Effect of CO2 on short-term human lymphocyte culture in vitro

There was no significant difference in the mitotic indices of the cultures maintained at different CO2 concentrations, i.e. 0 percent, 5 percent and 10 percent. However, considerable variation was recorded among different individuals.     

Improved fish lymphocyte culture for chromosome preparation

Cytogenetic methodology is still underdeveloped in fishes compared with mammals. Culture condition for fish lymphocytes was optimized to improve chromosome preparation using the rainbow trout (Oncorhynchus mykiss) as a model after changing the combination of parameters such as mitogens, incubation periods, media, cell components, and freshness of blood. The optimized culture condition included isolation of lymphocytes from fresh blood by a stirring method, their culture in medium 199 supplemented with 10% FBS, 18g/ml of phytohemagglutinin (PHA-W) and 100g/ml of lipopolysaccharide (LPS) as mitogens, and harvested at 6 days after culture. This condition provided a notably increased mitotic index (MI) of 4.3–10.0% in rainbow trout lymphocytes. In addition, the condition was highly reproducible as shown by the similar level of MI in cultured lymphocytes from 181 individuals without failure. Applicability of this method in a wide range of fish groups was also proven with MI of 1.1–13.3% in cultured lymphocytes from other 16 freshwater species of Acipenseridae, Anguillidae, Salmonidae, Cyprinidae, and Centrarchidae, and five marine species of Sparidae, Kyphosidae, Paralichthyidae, and Scorpaenidae. Chromosome preparations of improved quality by the present method were successfully applied for the replication R-banding with incorporation of 5-bromo-2-deoxyuridine and direct R-banding fluorescence in situ hybridization.     

Effect of aluminum chloride on mitogenesis,mitosis, and cell cycle in human short-term whole blood cultures: Lower concentrations enhance mitosis

Aluminum, the third most common element in the earth's crust (second to oxygen and silicon) and recently suspected by some investigators to be implicated in Alzheimer disease etiology, has been studied in relation to its effect on mitogenesis, mitosis, and cell cycle. We have observed that 2–4 mM concentrations of AlCl₃ have decreased the number of cells that undergo mitogenesis (PHA-induced blast transformation) and mitosis in human short term whole blood cultures. We have also shown that the rate of the cell cycle was slowed down, i.e., cell cycle time was increased in the presence of AlCl₃. Also, we have demonstrated a reversible effect on aluminum-induced reduced mitotic index in long-term EBV-transformed lymphoblastoid cultures. Although safeguards such as limiting aluminum serum concentrations have been recommended to protect individuals undergoing dialysis, it should be realized that concentration accumulations of aluminum may increase over chronic exposures. Accordingly, if the number of cells stimulated by PHA is reduced in the presence of AlCl₃, there may be a reduction of immune competence, since the degree of PHA stimulation has been used as an indicator of immune response. Similar reductions in mitotic index could affect every tissue involved with cell division. Although it may not be the same for higher concentrations, from our results, we have also shown that decreased mitotic rates were reversible in long-term EBV-transformed lymphoblastoid cultures. Increased numbers of mitoses were observed in human short-term whole blood cultures that were exposed to 2 μM concentrations of aluminum chloride. The concentration is close to those found in normal human serum and within the “safeguard” range recommended for dialysis patients. A similar trend for aluminum sulfate was also observed, while preliminary results for three other aluminum species, lactate, citrate, and maltol, were also reported. Although previous reports have indicated a positive effect of aluminum on mitosis in vitro or in vivo, this is the first such report involving human material. It is clear that higher concentrations of aluminum chloride at 2.0–4.0 mM reversibly inhibit mitosis while more dilute concentrations of 1–2 μM, closer to those found in normal serum, enhance mitosis. The present results, as well as those in the literature, suggest that aluminum may be an essential element in cellular processes for optimal growth, development, and health maintenance. Future research will further test this hypothesis.     

In vitro differential effects of sodium selenite on the growth of human hepatoma cells and human embryonic liver cells

The effects of various concentrations of Na₂SeO₃ on human hepatoma cells and human embryonic liver cells was investigated in vitro. For human hepatoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL Na₂SeO₃, mitotic activity of human hepatoma cells were partially arrested. In human embryonic liver cells continuously treated with Na₂SeO₃, (1 μg/mL) cell count of the treated group decreased only by d 7; mitotic index, labeled index, and mean silver grain number per 50 labeled nuclei were the same as in the control group on exposure to 1, 3, and 5 μg/mL for up to 72 h. In mixed cultures of human hepatoma and embryonic liver cells treated with 3 and 5 μg/mL of Na₂SeO₃ for 24 h, hepatoma cells showed vacuolated cytoplasms, distorted nuclei, condensed chromatin, and even pyknosis, whereas the embryonic liver cells retained a normal morphology under the same treatment.     

Interchromatidal central ridge and transversal symmetry in early metaphasic human chromosome one

The topographic structure of Giemsa-banded (G-banded) early metaphase human chromosomes adsorbed on glass was analyzed by atomic force microscope using amplitude modulation mode (AM-AFM). Longitudinal height measurements for early metaphasic human chromosomes showed a central ridge that was further characterized by transversal height measurements. The heterochromatic regions displayed a high level of transversal symmetry, while the euchromatic ones presented several peaks across the transversal height measurements. We suggest that this central ridge and symmetry patterns point out a transitional arrangement of the early metaphase chromosome and support evidence for interchromatidal interactions prior to disjunction.     

Effect of low-level, 60-Hz electromagnetic fields on human lymphoid cells: I. Mitotic rate and chromosome breakage in human peripheral lymphocytes

Dividing human peripheral lymphocytes from 10 normal adults (5 males and 5 females) were exposed in vitro to low level 60-Hz electromagnetic fields for 69 hours. The current density of the electrical field was 30 microA/cm2, while the magnetic field was either 1 or 2 gauss. The cytological endpoints measured were mitotic rate and chromosome breakage. No statistically significant differences, indicative of a field effect, were observed between treated and control cells whether exposed to an electric field, a magnetic field, or to various combinations of the two.     

Extension of lymphocyte viability for radiation biodosimetry: Potential implications for radiological/nuclear mass casualty incidents

Dicentric chromosome assay (DCA) is routinely used for estimating the absorbed radiation dose in exposed humans. Optimal lymphocyte viability is crucial for reliable dose estimation and most cytogenetic laboratories prefer the receipt of blood samples within 24 to 36 hours after collection. Delays in the shipment/receipt of samples can occur sometimes under certain unforeseen circumstances: (1) Adverse weather conditions, (2) distant location of blood collection sites, and (3) shipping and handling of a large number of samples after radiological/nuclear mass casualty incident(s). To circumvent some of these limitations, we evaluated the suitability of ex vivo irradiated blood samples stored in the presence of phytohemagglutinin (PHA) for 7 days at ambient temperature (22-24°C) for radiation biodosimetry. Blood samples stored in the presence of PHA for up to 7 days showed a higher mitotic index than blood samples stored without PHA. To verify the use of stored blood samples for DCA, frequencies of X-rays induced dicentric chromosomes were analyzed in the blood samples that were cultured either 24 hours after exposure or 7 days later after storage. Our results indicate that storage of ex vivo irradiated blood samples in the presence of PHA at ambient temperature was found optimal for DCA and that the radiation doses estimated by dicentric chromosome frequencies were grossly similar between the fresh and stored blood samples. Our study suggests that reliable and accurate biodosimetry results can be obtained for triage using blood samples stored for up to a week at ambient temperature in the presence of PHA.     

Syrian hamster lymphocyte cytogenetic technique and effects of blood storage on mitotic index

Summary The Syrian hamster has a diploid chromosome complement similar to humans in both number (2N=44) and morphology. For comparative mutagenic studies with humans, a repeatable lymphocyte chromosome technique involving laboratory mammals is desirable. The reported hamster lymphocyte cytogenetic technique appears technically uncomplicated and repeatable while providing a sufficient number of metaphase cells for quantiative analysis. In order to determine the effects of storing whole-blood (in culture media) for varying time periods at 8°C prior to adding a mitogen, the mitotic index was calculated following a 48-h culture period. Results indicated the storage up to six days can still result in a mitotic index adequate for cytogenetic analyses.     

乙酸铜对蚕豆根尖细胞致畸效应

采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的乙酸铜为诱变剂,选择不同的处理时间,测定蚕豆根尖细胞的有丝分裂指数、微核率和染色体畸变率。结果表明：乙酸铜能诱发较高频率的微核率,处理6h、12h时微核率均随着乙酸铜浓度的升高而增加,具有明显的剂量效应;处理24h时在实验浓度范围内,其微核率随乙酸铜浓度的升高而增加,但高于一定浓度后反而呈下降趋势。不同浓度的乙酸铜在不同处理时间均使蚕豆根尖细胞有丝分裂指数增大。乙酸铜还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。因此,乙酸铜对蚕豆根尖细胞具有明显的致畸效应。     

In vitro culture of Cryptosporidium muris in a human stomach adenocarcinoma cell line

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.     

Differential Effects of selenium on the proliferation of human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro

The effect of different concentrations of Na₂SeO₃ on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 μg/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1–5μg/mL sodium selenite for 1–3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 μg/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei.     

硫酸铜对蚕豆根尖细胞有丝分裂的影响

以蚕豆根尖为材料,研究硫酸铜对蚕豆根尖细胞的遗传毒性效应。采用蚕豆根尖细胞的微核试验方法和染色体畸变试验方法,以不同浓度的硫酸铜为诱变剂,测定蚕豆根尖细胞的有丝分裂指数、微核率和染色体畸变率。结果表明:不同浓度的硫酸铜均能使蚕豆根尖细胞有丝分裂指数明显增加,即5个实验组的分裂指数均明显高于对照组(P<0.01或P<0.001);不同浓度的硫酸铜对蚕豆根尖细胞有丝分裂各期百分数的影响有异;能诱发较高频率的微核率,即在一定浓度范围内,其微核率随硫酸铜处理浓度的升高而增加,但随着硫酸铜浓度的进一步升高而呈下降趋势;硫酸铜还能诱导染色体产生多种类型的畸变,染色体畸变率随硫酸铜处理浓度的升高而增加,随着硫酸铜浓度的进一步升高而呈下降趋势,但均明显高于对照组(P<0.001)。结论是硫酸铜对蚕豆根尖细胞具有明显的遗传毒性效应。     

Photoautotropic in vitro culture of Chrysanthemum under CO2 enrichment

Photoautotrophic culture of Chrysanthemum was established under CO2 enrichment (2% v/v). The shoot length and number of leaves were almost equal (2.7 cm and 14 respectively) both under photoautotrophic and photomixotrophic cultures, recorded after four weeks of incubation. Similarly, average dry mass of the plantlets were comparable (31 and 28 mg respectively) under both conditions. The number of branches and internode length which influence the number of propagule potential for mass propagation, were also identical. Nevertheless, photoautotrophic cultivation minimized the risk of contamination in cultures, which in turn will reduce the production cost. © Rapid Science Ltd. 1998     

Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34^cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis.     

Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34^cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis.     

丝裂霉素致小鼠骨髓淋巴细胞遗传损伤及西洋参的干预作用研究

目的:研究西洋参(Panax quinguefolium L)抗突变作用和对细胞增殖的影响.方法:采用小鼠骨髓淋巴细胞有丝分裂指数试验、染色体畸变试验和微核试验,观察西洋参对小鼠骨髓淋巴细胞遗传损伤效应的影响.结果:丝裂霉素(Mitomycin-c MMC)单独作用时,能抑制小鼠骨髓淋巴细胞的增殖(P<0.01),提高小鼠骨髓淋巴细胞的染色体突变(P<0.01)和微核率(P<0.01).西洋参高、中、低剂量组与MMC联合作用时,有丝分裂指数明显比模型组提高(P<0.01),并且均能显著地抑制由于MMC引起的染色体突变,其抑制率分别为68.25%、34.97%和14.29%,同时对MMC诱发的微核率也有明显的抑制效应.结论:西洋参能抑制MMC诱发的染色体畸变率和微核率,促进细胞的增殖能力.     

Effect of matrine on primary human hepatocytes in vitro

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO₂⁻ induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9680-1) contains supplementary material, which is available to authorized users.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.