The effects of temperature and adrenergic agonists on cardiac myocytes of perch (Perca fluviatilis) in cell culture conditions

1. 1. Isolated cardiac myocytes of perch, Perca fluviatilis, were kept in culture conditions for 1–2 months at 12 or 22°C. In the culture most myocytes flattened, lost their spindle-shaped morphology, protruded pseudopod-like branches and many of them started visible contractions in 1–2 weeks and continued beating for several months. Myocytes did not divide in the sparse cell population used. Typical intracellular structures could be seen in electron micrographs still after 1–2 months, but the sarcoplasmic organization became gradually more irregular in the culture.

2. 2. Beat rates showed linear temperature relationship on the Arrhenius plot. Myocytes cultivated at 22°C showed higher frequencies and slightly less dependence on temperature than myocytes cultivated at 12°C (apparent activation energies (E_a) 86 and 107 kJ/mol, respectively).

3. 3. Temperature dependence of frequencies was related to the presence of added serum or adrenergic agonists: β-adrenergic agonists increased the frequencies and rendered the cells less dependent on temperature; apparent activation energy was 43 kJ/mol for isoprenaline or adrenaline and 108 kJ/mol for noradrenaline and control group.

4. 4. Heat tolerance was greater in myocytes cultivated at 22°C than in myocytes cultivated at 12°C, and the change in tolerance appeared in 12 h after the alteration of culture temperature and the increased tolerance was persistent after that.

5. 5. It is suggested, that the processes of quick heat-hardening and of slower but persistent heat resistance acclimation developed in these cells in culture conditions but not the capacity acclimation, which seems to be dependent on adrenergic regulation of beat rate.

New separation-free assay technique for SNPs using two-photon excitation fluorometry

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia™ TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot™ assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.     

Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

Phosphorescent porphyrin probes in biosensors and sensitive bioassays

Platinum(II) and palladium(II) complexes of porphyrins and related tetrapyrrolic pigments emit strong phosphorescence at room temperatures, which is characterized by long lifetimes falling into the sub-millisecond range and long-wave spectral characteristics. These features make the dyes useful as probes for a number of bioanalytical applications, particularly those employing time-resolved fluorescent detection. They can provide high sensitivity and selectivity, together with rather simple instrumental set-up. A number of analytical systems are now under development that are based on the use of phosphorescent porphyrin probes. Experimental results are presented on the following systems: (i) fibre-optic phosphorescence lifetime-based oxygen sensor on the basis of hydrophobic platinum-porphyrins and development of advanced sensing materials and prototype instrumentation; (ii) practical applications of the optical oxygen sensor, including a sensitive immunosensor that employs glucose oxidase labels, a rapid screening method for cell viability in microtitre-plate format, non-destructive measurement of oxygen in packaged foods and reagentless biosensors for metabolites (glucose, lactate); and (iii) the use of water-soluble platinum- and palladium-porphyrins as labels for ultra-sensitive time-resolved phosphorescence immunoassays.     

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

Genes involved in systemic and arterial bed dependent atherosclerosis--Tampere Vascular study

Background

Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed.

Methodology/Principal Findings

We characterized the genes generally involved in human advanced atherosclerotic (AHA type V–VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25).

Conclusions

This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.     

Particle classification from light scattering with the scanning flow cytometer.

BACKGROUND:The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. METHODS:The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. RESULTS:The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm. CONCLUSIONS:The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.