The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step.

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp.

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA–oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.

This report describes a novel approach to the detection of acetylcholine using DNA aptamers. Aptamers were developed by eight rounds of acetylcholine affinity column chromatography and polymerase chain reaction (PCR) amplification. Sequences from rounds 5 and 8 were screened by colorimetric enzyme-based microtiter plate assays and found to bind acetylcholine and related compounds, but not unrelated compounds. One of the highest affinity aptamers, designated ACh 6R, was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. Using Neuro-2a murine neuroblastoma cells induced to differentiate in the presence of 1 μM all-trans-retinoic acid for 5–7 d, ACh 6R detected cholinergic cells by both the peroxidase and fluorescence methods. Unrelated DNA aptamers did not stain the cells using either method. Fixation with cold 2% paraformaldehyde was compared to cold alkaline allyl alcohol plus glutaraldehyde for immobilization of acetylcholine in situ and appeared to enable detection of greater numbers of cholinergic cells, although differences in levels of differentiation may have been a factor as well. Acetylcholine generally appeared to be distributed throughout the differentiated Neuro-2a cell bodies. However, in some cells, punctate staining along neurite outgrowths and near the termini of cellular processes suggested detection of acetylcholine in discrete vesicles.

This study modeled, in vitro, the potential effect of conjugative (phase II) metabolism on the cytoprotective capacity of fruit flavonoids against oxidative stress. Flavonoid aglycones were compared with their corresponding isomeric mixtures of glucuronides for their ability to enhance the survival of cultured human Jurkat T and neuroblastoma cells stressed with hydrogen peroxide. Various polyphenolic compounds were tested as substrates in vitro for an ovine liver glucuronyl transferase preparation. Flavonoids and their glycoside derivatives were found to be good substrates, whereas phenolic acids were either poor or nonsubstrates. Five common flavonoids were glucuronidated to prepare mixtures for bioassay testing. Glucuronidation generally weakened the cytoprotective capacities of flavonoids (in the presence of H₂O₂), but some compounds were weakened much more than others. The concentration that halved cell death was well below 0.5 μM for most flavonoids tested, but glucuronidation increased median effective concentration values to a range of 1–16 μM. This compares with the generally accepted physiological range (0.1–10 μM) for circulating dietary polyphenolics detected in the body. Therefore, some flavonoids may retain a reduced cytoprotective capacity in vitro, after glucuronidation, whereas others may be effectively inactivated.

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration–viability curve. Results show that cell survival is significantly higher in cooled cells (T<22° C) than in non-cooled cells (T=37° C), but no significant differences are visible between T=10° C and T=22° C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided.

Osteosarcoma is the most common form of primary bone cancer. In this study, we established a human osteosarcoma cell line (OS 99-1) from a highly aggressive primary tumor. G-banding karyotype analysis demonstrated a large number of clonal abnormalities, as well as extensive intercellular heterogeneity. Through the use of immunologic, molecular, and biochemical analyses, we characterized protein and gene expression profiles confirming the osteogenic nature of the cells. Further evaluation indicated that OS 99-1 cells maintain the capacity to differentiate in an in vitro mineralization assay as well as form tumors in the in vivo chicken embryo model. This cell line provides a useful tool to investigate the molecular mechanisms contributing to osteosarcoma and may have the potential to serve as a culture system for studies involving bone physiology.

Summary The effect of cortisol on calcium (Ca² ) transport across cultured rainbow trout gill epithelia composed of both pavement cells (PVCs) and mitochondria-rich cells (MRCs) was examined. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), cortisol had subtle effects on gill epithelial preparations. Both control and cortisol treated epithelia exhibited Ca² influx and efflux rates (measured radioisotopically using ⁴⁵Ca) that were approximately balanced, with a slight inwardly directed net Ca² flux. Ussing flux ratio analysis indicated active Ca² transport in the inward direction across epithelia bathed symmetrically regardless of hormone treatment. In contrast, under asymmetrical conditions (freshwater apical/L15 media basolateral) control epithelia exhibited active Ca² transport in the outward direction (basolateral to apical) throughout experiments conducted over a 24-h period, whereas cortisol-treated preparations exhibited active transport in the inward direction (apical to basolateral) during the early stages of an asymmetrical culture period (e.g., T0–6 h) and passive transport during the later stages (e.g., T18–24 h). When soft freshwater (with tenfold lower [Ca² ]) was used for asymmetrical culture instead of freshwater, control epithelia developed outwardly directed active Ca² transport properties, whereas cortisol-treated preparations did not. The results of this study support a hypercalcemic role for cortisol in rainbow trout and demonstrate that treating cultured gill epithelia composed of both PVCs and MRCs with cortisol can stimulate active Ca² uptake under circumstances that more closely resemble natural conditions for fish gills (i.e., freshwater bathing the apical side of the epithelium).

Identification of cellular and morphological changes in myoblasts during three-dimensional (3D) culture may provide novel insight into skeletal muscle morphogenesis. One particular morphological change that occurs during the transition from monolayer culture to the 3D environment is the appearance of cytoplasmic projections (podia). The purpose of these studies was to determine if: (1) 3D culture increased podia formation in single cells, and (2) podia were F-actin dependent. C2C12 cells were grown in 3D conditions using a rotary cell culture system (RCCS) for 3, 6, and 9 h, fixed, and stained. Analysis of confocal images revealed that podia were significantly more numerous on RCCS cultured cells than those on suspension controls. Further, the podia of RCCS cultured cells decreased in number and increased in length during the time intervals examined. RCCS cultured cells showed no significant changes in viability, Annexin V staining, and activated Caspase 3 expression over time. In contrast, significant decreases in viability of suspension controls occurred. The application of 2 μM Latrunculin A (Lat A), an actin depolymerizing agent, significantly reduced the number of cells with podia. The number of cells with podia recovered with Lat A removal. Changes in viability and apoptosis markers were not significant during Lat A application or washout experiments. These observations reveal that: (1) culture conditions in the RCCS increase the quantity of podia formation; (2) these podia increase in length with time; and (3) F-actin plays a role in podia formation.

Numerous previous studies demonstrated that gene expression was influenced by histone modifications. However, little information is available about the relation of histone methylation with embryonic gene expression. Here, we examine the significance of histone H3 dimethyl-lysine 4 (H3K4me2) during mouse zygotic genome activation (ZGA) by inhibiting demethylation with the specific histone H3 lysine 4 demethylase inhibitor bisguanidine 1c (1c). A 1c treatment of one-cell embryos did not significantly affect the level of eIF-4C transcripts but did affect Oct4 levels by the two-cell stage. Furthermore, 1c treatment significantly inhibited cleavage of the embryos to the four-cell stage (from 82.7% to 18.2%), and the inhibitory effect was identified to be irreversible. These results suggest that histone methylation may be closely correlated with the formation of a transcriptionally repressive state during ZGA and that the repressive state actually dictates the appropriate pattern of gene expression required for further development.

The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phospha-tase (ALP) activity decreased in infected cells.