A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis

Establishing flow cytometric DNA analysis as a clinical routine procedure requires adequate and proven guidelines, by which the data can be obtained and interpreted to directly influence management of the individual patient with a specific neoplasm. The present paper is intended as a contribution to such guidelines, of which only fragments are available today. We have previously described a system of methods, designed for routine flow cytometric DNA analysis. In the present status report our experience, based on approximately 18,000 samples (clinical and experimental) is summarised. Sample acquisition with fine-needle aspiration, storage at -80 degrees C, internal standardization by chicken (CRBC) and trout red blood cells (TRBC), staining with propidium iodide (PI), and analysis in the flow cytometer is recapitulated, with emphasis on previously unpublished aspects. The method of statistical analysis which has an integrating role is described in some detail. A lack of linearity between channel number and DNA content was determined experimentally, and the coefficient of variation (CV) was found to decrease with increasing channel number. The corrections in the algorithm of deconvolution made necessary by these findings are fundamental for estimating the end results. The zero point adjustment and procedures for changing from one batch of standards to another are described. A systematic approach to interpretation of DNA histograms is attempted and illustrated by data from clinical specimens of malignant lymphoma, breast cancer, small cell lung cancer, cancer of the oral cavity, and bladder cancer. Some problems are still unsolved and visual inspection is required to determine if the quality of the individual histogram is satisfactory. Inspection of the fluorescence/light scatter dot-plot provides additional information for the recognition of artifacts. The results stress that good quality DNA histograms with as small CVs as possible are important for interpretation of the data. It is essential that statistical methods are employed to extract the key end-point results. These are the number of subpopulations and their relative representation, and for each subpopulation the DNA index (DI) and the fractions of cells in the cell cycle phases. For the DNA data to have any rationally based impact on clinical decision making, it must be demonstrated that they have an independent prognostic value. Strategies for final evaluation are discussed. Multicenter trials on fresh material, to accrue quickly the number of patients necessary for firm conclusions, are suggested.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Differences in tetranectin immunoreactivity between benign and malignant breast tissue

Summary Tetranectin (TN) is a human, plasminogen kringle 4 binding plasma protein with ubiquitous cellular distribution and lectin-like characteristics. By means of the peroxidase-antiperoxidase staining technique a polyclonal and a monoclonal antibody were used to demonstrate TN within the intracellular as well as the extracellular compartment of invasive breast carcinoma. Whereas cell associated TN was universal showing only quantitative differences depending of the growth pattern of the tumor, 78 of 133 tumors displayed TN extracellularly as well. The occurrence of this stromal TN immunoreactivity was closely associated with desmoplasia, recognized morphologically by an increase in fibroblastic cells and immunohistochemically by an intense staining for the connective tissue glycoprotein fibronectin (FN). Benign breast tissue displayed a universal, intense cytoplasmic but no extracellular reaction for TN, with the exception of rare foci of granulation tissue and around dilated cysts. Functional studies have shown that human embryonal lung fibroblasts increase their release of TN to the growth medium upon stimulation. The presence of TN extracellularly within fibroblast-rich foci of desmoplasia (and granulation tissue) suggests that a similar increased release of the protein takes place in vivo during active states. Desmoplasia has been found to have a protective effect on tumor cell propagation and metastasis in a murine model. The molecular interactions, which are responsible for this effect, are undoubtedly complex. However, TN may, by its specific binding to kringle 4 of plasminogen and its high affinity for sulphated polysaccharides, add to the understanding of how plasminogen activation is modulated at the local extracellular level.     

Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects

Immobilized a-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.     

The fifteenth- and twentieth-century colonization of the Basin of Mexico by the Great-tailed Grackle (Quiscalus mexicanus)

Historical evidence indicates that Great‐tailed Grackles colonized the Basin of Mexico from the Gulf Coast lowlands in the fifteenth century. They were probably assisted by an intentional introduction, but colonization succeeded because of anthropogenic habitat alterations over the previous two centuries. During the Colonial period, grackles withdrew from the Basin, only to recolonize it in recent decades. This withdrawal was also due probably to changes in land use, including drainage of much of the water from the Basin's lakes.     

Role of pagL and lpxO in Bordetella bronchiseptica Lipid A Biosynthesis

PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

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