Combination of mRNA and protein microarray analysis in complex cell profiling.

UNLABELLED: This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.

Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.

This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.

Microarray analysis using a limited amount of cells.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

[Serum ferritin, its clinical significance and our personal experience]. / Sérový feritín, jeho klinický význam a nase skúsenosti.

The paper presents a survey of the information on serum ferritin and its clinical value along with the authors' own experience.

[Densitometric determination of dithiadenoxide in blood]. / Densitometrické stanovení dithiadenoxidu v krevní plazme.

The present paper describes a method of dithiadenoxide determination in blood plasma. Dithiadenoxide was reduced to dithiaden with zinc dust in acidified plasma. The solution was alkalified and dithiaden extracted into n-hexane and isolated on a thin layer of silica gel using the system chloroform--n-heptane-methanol (60:22:18). After spraying the plate with a solution of m-nitrophenyldiazoniumfluoroborate in acid medium, dithiaden stains were evaluated densitometrically in the absorption maximum of the reaction product at 565 nm (RF = 0.37). Detection limit of dithiadenoxide in the proposed method is 0.004 microgram in an applied amount.

[Densitometric determination of piroxicam in plasma]. / Densitometrické stanovení piroxicamu v plazme.

Piroxicam was extracted from acid medium into chloroform and isolated on a thin layer of silica gel with the use of the system chloroform - absolute ethanol - benzene - aqueous ammonia (20:15:15:0.5). The intensity of fluorescence of stains was measured densitometrically (an Hg lamp, a filter FL - 39, 330 nm). The limit of detection of the stain is 0.020 microgram/ml of plasma.

Interspecies pharmacokinetic scaling of metazosin, a novel alpha-adrenergic antagonist.

Based on pharmacokinetic data from mice, rats, and rabbits, the prediction of pharmacokinetics of intravenous metazosin in man has been performed. The correlations were based upon allometric scaling of plasma clearance and the volume of distribution at steady-state. A one-compartment body model approximating clinical pharmacokinetics fits well the elimination phase of subsequently measured metazosin concentrations in volunteers. Fitting human pharmacokinetic data to allometric equations enabled us to superimpose pharmacokinetic curves from different species.

Pharmacokinetics and pharmacodynamics of conventional and controlled-release formulations of metipranolol in man.

The pharmacokinetics and beta-adrenoceptor blocking effects of conventional and sustained-release metipranolol have been studied in 6 healthy male volunteers given a single oral dose of 40 mg. Plasma drug concentrations determined by TLC and a radioreceptor assay, and the inhibition of exercise-induced tachycardia, were monitored for 48 h. Relevant amounts of active metabolites other than deacetylmetipranolol were not found. Compared to conventionally formulated metipranolol, the controlled-release product had a prolonged mean residence time (10.7 vs 5.5 h), the peak drug concentration was halved and the time to peak drug concentrations was delayed. Relatively constant plasma concentrations (cideal = 6.5 ng/ml) and a clinically significant reduction of exercise-induced tachycardia were maintained throughout a 24 h dosing interval. An individual deacetylmetipranolol plasma concentration-effect relationship was evaluated using the Emax model. Mean parameters were Emax 26% and C50 2.9 ng/ml.