Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.     

Expression of thyrotropin receptor on clonal osteoblast-like rat osteosarcoma cells

OBJECTIVE: To assess (1) pediatricians' attitudes toward and practice of complementary and alternative medicine (CAM) for their patients; (2) their knowledge, experience, and referral patterns for selected CAM therapies; and (3) their desire for continuing medical education courses on CAM therapies. METHOD: An anonymous, self-report, 25-item questionnaire was mailed to fellows of the Michigan chapter of the American Academy of Pediatrics. RESULTS: Of 860 pediatricians, 348 (40.5%) responded; their median age ranged from 35 to 45 years, 54.3% were men, 67.6% were white, 67.9% were general pediatricians, and 65.2% were trained in the United States. Of the respondents, 83.5% believed their patients use CAM therapies, but 55.1% believed this constituted less than 10% of patients. Of the pediatricians who talked about CAM (53.8%), 84.7% said the discussion was initiated generally by the patient's family. More than half of the physicians (55.2%) said they would use CAM therapies personally, and 50.3% would refer for CAM therapies. Therapies referred for were biofeedback (23.6%), self-help groups (23.3%), relaxation (14.9%), hypnosis (13.8%), and acupuncture or acupressure (10.9%). Of the physicians who responded, 54.1% were interested in continuing medical education courses on CAM therapies. White respondents, US medical school graduates, and general pediatricians were most likely to believe their patients use CAM and discuss or refer for CAM therapies (P<.01). Female pediatricians were most likely to discuss or refer for CAM and to want more continuing medical education on CAM therapies (P<.05). CONCLUSIONS: A majority of pediatricians sampled believed a small percentage of their patients were seeking alternatives to conventional medicine. Half would consider referring patients for CAM, and most were interested in continuing medical education courses on CAM. Larger studies surveying pediatricians, along with more education and research on CAM therapies, need to be considered for the future.     

Effects of ionizing radiation on proliferation and differentiation of osteoblast-like cells

Diagnostic radiation for immediate post-surgical assessment of osseointegrated dental implants has been discouraged, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. To assess this possibility, we investigated the effects of ionizing radiation on proliferation and differentiation of osteoblasts using osteoblast-like cells isolated from the calvariae of newborn rats (ROB) and a clonal osteoblastic cell line (MC3T3-E1). The cells were exposed on day 3 to a single dose of x-rays at either 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40-mGy dose from a diagnostic chest x-ray machine. The effects of radiation on cell growth and alkaline-phosphatase-specific (ALP) activity were evaluated at three-day intervals after irradiation up to day 12 in ROB cells, and evaluated at day 12 in MC3T3-E1 cells. At the culture end-point, the effects on formation of bone-like nodules were also evaluated in both ROB and MC3T3-E1 cells. Exposure of 4000 mGy differentially affected the two cell types. It inhibited cell growth and alkaline phosphatase activity, and inhibited DNA content in MC3T3-E1 cells. This irradiation also strongly inhibited the formation of bone-like nodules in ROB cells. On the other hand, exposure of 40-, 100-, and 400-mGy (Linac) and 40-mGy (diagnostic quality) irradiation induced no significant changes in cell growth, alkaline phosphatase activity, and formation of bone-like nodules in ROB cells. These doses also induced no significant changes in DNA content and ALP activity in MC3T3-E1 cells. These results indicate that ionizing radiation at a single dose of up to 400 mGy induces no significant changes in cell growth and differentiation of osteoblast-like cells, at least in vitro. Higher radiation doses (4000 mGy) may exert different effects on cell proliferation and cell differentiation of osteoblasts, depending on the cell types affected. Thus, diagnostic radiation seems to have less effect on proliferation and differentiation of osteoblasts.     

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

Control of expression of differentiated functions in neuroblastoma cell hybrids

Cell hybrids have been extensively utilized for gene mapping; more than 50 enzymes and nonenzyme proteins have been assigned to individual human chromosomes. Hybrids have also been used in the study of differentiation; fusions involving mouse or human neuroblastoma cells and various nonneuronal lines resulted in hybrid cells that continued to express neuronspecific functions. The expression of the differentiated state is, however, not an all-or-none phenomenon: One neuronal trait may be evident in such hybrids, in the absence of others. The potential usefulness of the human neuroblastoma hybrids for the assignment of genes involved in the expression of differentiated functions to specific chromosomes is discussed.     

Time-course effects of human recombinant luteinizing hormone on porcine Leydig cell specific differentiated functions

Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.     

Cloning of a novel retinoic acid-inducible mRNA from human osteoblast-like cells

Retinoids have long been known to influence skeletal development and bone remodeling. Cells of the osteoblastic lineage play a key role in these processes. In this study we have used the differential display PCR technique to identify retinoic acid (RA)-induced mRNAs in human osteoblast-like cells. We report the cloning and sequencing of one such mRNA, AT-RA 6, which was specifically induced by all-trans RA both in normal human osteoblast-like cells and in MG-63 osteosarcoma cells. Maximal expression was found after 60 min, suggesting that this may be an early response gene. Expression was found in all tissues examined. No homology to known mRNA sequences was detected.     

Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering

As the field of dental implants continues to grow at a rapid rate so does our quest to find new techniques to enhance bone grafting. Tissue engineering is an exciting new technique in bone grafting. Therefore, the purposes of this study were to develop a simple, reproducible method to isolate human osteoblast-like cells (HOBs) and to evaluate in vitro cell proliferation within 2 different 3-dimensional (3-D) constructs targeted for tissue engineering applications. Ultimately, HOBs that have been amplified within 3-D constructs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol employed human fetal calvaria tissue sequentially digested with trypsin and collagenase. The HOB cells from only the third and fourth digests were obtained, cultured and evaluated within the constructs. An osteoblast-like phenotype was in part verified for these HOB cells by demonstrating a significantly higher alkaline phosphatase activity than for human gingival fibroblasts, and a comparable level to the osteoblast cell line MG-63. The HOB cells were cultured within either poly (D,L-lactide) (PLA) or a fused fiber ceramic and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying cells throughout the 3-D constructs. Continuous cell viability was demonstrated for the duration of the 33-day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vitro amplification results further indicate that tissue engineering strategies with either the PLA or fused fiber construct may be suitable for bone regeneration therapy for dental implants.     

Transforming growth factor--alpha stimulates chemotaxis of osteoblasts and osteoblast-like cells in vitro

Homeostatic turnover of bone is believed to be regulated in part by growth factors present in the surrounding environment. The first step during the bone formation process is the recruitment of osteoblasts from the surrounding intact bone to the resorption site. In this study, the effects of TGF-alpha on osteoblast chemotaxis was investigated. Cultures of rat osteoblasts and human osteoblast-like cells were examined for chemotaxis in response to increasing concentrations of TGF-alpha utilizing a modified Boyden Chamber technique. TGF-alpha stimulated a dose-dependent increase in chemotaxis by both cell populations. These results indicate that TGF-alpha may be playing an important role in the early stages of bone matrix regeneration by stimulating osteoblast chemotaxis.     

Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro

To study the direct effects of thyroid hormones on human osteoblasts we examined the effects of triiodothyronine (T3) on proliferation and differentiation of human osteoblast-like (hOB) cells in vitro. T3 increased 3H-thymidine incorporation in DNA of hOB cells (p < 0.05, n = 10). Half maximal effects obtained at a T3 concentration of 1-10 nM which lies within the physiological concentration of the hormone. In addition, T3 increased alkaline phosphatase production (p < 0.05, n = 13) and inhibited procollagen type I carboxyterminal propeptide (PICP) production (p < 0.05, n = 13). T3 interaction with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was also studied. 1,25-(OH)2D3 (10(-9)M) alone doubled AP production and induced osteocalcin expression by hOB cells. Concurrent addition of T3 and 1,25-(OH)2D3 did not further increase production of AP, PICP or osteocalcin by hOB cells. In conclusion, T3 exerts significant effects on osteoblast proliferation and differentiation, suggesting that human osteoblasts are targets for thyroid hormones.     

Differential expression of plasma membrane calcium pump mRNA isoforms in rat osteoblast-like cells

Cryptococcal meningitis is a common infection in patients with AIDS. Using a murine cryptococcosis model, we compared treatment with a new triazole, D0870, and fluconazole. Groups of ICR (Institute for Cancer Research) mice were infected intracerebrally with eight different isolates of Cryptococcus neoformans variety neoformans with different in vitro susceptibilities to fluconazole. For survival studies mice were challenged with two to four times LD(50) or six to nine times LD(50). Treatment was given for 10 days. Mice were observed through to day 30. To assess the effect of treatment on fungal tissue burden, mice received a three to five times LD(50) inoculum and treatment for 10 days. They were sacrificed on day 12 and serial dilutions of brain homogenates were cultured. Fluconazole prolonged survival primarily in isolates which were susceptible in vitro. D0870 prolonged survival in all isolates except one, which was also resistant in vitro to D0870 and fluconazole. Both drugs reduced colony counts of all isolates. D0870 warrants further development for use in cryptococcosis, and appears effective for isolates relatively resistant to fluconazole. There is a relative correlation of in vivo and in vitro susceptibility to D0870 as well as fluconazole.     

In vitro biomineralization by osteoblast-like cells. II. Characterization of cellular culture supernatants

The quantification of total calcium, phosphorus, iron, chromium and nickel in cell culture medium by electrochemical or spectroscopic means may require digestion of samples. Nevertheless, when pH adjustment is performed for values higher than about 6.5, the formation of two phases occurs: a white precipitate and a clear solution. Analysing both phases using microelectrodes, atomic absorption spectrometry (AAS), diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, X-ray dispersive (XRD) analysis, scanning electron microscopy (SEM) and energy dispersive spectroscopic (EDS) analysis, it was observed that iron, chromium and nickel are not co-precipitating with the white solid phase. If quantification of calcium, phosphorus and magnesium is intended, a ten-fold dilution at least, must be performed to avoid most of these elements going into the precipitate. This knowledge is crucial if a mineralization study is going to be made.     

Deposition and dispersion of 1-micrometer aerosol boluses in the human lung: effect of micro- and hypergravity

We performed bolus inhalations of 1-micrometer particles in four subjects on the ground (1 G) and during parabolic flights both in microgravity (microG) and in approximately 1.6 G. Boluses of approximately 70 ml were inhaled at different points in an inspiration from residual volume to 1 liter above functional residual capacity. The volume of air inhaled after the bolus [the penetration volume (Vp)] ranged from 200 to 1,500 ml. Aerosol concentration and flow rate were continuously measured at the mouth. The deposition, dispersion, and position of the bolus in the expired gas were calculated from these data. For Vp >/=400 ml, both deposition and dispersion increased with Vp and were strongly gravity dependent, with the greatest deposition and dispersion occurring for the largest G level. At Vp = 800 ml, deposition and dispersion increased from 33.9% and 319 ml in microG to 56.9% and 573 ml at approximately 1.6 G, respectively (P < 0.05). At each G level, the bolus was expired at a smaller volume than Vp, and this volume became smaller with increasing Vp. Although dispersion was lower in microG than in 1 G and approximately 1.6 G, it still increased steadily with increasing Vp, showing that nongravitational ventilatory inhomogeneity is partly responsible for dispersion in the human lung.     

关于培养和造就高层次人才的思考

21世纪是全球化、市场化、信息化的世纪 ,是知识经济的时代 ,知识生产将成为现实生产的强大推动力 ,成为经济发展的关键因素 ,社会经济的增长 ,将主要通过智力因素来实现。所以 ,作为掌握知识、运用知识的人才资源将成为最重要的资本 ,而且将成为战略性资本。面向 2 1世纪 ,必须确立“人才资源是第一资源”的观念 ,把强化人才资源的开发和利用 ,作为促进社会发展的一项战略任务抓实抓好。一、确立“人才资源是第一资源”的观念 ,尊重知识 ,尊重人才2 1世纪的竞争 ,是人才的竞争 ,人才是科技进步和经济社会发展中最重要的资源 ,谁拥有了人才…     

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

Two experiments investigated a dating bias in which people tend to estimate recent dates too remotely and remote dates too recently. Experiment 1 examined and upheld a prediction of the hypothesis that bias arises because events whose time of occurrence is unknown are dated through associated events for which some time information is available. Experiment 2 required some subjects to date prototype events while others dated specific events. Prototype events were dated more recently, and the errors in dating specific events were related to differences in the way the prototype events were dated. Both sets of results were predicted by the association hypothesis, according to which events whose dates are well known are dated with reference either to specific associated events or to prototype events.     

Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells

Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI-DNA-adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI-adenofection.