An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

[Assessment of microbiological quality of food served in dining rooms of private enterprises]. / Determinación de la calidad microbiológica de alimentos servidos en comedores de empresas privadas.

The aim of this study was to evaluate foods microbiological quality in food-service establishments. A total of 620 food samples were obtained from four establishments and analyzed for aerobic mesophilic (AM), yeasts, moulds, Staphylococcus aureus and Escherichia coli counts and the presence of Salmonella spp. Drinking water, equipment, surfaces, environment and food handlers were also sampled. E. coli was found in raw vegetables (76.2%), cooked vegetables (15.2%), beef and pork (15.9%), poultry (16.7%), fish (11.8%), desserts (27.3%), equipments (57.9%), surfaces and environment (53.6%) and in 21.9% of food handlers. Survey results were compared with the recommended maximum microbial levels. Our results demonstrate the need for the adoption of more effective hygienic measures in this kind of establishment in order to avoid any risk to consumers.

Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1.

Native LDL (n-LDL) increased human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL-treated ECs bound nearly threefold more phorbol myristate acetate (PMA)-stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti-cellular adhesion molecule-1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL-treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n-LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule-1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis.

Temporal restriction of neural crest development in vitro: changes in the adrenergic differentiation of neural crest clusters in the presence and absence of an overlay of reconstituted basement membrane-like matrix.

Neural crest clusters were isolated after 18 or 42 h of growth in suspension and plated on a fibronectin substrate in the presence or absence of an overlay of a reconstituted basement membrane-like (RBM) matrix. After 1 week in vitro, cultures of 18-h clusters grown with the RBM gel overlay exhibited a 60-fold increase in the number of catecholamine-positive cells, while the number of melanocytes was decreased to one-quarter the control value. In contrast, clusters isolated after 42 h in suspension developed few adrenergic cells in the presence or absence of the RBM gel overlay. Melanocyte cell number was not altered in 42-h clusters grown in the presence of the RBM gel overlay. Total cell number after 1 week in vitro was similar under all conditions for both 18- and 42-h clusters. The distribution of tyrosine hydroxylase-immunoreactive cells among the experimental conditions was similar to that of catecholamine-positive cells. The presence of the neural tube was not necessary to generate the different developmental properties of clusters isolated at 18 h versus 42 h. These results show that the RBM gel can enhance adrenergic cell development and inhibit melanogenesis in young neural crest cluster cultures. Also, they demonstrate that the capacity of these neural crest cluster cell populations to differentiate and to respond to environmental cues changes as a function of time. These results are compatible either with a model in which the survival of adrenergic precursors within the clusters declines with time or one in which the adrenergic precursors switch their fate to another phenotype.