deprivation and have been maintained in culture for more than 14 mo. Two cell types were identified by phase microscopy: uninucleated trophoblastic cells and trophoblastic giant cells, which were predominantly binucleate cells. Binucleate cells were present in small numbers through all passages. This procedure provides a reliable method to obtain trophoblastic cell lines for studies of trophoblastic cell physiology and susceptibility to infectious and toxic agents.     

Cytotoxic effects of expression of human superoxide dismutase in bovine adrenocortical cells

Oxygen radicals and the cellular antioxidant enzymes may play a role in cellular senescence. We studied the feasibility of altering oxygen radical metabolism in a normal differentiated cell that undergoes senescence in culture by transfection of an expression vector containing human CuZn-SOD cDNA. Plasmid pRSV-2-cSOD was constructed to contain the cDNA for human CuZn-SOD under the regulation of the Rous sarcoma virus long terminal repeat. Early passage cultures of bovine adrenocortical cells were cotransfected with pRSV2-cSOD and a plasmid (pSV3neo) allowing initial selection and continued growth of transfectants. Three passages after isolation of the polyclonal population, as cells grew to confluence, cultures showed focal cell death that spread outward to affect neighboring cells, so that by 72 h most cells had detached from the culture dish. Long term growth of the polyclonal population of transfectants without extensive cell death was achieved by continuous maintenance of low cell density during growth. Southern blot analysis of DNA from the pooled polyclonal population of transfected cells showed the presence of the expected 625 bp band from human CuZn-SOD. However, the intensity of this band indicated that only a minority of cells in the population had integrated the SOD plasmid, and DNA isolated from cells after 25 passages at low cell density showed plasmid sequences only of an altered form, suggesting that cells containing intact human SOD cDNA had been selectively lost from the population. When early passage low density transfectants were allowed to grow back to high cell density, cell death foci were again observed. Additionally, cell fusion with the formation of giant cells with massive multinucleation was observed by flourescence microscopy after staining cultures with a DNA binding dye. In later stages of this process, the large nuclear mass in such a giant cell became fragmented as the cell detached from the dish and formed the center of a focus of cell death in the surrounding cells. Because cell death prevented the growth of large numbers of transfected cells, it was not possible to demonstrate the involvement of CuZn-SOD in the cytotoxic effect by direct means, but the control plasmid without the CuZn-SOD cDNA insert had no cytotoxic effect. Thus, the introduction of a vector for human CuZn-SOD in a normal differentiated cell caused a cytotoxic effect involving cell death, cell fusion, and nuclear fragmentation.     

Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture.

Bovine erythrocytes infected with Anaplasma marginale were cultured for 1 to 5 days in a CO2 incubation chamber, pulse-labeled with [3H]thymidine and [14C]methionine, lysed, and fractionated by differential centrifugation and continuous density gradient centrifugation in Renografin. Anaplasma and associated fragments of stroma formed two distinct bands in the dense region of the gradient. Electron microscopic examination of pelleted material from the bands from cells cultured for 1 day revealed the presence of organisms that were morphologically intact or in various states of degeneration. Examination of fractions from the gradient for incorporation of label revealed that analplasma present in erythrocytes can incorporate both [3H]thymidine and [14C]methionine. Subsequent experiments demonstrated that organisms cultured for 3 and 5 days incorporated the radiolabeled compounds also, but to a lesser extent. The experiments demonstrate that it is possible to culture analplasma in vitro for short periods of time and monitor their growth characteristics.     

Chromatin reorganization during senescence of proliferating cells

It was previously proposed (Macieira-Coelho, 1979) that aging of proliferating cells is the result of genome reorganization taking place during the division cycle. This hypothesis was investigated and a reorganization could indeed be ascertained in the different hierarchical orders of DNA structure; a correlation was found between changes in chromatin organization and the impairment of cell cycle-related events. Indeed, like the latter, the reorganization of chromatin structure is characterized by a succession of subtle changes through the cell population life span, and a final short stage with abrupt events. The final events seem to concern mainly the organization of heterochromatin.The reorganization in the genome is accompanied by structural changes in the cellular scaffold and an evolution of cell morphology.The remodeling occurring in the cell through serial divisions seems to take place in such a way as to decrease the probability of further reorganizations, tending to a limit. The decline of the proliferative activity seems to be the result of the tendency to reach this limit.     

Fragmentation of DNA and chromatin during senescence in barley leaves

DNA or chromatin from young (7 day) first seedling leaves of barley showed only one component which migrated little into 1% agarose gels on electrophoresis. However, DNA or chromatin from senescent (17-, 19-, and 23-day-old) leaves showed additional dispersed components migrating throughout the length of the gel which increased with age. These low molecular weight components increased even more on autodigestion of chromatin from senescent leaves by its associated DNase or by digestion of DNA from senescent leaves with partially purified chromatin DNase. DNA or chromatin from young leaves also produced gel pattern similar to old leaves on digestion with partially purified chromatin DNase from old barley leaves. Thus, fragmentation of DNA and chromatin by chromatin associated DNase, previously shown to increase 4000% on aging, occurs during senescence in barley leaves.     

肌组织工程的基础研究—卫星细胞培养及鉴定

目的 :为肌组织工程的研究作技术上的准备 ,建立大鼠肌卫星细胞的培养方法。方法 :采用成年Wistar大鼠 ,利用二步消化法成功地分离出卫星细胞 ,在体外培养 ,观察其生长、分化的过程 ,并用电镜及免疫组化法进行鉴定。结果 :二步消化法分离的细胞成活率较高 ,生长良好 ,卫星细胞在体外增殖后 ,最终会经过由单核细胞变为多核细胞的过程 ,即产生了质的改变。结论 :本研究成功地建立了卫星细胞培养体系 ,a sarcometricactin免疫细胞化学鉴定方法对卫星细胞具有特异性     

P> 不同年龄小鼠脂肪源间充质干细胞体外原代培养衰老和凋亡的变化/P>

目的 探讨小鼠脂肪源间充质干细胞(ADMSCs)体外原代培养过程中衰老和凋亡变化。方法 用差速贴壁法对1月和24月龄小鼠（各30只）腹股沟脂肪组织的ADMSCs进行原代培养,免疫荧光技术检测第3代ADMSCs表面抗原CD73、CD90和CD105的表达,比较两组细胞的形态差异和生长情况,β-半乳糖苷酶(SA-β-Gal)染色检测ADMSCs衰老情况,PI-Hoechst33342双染色观察ADMSCs的凋亡变化。 结果 两组体外原代培养的ADMSCs均贴壁生长,具有典型干细胞的形态和生长特征,两组ADMSCs CD73、CD90和CD105的表型一致。SA-β-Gal染色显示：原代培养的老年组ADMSCs衰老比例比幼年组多,1月组原代培养2~7d的ADMSCs阳性率分别为（3.87±1.34）%、（4.21±1.22）%、（9.00±0.98）%、（11.20±1.24）%、（9.50±2.19）%和（13.20±2.93）%;24月组分别为（8.67±1.05）%、（10.23±0.98）%、（12.80±0.78）%、（17.10±1.13）%、（19.80±1.59）%和（24.70±1.86）%。PI-Hoechst 33342染液双染显示：1月组原代培养2~7d的ADMSCs凋亡率分别为(22.1±1.4)%、(36.7±1.62)%、(11.7±1.45)%、(38.4±1.57)%、(6.5±1.32)%和(11.3±1.63)%;24月组4~7d的ADMSCs凋亡率分别为(29.4±1.72)%、(37.6±1.64)%、(19.4±1.29)%和(18.9±1.25)%。 结论 老龄ADMSCs增殖能力下降,衰老、凋亡和坏死的细胞比例增加;随着ADMSCs原代培养时间的延长,其衰老增加,但凋亡和坏死无规律性变化。     

Artificial senescence of bovine retinal pigment epithelial cells induced by near-ultraviolet in vitro

RPE cells irradiated by near-ultraviolet (NUV) were characterized at cellular, biochemical and molecular levels in order to determine whether light-induced RPE changes contribute to the senescence of RPE cells in vitro. Biochemical and molecular parameters of cellular senescence were studied by using both bovine RPE cells at confluence repeatedly irradiated by NUV (peaking at 365 nm) and RPE cells at different levels of population doubling (PDL). After repeated NUV irradiation, RPE proliferation was markedly suppressed. In parallel, the BrdU index significantly reduced to a minimum level, similar to RPE cells undergoing multiple population doublings. NUV irradiation resulted in a decrease in cellular alkali-soluble melanin and an increase in lipofuscin-like fluorophores. The lipofuscin-like fluorophores, isolated from RPE cells exposing repeated NUV irradiation, represented a gradual hyperchromic change and red-shift, reaching the wavelength maxima (560–572 nm), at excitation wavelength of 365 nm, a typical range of ‘age pigment’. These phenomena were substantially eliminated in oxygen-free conditions. Both the NUV-irradiated RPE cells and RPE cells at 20 Pd expressed 4 to 8-fold and 2 to 4-fold less PEDF and TIMP-3 genes, respectively. As result of experiments using chronic photochemical treatment, RPE cells represented several characteristics of cellular senescence. In addition to alterations of the melanin/lipofuscin system, DNA synthesis was greatly suppressed in NUV-irradiated RPE cells, indicating replicative senescence. The phenomena of downregulation of the possible senescence markers imply that photochemical reactions of RPE cells accelerate the process of RPE senescence.     

cAMP and excitability in neuroendocrine cells during reproductive senescence

Excitability changes during reproductive senescence were investigated in the neurosecretory caudodorsal cells (CDCs) that control egg laying in the mollusc Lymnaea stagnalis. CDCs in the isolated central nervous system (CNS) were exposed to different discharge inducing treatments. Senescent CDCs (of animals 8 weeks after laying their last egg mass) and inhibited (I-) state CDCs (of egg-laying (EL) animals) were used. We showed that senescent and I-state CDCs closely resemble each other electrophysiologically. Electrical stimulation did not induce an afterdischarge in either type of CDC but exposure to release products of CDCs from EL animals or to saline with high potassium concentration did induce discharge activity. Also, 8-chlorophenylthio (8-CPT)-cAMP (10(-5) M) induced discharge activity. Exposure to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (10(-3) M) or to the adenylate cyclase activator Forskolin (10(-4) M), restored afterdischarge induction by electrical stimulation. Application of IBMX (10(-3) M) and Forskolin (10(-4) M) together induced discharges in the absence of electrical stimulation. Our results suggest that in senescent CDCs changes in the intracellular cAMP pathway may underlie afterdischarge failure.     

Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparaginelinked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.     

Primary tissue culture of normal adult rabbit adrenocortical cells fromzonae fasciculata andreticularis

Summary Parenchymal cells isolated from decapsulated adrenal cortices of adult New Zealand white female rabbits by means of enzymatic (trypsin plus collagenase and hyaluronidase) predigestion at 37° C followed by mechanical dissociation within a calcium (Ca²⁺)-free buffer were set up in primary monolayer cultures onto very thin, porous polyethylene discs floating on the top of a growth medium contained inside the wells of a plastic cluster plate. In this setting, adrenal cortical cells, being at the interface between air and growth medium, benefit from both the respiratory and metabolic standpoint. Therefore, they fully respond to anabolic and growth-promoting stimuli which may increase cellular oxygen consumption significantly. Hence, this in vitro model is well suited for studying the fairly complex regulatory mechanisms of mammalian adult adrenal cortical cell proliferation and differentiation, as it allows one to take full advantage of an artificially controlled environment and of completely synthetic growth media.     

Ribonucleotide reductase activity during the senescence of normal human diploid fibroblasts in culture

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity by dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, deoxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.     

Excitability and branching of neuroendocrine cells during reproductive senescence

In the mollusc Lymnaea stagnalis, neuroendocrine caudodorsal cells (CDCs) were studied physiologically and morphologically from egg layers (EL) (aged 154–400), and animals 4 weeks (CEL-4) (342–455 days), and 8 weeks (CEL-8) (477–660 days) after production of their last egg mass. After recording chemical transmission, electrical coupling and stimulation induced afterdischarges (ADs), CDCs then were filled with Lucifer Yellow. Based on the axonal branching revealed by Lucifer Yellow, CDCs were classified as extensively, moderately, or minimally branched. In EL-CDCs, induction of AD activity, which normally (9) precedes egg-laying, only was initiated in the resting state. CEL-4 CDCs exhibited ADs whereas CEL-8 CDCs did not. CEL-8 CDCs exhibited significantly reduced chemical and electrical transmission, and CEL-4 CDCs did not differ from resting state EL-CDCs. CDC branching was significantly reduced with both increasing age and declining egg-laying. Minimally branched CDCs most frequently failed to exhibit an AD and exhibited reduced electrical coupling. We conclude that both physiology and morphology of CDCs are related to age and reproductive state.