An improved procedure for quantitating mitochondrial DNA in cultured mammalian cells

A new improved method that reproducibly measures small perturbations of mitochondrial DNA in populations of cells has been developed. It is based on first obtaining a cell count and then analyzing three aliquots of cells: one for total DNA per cell by fluorometry, one for total protein per cell and one for the amount of mitochondrial DNA per microgram of total cell DNA. To quantitate mitochondrial DNA, 0, 1, 2, and 3 nanograms of mouse mtDNA purified from a plasmid are added as internal standard DNA to four 1.0-microgram samples of purified total cell DNA containing an unknown amount of mitochondrial DNA (a sample set). Three sample sets are electrophoresed in an agarose gel devoid of ethidium bromide. Following Southern transfer to nitrocellulose and hybridization to purified 32P-labeled mouse mitochondrial DNA, an autoradiogram is prepared for use as a template to locate the mitochondrial DNA bands. These bands are cut out of the nitrocellulose filters, and their 32P-content is determined using a liquid scintillation counter. For each sample set, the counts per minute is plotted against the amount of mitochondrial DNA added. The plot is linear and the negative average of the values for the three intercepts on the x-axis yields the amount of nanograms of mitochondrial DNA per microgram of total cell DNA. The method is highly reproducible with a standard deviation of approximately 9 percent. The advantages of using this method over others that have been reported are discussed.     

An image processing system for digital chest X-ray images

This paper investigates the requirements for image processing of digital chest X-ray images. These images are conventionally recorded on film and are characterised by large size, wide dynamic range and high resolution. X-ray detection systems are now becoming available for capturing these images directly in photoelectronic-digital form. In this report, the hardware and software facilities required for handling these images are described. These facilities include high resolution digital image displays, programmable video look up tables, image stores for image capture and processing and a full range of software tools for image manipulation. Examples are given of the application of digital image processing techniques to this class of image.     

An image processing system for digital chest X-ray images

This paper investigates the requirements for image processing of digital chest X-ray images. These images are conventionally recorded on film and are characterised by large size, wide dynamic range and high resolution. X-ray detection systems are now becoming available for capturing these images directly in photoelectronic-digital form. In this report, the hardware and software facilities required for handling these images are described. These facilities include high resolution digital image displays, programmable video look up tables, image stores for image capture and processing and a full range of software tools for image manipulation. Examples are given of the application of digital image processing techniques to this class of image.     

Analysis of pigmentation in individual cultured plant cells using an image processing system

Anthocyanin accumulation in strawberry (Fragaria ananassa) cells cultured on a solid medium was monitored using an image-processing system that did not require direct sampling or destruction of the cells. Because of the intercellular heterogeneity of secondary metabolite production in plant cell cultures, the maximum metabolite concentration in individual cells is often more than 10 times higher than that of the average concentration. An image-processing based method enabled the growth and the pigmentation behavior of individual cells to be traced. Changes in the time courses of the anthocyanin content of individual cells differed from each other, although the average anthocyanin contents increased gradually with time in a batch culture. However, these various changing patterns in the anthocyanin content of each cell were independent of the cell cycle. In addition, image analysis revealed that the two cells just after cell division were almost identical to each other both in size and anthocyanin content. The proposed method which uses an image-processing system provides a useful tool for analyzing the secondary metabolism in individual cultured plant cells.     

A method for enucleating cultured mammalian cells

A method is described for enucleating cells which normally could not be enucleated due to their poor adhesion to the growth surface. The technique consists of linking ConA to the surface and then applying the cells. This results in cell adhesion firm enough to withstand the centrifugal forces necessary to enucleate. The method has been applied to fibroblastic, epithelioid and lymphoid cell lines.     

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.     

A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

A fluorescent assay of proteinases in cultured mammalian cells

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.