Establishment and characterization of a fibroblast line from Simmental cattle

A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8 h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2n = 60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N₃, pEYFP-N₁ and pDsRed1-N₁) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.     

Establishment and characterization of a fibroblast cell line derived from Jining Black Grey goat for genetic conservation

An ear marginal fibroblast cell bank was established from the Jining Black Grey (JBG) goat using attachment culture and freezing biotechniques. This bank included 32 ear samples (15 males and 17 females) and has stocks of 168 cryogenically preserved vials, each vial contained 4.0 × 10⁶ cells per milliliter. The cells of the bank that were checked for the quality and the biological characteristics showed a typical fibroblast morphology when they cultured in vitro. The growth curve consisted of a growth curve consisting of a latent phase, logarithmic growth phase and stationary phase, cell population doubling time (PDT) of 48 h. The chromosome analysis showed that the frequency of cells having the diploid number of chromosomes (60) was 98.65 ± 2.89%, and no microbe contamination (bacteria, epiphyte, virus or mycoplasma) was detected. In addition, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) zymography indicated that this cell bank was free of cross-contamination. At 24, 48 and 72 h after transfection, the expression efficiency of pEGFP-C1, pEGFP-N3, pEYFP-N1, pECFP-N1, pECFP-mito and pDsRed1-N1 were between 11.8% and 56.3%. The fluorescence could be observed well-distributed in cytoplasm and nucleus except for some cryptomere vesicles at 24 h after transfection. These newly established cell lines meet all the quality control standards established by the American Type Culture Collection. We have employed a new method for conserving the genetic resources of an important and endangered animal breed. The fibroblast bank that we have established from the JBG goat also provides an invaluable material resource for future studies that will utilize molecular and cell biology applications.     

Establishment and biological characteristics of Ujumqin sheep fibroblast line

A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 × 10⁶ cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.     

Ear fibroblasts derived from Taiwan yellow cattle are more heat resistant than those from Holstein cattle

The objective of this study was to compare the thermotolerances of ear fibroblasts derived from Holstein (H) and Taiwan yellow cattle (Y) and their apoptosis-related protein expressions with (1, 3, 6, 12, and 24 h) or without heat shock treatment. The results showed that the vaginal temperatures of Y (38.4–38.5 °C) were (P<0.05) lower than that of H (38.8 °C) during the hot season. The apoptotic rates of ear fibroblasts derived from Y (6 h: 1.1%; 12 h: 1.6%; 24 h: 2.6%) were lower (P<0.05) than those of cells derived from H (6 h: 1.8%; 12 h: 4.0%; 24 h: 6.9%), respectively, after heat shock (42 °C). The expression level of apoptosis inducing factor (AIF) in ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 6 h and 12 h, respectively. The level of cytochrome c of ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 1–12 h, respectively. The abundances of Caspase-3, Caspase-8 and Caspase-9 of ear fibroblasts derived from H were higher (P<0.05) than those of cells derived from Y after 12 h and 24 h of heat shock, respectively; the Bcl-2/Bax ratios of ear fibroblasts derived from H were lower (P<0.05) than those from Y-derived fibroblasts after heated for 1–24 h. The expression level of HSP-70 of Y-derived ear fibroblasts was also higher (P<0.05) than that from H after the same duration of heat shock treatments. Taken together, the thermotolerance of ear fibroblasts derived from Taiwan yellow cattle was better than that of cells derived from Holstein cattle.     

Dexamethasone protects cultured rat hepatocytes against cadmium toxicity: involvement of cellular thiols

In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William’s E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 μM) in the absence or presence of 0.1 μM dexamethasone. Cadmium chloride, 5 or 10 μM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 μM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 μM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.     

Interactions between Cryptosporidium parvum and bovine corona virus during sequential and simultaneous infection of HCT-8 cells

Neonatal diarrhoea in calves is one of the major health problems in the cattle industry. Although co-infections are often associated with greater severity of disease, there is limited information on any impact on the pathogens themselves. Herein, we studied Cryptosporidium parvum and bovine coronavirus (BCoV) in human HCT-8 cells, inoculated either sequentially or simultaneously, to investigate any influence from the co-infections. Quantitative results from (RT)-qPCR showed that prior inoculation with either of the two pathogens had no influence on the other. However, the results from simultaneous co-inoculation showed that entry of viral particles was higher when C. parvum sporozoites were present, although elevated virus copy numbers were no longer evident after 24 h. The attachment of BCoV to the sporozoites was probably due to specific binding, as investigations with bovine norovirus or equine herpes virus-1 showed no attachment between sporozoites and these viruses. Flow cytometry results at 72 h post inoculation revealed that C. parvum and BCoV infected 1–11% and 10–20% of the HCT-8 cells, respectively, with only 0.04% of individual cells showing double infections. The results from confocal microscopy corroborated those results, showing an increase in foci of infection from 24 to 72 h post inoculation for both pathogens, but with few double infected cells.     

Genetic Structure and Differentiation of Three Chinese Indigenous Cattle Populations

Levels of genetic differentiation, gene flow, and genetic structure of three indigenous cattle populations (Luxi, Bohai, and Minnan) and two reference cattle populations (Chinese Holstein and Qinhai yak) in China were estimated using the information from 12 microsatellites, and 141 microsatellite alleles were identified. The mean number of alleles per locus ranged from 2.9005 in yak to 4.9722 in Holstein. The observed heterozygosity ranged from 0.5325 (yak) to 0.7719 (Holstein); 29 private alleles were detected. The global heterozygote deficit across all populations amounted to 58.5% (p < 0.001). The overall significant (p < 0.001) deficit of heterozygotes because of inbreeding within breeds amounted to 43.2%. The five cattle populations were highly differentiated (F _st = 26.9%, p < 0.001) at all loci. The heterozygote deficit within the population was highest in Luxi cattle and lowest in yak. The average number of effective migrants exchanged per generation was highest (1.149) between Luxi and Holstein, and lowest (0.509) between Luxi and yak. With the application of prior population information, cluster analysis achieved posterior probabilities from 91% to 98% of correctly assigning individuals to populations. Combining the information of cluster analysis, gene flow, and Structure analysis, the five cattle populations belong to three genetic clusters, a taurine (Luxi and Chinese Holstein), a zebu (Bohai and Minnan), and a yak cluster. This indicates that Bohai black is closer to Bos indicus than Luxi cattle. The evolution and development of three indigenous cattle populations are discussed.     

Effects of industrial storage on the bioreduction capacity of brewer’s yeast

The effects of industrial storage on the changes of the cell viability and the activities of intracellular alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in brewer’s yeast, and the corresponding capacity for the bioconversion of ethyl-3-oxobutanoate (EOB) to ethyl (S)-3-hydroxybutanoate ((S)-EHB), were investigated. The viability of fresh brewer’s yeast cells stored in industrial circulating cooling water at 1–2°C showed 4 and 15% drop after the storage of 7 and 15 days, respectively, after which cells died rapidly. The pretreatment of the stored brewer’s yeast cells by washing and screening significantly enhanced cell viability during industrial storage. The intracellular levels of ADH and G6PDH after permeabilization of these stored cells with cetyltrimetylammonium bromide (CTAB) were much higher, which showed only slight decrease within 2 weeks during the industrial storage. When the stored cells after the permeabilization treatment was used as the biocatalyst at 90–120 g/L, EOB was converted almost completely into enantiopure (S)-EHB with an enantiomeric excess (ee) more than 99% and a yield of over 96%, by fed-batch bioconversion of 560 mM EOB within 6 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional Characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum</Emphasis> F1

In this study, Lactobacillus fermentum (L. fermentum) F1 reduced cholesterol 48.87%. The strain was screened from cattle feces using an API 50 CHL system and the 16S rRNA sequence contrasting method. L. fermentum F1 showed acid and bile tolerance, and antimicrobial activity against pathogenic Escherichia coli and Staphylococcus aureus. L. fermentum F1 deconjugated 0.186 mM of free cholalic acid after it was incubated at 37°C in 0.20% sodium taurocholate (TCA) broth for 24 h. Heat-killed and resting cells of L. fermentum F1 showed small amounts of cholesterol removal (6.85 and 25.19 mg/g, respectively, of dry weight) compared with growing cells (33.21 mg/g of dry weight). The supernatant fluid of the broth contained 50.85% of the total cholesterol, while the washing buffer and cell extracts had 13.53 and 35.39%, respectively. These findings suggest that L. fermentum F1 may remove cholesterol by co-precipitating with deconjugated bile salt, assimilating with cells and by incorporation into cellular membranes. Cholesterol assimilated by cells held 72.0% of the reduced cholesterol, while 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells.     

Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (MSC) have previously been reported to be susceptible to cryopreservation-induced apoptosis. A significant fraction of MSC lose their viability during freeze-thawing, which represent a major technical barrier in attaining adequate viable cell numbers for optimal efficacy in transplantation therapy. Recently, it was reported that a Rho-associated kinase (ROCK) inhibitor Y-27632 could enhance the post-thaw viability and physiological function of cryopreserved human embryonic stem cells (hESC). Hence, this study attempted to investigate whether Y-27632 can exert a similar beneficial effect on the post-thaw viability of cryopreserved MSC. A concentration range of 1–100 μM Y-27632 was supplemented in both the cryopreservation medium (10% (v/v) dimethyl sulfoxide), as well as the post-thaw culture medium. The supplementation of Y-27632 had no significant effect on the immediate post-thaw viability, as assessed by trypan blue exclusion. However, 24 h after the frozen-thawed cell suspensions were re-plated on new cell culture dishes (with varying concentrations of Y-27632 within the post-thaw culture media); the MTT assay subsequently showed significant differences in the proportion of adherent viable cells over the concentration range of Y-27632 examined, with a peak at between 5 and 10 μM. At zero concentration of Y-27632, the proportion of viable adherent cells was 39.8 ± 0.9%; and this value peaked at 48.5 ± 1.7% with 5 μM Y-27632 and 48.4 ± 1.8% with 10 μM Y-27632, prior to decreasing to 36.0 ± 0.6% with 100 μM Y-27632. Additionally, it was observed that Y-27632 induced morphological changes in the frozen-thawed MSC. With increasing Y-27632 concentration, the cells displayed more extensive branching of cytoplasmic extensions that gave a ‘web-like’ appearance. This is consistent with previous reports of Y-27632 stimulating neuronal differentiation of MSC.     

GRP78 from grass carp (Ctenopharyngodon idella) provides cytoplasm protection against thermal and Pb2+ stress

Glucose regulated protein (GRP) located in endoplasmic reticulum (ER) was a member of heat shock protein (Hsp) family. The protective mechanism adapted to ER stimuli was closely related to GRP. GRP78, known as BiP, was one of central regulator responded to stress in ER. Grass carp (Ctenopharyngodon idella) GRP78 (CiGRP78) was up-regulated in almost tissues, especially in liver, under heat shock (34 °C), cold stress (4 °C) or lead nitrate (0.25 mmol/L) stress. In order to understand the function of CiGRP78 in cellular protection, CiGRP78 ORF cDNA was inserted into the plasmid of pET-32a(+) or pEGFP-C1 respectively, then the recombinant plasmids were transformed or transfected into Escherichia coli cells, mouse myeloma cells (SP2/0) or grass carp kidney cells (CIK). In the cells, CiGRP78 was over-expressed following thermal, cold or Pb²⁺ stress. Results showed that CiGRP78 not only contributed to protecting prokaryotic cells against thermal or cold extremes, but also played the same role in SP2/0 and CIK cells. After treatment with heat stress at 42 °C for 1 h, although the viability of the cells declined a lot, CIK cells with pEGFP-C1/CiGRP78 exhibited a higher survival rate (28%) than wild-type cells (7%) or cells with only pEGFP-C1 (5.1%). When the time lag extended to 2.5 h, the survival rates were 19%, 5.7%, 4.8% respectively. In addition, CiGRP78 would also provide a transient cytoplasm protection against Pb²⁺ stress in a dose- and time-dependent manner. After treatment with lead nitrate at concentration of 10 μmol/L for 12 h, 24 h or 36 h, the survival rates of cells with pEGFP-C1 or wild-type cells were 46.7% or 46.7% (12 h), 25% or 22% (24 h), 10% or 11% (36 h) respectively. When the cells were treated with lead nitrate at the concentration of 25 μmol/L, the survival rates of cells with pEGFP-C1 or wild-type cells were 45.5% or 30% (12 h), 16.7% or 25% (24 h), 6.5% or 8% (36 h), respectively. CiGRP78 provided a distinct protection in CIK cells at the low concentration for 24 h. The survival rates of CIK cells with pEGFP-C1/CiGRP78 treated with lead nitrate at concentration of 10 μmol/L or 25 μmol/L were 65.9% or 58.8% respectively. When the cells were treated with lead nitrate at concentration of 50 μmol/L for 24 h, the survival rate of the CIK cells was only about 30%. If the process-time was extended to 36 h, CiGRP78 could not provide any cytoplasm protection for CIK cells.     

MyoD基因对肉牛胴体性状影响的分析

用PCR技术克隆到MyoD基因的第二内含子, 采用PCR-SSCP方法研究了3个黄牛品种(鲁西牛、晋喃牛、秦川牛)及4个杂交肉牛(夏洛莱×鲁西牛、安格斯×鲁西牛、利木赞×鲁西牛、西门塔尔×鲁西牛)群体MyoD基因的多态性, 并分析了基因位点多态性与肉牛肉质性状的相关性。实验结果,在国内首次扩增出肉牛MyoD基因的第二内含子的全部序列, 共261 bp。用SSCP方法检测到MyoD基因内含子2有A和B两个等位基因。测序结果表明该座位的多态性是由于内含子二39 bp处C-T的突变和112 bp处C→G的突变造成的。等位基因B在中国地方品种的分布频率高于引进品种的杂交牛群体。c2检验的结果表明, 在该位点的除夏洛莱和安格斯杂交牛外, 其余五个群体(晋南、鲁西、秦川、西门塔尔杂交牛和利木赞杂交牛)均处于Hardy-Weinberg不平衡状态(P>0.05)。实验群体不同基因型与肉牛的宰前活重、胴体重、净肉重、高档肉重、眼肌面积等性状的影响差异极显著或显著(P<0.01或P<0.05), 并且AA型个体均高于AB型个体。     

Cell cycle arrest induced by Pisosterol in HL60 cells with gene amplification

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 μg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 μg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 μg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 μg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.     

Carnosine Inhibits Growth of Cells Isolated from Human Glioblastoma Multiforme

The present study evaluates the effect of the naturally occurring dipeptide carnosine on primary cell cultures established from patients with glioblastoma multiforme. Surgically removed tumors were used to establish primary cell cultures that were incubated for 96 h with medium supplemented with carnosine at concentrations of 20, 40 and 50 mM. Following incubation, dehydrogenase activity, cellular adenosine triphosphate concentration (ATP), caspase activity, lactate dehydrogenase (LDH) release and the rate of DNA synthesis were determined. After 96 h of carnosine treatment a significant reduction in cellular ATP and dehydrogenase activity was detected already at a concentration of 20 mM carnosine. Carnosine (50 mM) reduced ATP concentration to 42.7 ± 13.5% (n = 6) and dehydrogenase activity to 41.0 ± 19.3% (n = 6) compared to untreated cells. Additional experiments revealed no sign of enhanced apoptosis or necrosis in the presence of carnosine. However, a quantitative bromo-desoxy-uridine-based proliferation assay demonstrated a clear effect of carnosine on DNA synthesis reducing its rate down to 50% (2 cultures) and 10% (4 cultures). Therefore, it can be concluded that carnosine is obviously able to inhibit proliferation of cells derived from glioblastoma. Since it is a naturally occurring substance that appears to be non-toxic to normal tissue and is able to penetrate the blood–brain barrier it may be a candidate for a therapeutic agent that may reduce proliferation of neoplastic cells even in vivo and especially in cases of glioblastoma multiforme.     

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P < 0.05). In contrast, CASP3 and CASP9 gene expression was significantly down-regulated during myogenesis (P < 0.05). Moreover, we put the CAPN1, CAPN3, CASP3, CASP7, CASP9, Myf5, and MyoG together to say that these genes expression had no significant correlation between the satellite cell model and the skeletal muscle tissue (P > 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.     

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to human lung epithelial cells

Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen. It is currently a major public health concern, there is widespread exposure to it in occupational settings and to the general public. However, despite the potential widespread exposure and the fact that the lung is its target organ, few studies have considered the toxic effects of particulate Cr(VI) in human lung cells. Accordingly, we used lead chromate as a model particulate Cr(VI) compound and determined its cytotoxicity and genotoxicity in cultured human bronchial epithelial cells, using BEP2D cells as a model cell line. We found that lead chromate induced concentration-dependent cytotoxicity in BEP2D cells after a 24 h exposure. Specifically, the relative survival was 78, 59, 53, 46 and 0% after exposure to 0.5, 1, 5, 10 and 50 μg/cm² lead chromate, respectively. Similarly, the amount of chromosome damage increased with concentration after 24 h exposure to lead chromate. Specifically, 0.5, 1, 5 and 10 μg/cm² damaged 10, 13, 20 and 28% of metaphase cells with the total amount of damage reaching 11, 15, 24 and 36 aberrations per 100 metaphases, respectively. Lead chromate (50 μg/cm² lead chromate) induced profound cell cycle delay and no metaphases were found. In addition we investigated the effects of soluble hexavalent chromium, sodium chromate, in this cell line. We found that 1, 2.5, 5 and 10 μM sodium chromate induced 66, 35, 0 and 0% relative survival, respectively. The amount of chromosome damage increased with concentration after 24 h exposure to sodium chromate. Specifically, 1, 2.5 and 5 μM damaged 25, 34 and 41% of metaphase cells with the total amount of damage reaching 33, 59 and 70 aberrations per 100 metaphases, respectively. Ten micromolar sodium chromate induced profound cell cycle delay and no metaphases were found. Overall the data clearly indicate that hexavalent Cr(VI) is cytotoxic and genotoxic to human lung epithelial cells.     

Development of cell line from the testicular tissues of crab <Emphasis Type="Italic">Scylla serrata</Emphasis>

This is the first report on development of a finite cell line from testicular tissues of crab, Scylla serrata. Both the explant and segregated tissues of testes yielded cells that could proliferate and grow. These cells ranged in size from 10 to 38 μm with distinct nuclei of varying shapes. The testicular cells survived and proliferated best in L-15-crab saline medium supplemented with epidermal growth factor (20 ng/mL) and glucose (1 mg/mL). The cell proliferation rate was assessed by Methyl tetrazolium assay in terms of change in optical density which clearly indicated a prominent increase in cell density. The testicular cells were subcultured at an interval of 4–6 days. These subcultured cells remained healthy and proliferated for 5 months with a minimum of ten subsequent passages. The finite cell line was characterized in terms of morphology, growth rate, lactate dehydrogenase release (for detecting health status) and 18S rRNA sequencing. This cell line could be a very useful tool for testing infections and replications of crustacean viruses. The present work provides a technique that could be extended for developing other crustacean cell lines.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

云南矮马耳缘组织成纤维细胞系的建立及其生物学特性

随着全球生物多样性及其遗传资源丧失程度的加剧 ,使得各国纷纷投入大量人力和财力 ,并以多种方式收集、保存和整理大自然赋予人类的宝贵财富 ,如美国典型培养物保藏中心 (AmericanTypeCultureCollection ,ATCC) ,欧洲动物细胞培养物保藏中心 (EuropeanCollectionofAnimalCellC     

The time course of Akt and ERK activation on XIAP expression in HEK 293 cell line

The cell growth is controlled by the interaction of survival and cell growth arrest pathways as well as apoptosis mechanisms which determine the outcome of cell faith as proliferation or apoptosis. In this study, we have studied the activity of survival pathways, i.e., Akt and ERK1/2 with regard to XIAP (inhibitor of apoptosis) in serum starved and stimulated conditions. The HEK-293 cells were cultured in RPMI + 10% FBS. The cells were serum starved by switching to medium with 1% FBS for 24 h and serum stimulated by changing the medium to 10% FBS following serum starvation. The expression of p-Akt, p-ERK, Akt, ERK and XIAP was studied in various time points using western blot. The apoptosis was evaluated by DNA condensation using Hoechst 33258 and Caspase-3 assay. In serum starved condition expression of p-Akt and XIAP is very low. Serum stimulation increases p-Akt and p-ERK within 5 min and sustains a high level for 30 min. The expression of total Akt and ERK1/2 has not changed significantly for 24 h. XIAP expression starts at 6 h after serum stimulation, reaches to maximum level at 12 h and decreases to baseline within 24 h. Furthermore, serum starvation for 24 h does not induced apoptosis and DNA condensation. Taken together, the results indicate that serum activates Akt and ERK pathways earlier than XIAP expression. Furthermore, XIAP expression is low in serum starvation unlike p-ERK which suggests a survival role for ERK in serums starvation. The expression pattern of XIAP indicates induction by Akt and/or ERK activation which requires further studies.