Long term culture of mouse hybridoma HB8852 cells in a protein free medium

Summary We developed a protein free medium based on ERDF medium containing 80 M FeSO₄, ethanolamine and selenite. Although this medium contained neither transferrin nor insulin, the medium could support long term culture of mouse hybridoma HB8852 without any significant changes in antibody production.     

Properties of the telomeric DNA-binding protein from Oxytricha nova

Telomeres of Oxytricha macronuclear DNA exist as discrete DNA-protein complexes. Different regions of each complex display characteristic DNA-protein interactions. In the most terminal region, binding of a 43- and a 55-kDa protein to the telomeric DNA appears to account for all the DNA-protein interactions that can be detected by chemical and nuclease footprinting. We have used gradient sedimentation and protein-protein cross-linking to establish that the 43- and 55-kDa proteins are subunits of a heterodimer. Both subunits are very basic, which is unexpected considering the resistance of the DNA-protein interaction to high concentrations of salt. It is extremely difficult to dissociate the two subunits either from telomeric DNA or from each other. Even after extensive treatment of protein preparations with nuclease, a fragment of the 3' tail from macronuclear DNA remains bound to the protein. A wide range of conditions was screened for dissociation of the subunits from the DNA and/or from each other. Dissociation was only obtained by using conditions that caused some inactivation of the DNA-binding capacity of the protein. The use of reagents that covalently modify sulfydryl groups during the purification procedure facilitates preparation of telomere protein with full DNA-binding activity.     

DNA-binding specificity of a chromatin non-histone protein fraction of HeLa cells.

The DNA-binding site of a previously characterized non-histone chromosomal protein antigen(s) from HeLa cells was investigated for its species specificity. Treatment with large amounts of micrococcal nuclease abolishes immunoactivity, which can then be recovered by the subsequent addition of human or HeLa DNA to reconstitute the immune complex. Neither rat nor calif DNA exhibits this property, but DNA from monkey cells gives considerable activity. The antigen is not, however, detectable in monkey chromatin.     

Growth of cells on solid culture medium

Summary For precise experiments with yeast (or other) cells stationary populations are produced by growth on the surface of a solid nutrient medium. The energy supply to these cells is well known from a former publication. The oxygen supply during growth is analysed here in detail. Different types of cell populations can be produced in this way dependent on the thickness of nutrient medium.If such cells are transferred into a liquid buffer solution cell multiplication can be initiated without any nutrient flux into the cell. This new type of initiation of the cell cycle of G₁-cells has to be distinguished from the usual initiation by nutrient supply and from the mechanism of meiotic cell division. The dependence of this cell growth on cell volume, pH-value, oxygen concentration and osmotic pressure is analysed and possibilities to avoid this kind of cell multiplication reaction are discussed.     

Primary culture of adrenal medullary chromaffin cells in a chemically defined medium

Primary cultures of bovine adrenal medullary chromaffin cells have been maintained in the absence of serum for up to 3 weeks. Chromaffin cell catecholamine and protein contents were maintained, after an initial loss at the time of plating, as were the functional properties of the cells, including nicotine-evoked, calcium-dependent catecholamine secretion. Important factors in the maintenance of chromaffin cells included the cell plating density, frequency of medium replacement, and extent of medium replacement, suggesting ‘conditioning’ of the culture medium. Initially, serum was used for the first 48 h of culture, but pretreatment of the tissue culture plates with fibronectin allows complete elimination of serum from the plating medium. The establishment of serum-free culture conditions for chromaffin cells should facilitate the study of their cell biology and biochemistry.     

Hepatitis C virus core protein is efficiently released into the culture medium in insect cells

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.     

Total purification of a DNA-dependent ATPase and of a DNA-binding protein from human cells

We have purified to near homogeneity the major DNA-dependent ATPase from human cells. The pure enzyme has a mol. wt. of 68,000 and a minimum specific activity of approximately 150 U/mg. When the properties of the pure enzyme are compared with those of a less purified preparation, significant differences are observed both in structure and in function. These can be ascribed to the interaction of the ATPase with a DNA-binding protein (mol. wt. 28,000) that we can also purify to near homogeneity from the same cells and which is present in the less purified preparations of the ATPase. The ability of the less purified ATPase to stimulate DNA polymerase alpha in helicase fashion is probably due to the presence of the DNA-binding protein.     

BRL条件培养基在ES细胞培养中的应用方法探讨

目的：探讨布法罗大鼠肝细胞条件培养基（Buffalo rat liver cell conditioned medium,BRL）在ES细胞培养中的应用方法。方法：ES细胞复苏后分别培养在BRL条件培养基、小鼠胚胎成纤维细胞饲养层（mouse enbryonic fibroblast,MEF）及合并应用BRL条件培养基和MEF饲养层的环境中,通过细胞计数、拟胚体计数和ES细胞集落边缘细胞分化状态比较ES细胞在三种培养基中生长和分化差异。结果：与BRL组比较,MEF组和BRL＋MEF组细胞生长较快（P＜0.01）,ES细胞集落边缘分化细胞较少;MEF组和BRL＋MEF组无明显差异。结论：在复苏后早期阶段ES细胞培养中,不宜单独应用BRL条件培养基,须用MEF饲养层或合并应用BRL条件培养基和MEF饲养层。     

Functional diversity of polypeptides in primary culture supernatant of thymus epithelial cells

Polypeptide fractions A-C (M.W., 7 kd, 4.7 kd, and 3 kd) were obtained from the primary culture supernatant of thymus epithelial cells from Wistar rats by high-pressure liquid chromatography with a gel-filtration column. Changes in the mitogen responses of rat thymocytes and their subpopulations with addition of a fraction were studied. One subpopulation was rich in non-rosette-forming cells (non-RFCs), and the other was cortisone resistant thymocytes (CRTs). These subpopulations were incubated with a fraction for 24 hrs. before mitogen stimulation. Fractions A and C increased the response of the non-RFCs to concanavalin A (Con A) and that of total thymocytes and CRTs to phytohemagglutinin (PHA). Fraction B inhibited Con A and PHA response of total thymocytes and CRTs. Fraction B was cytotoxic toward total thymocytes and CRTs when viability was evaluated by [3H]uridine prelabelling. This cytotoxicity was suppressed by treatment with trypsin. Subfractions B3 and B4 obtained by reversed-phase column chromatography were cytotoxic toward CRTs. The effects of the fractions on thymocyte maturation were different, showing their functional diversity.     

Properties of barnacle photoreceptor cells in culture

1. Properties of median photoreceptor cells in cultured ocelli from the giant barnacle (Balanus nubilus) were compared in isolated ocelli, ocelli maintained with the supraesophageal ganglion, and fresh ocelli. 2. Cultured photoreceptor cells exhibited slight deterioration after 2-4 weeks. Cell bodies maintained their structure but apparently lost some dendrites. Electron micrographs revealed fewer rhabdomeres. Axons did not degenerate. 3. Intracellularly recorded responses to light in both cultured preparations were qualitatively normal with a small decrease in sensitivity and increase in input resistance. The waveforms of the light responses were normal. 4. The characteristic shadow reflex was maintained after 6 weeks.