A single-tube flow cytometric procedure for enhancing the diagnosis and prognostic classification of patients with myelodysplastic syndromes.

INTRODUCTION: We created a simple and effective flow cytometry scoring system (FCSS) for suspected Myelodysplastic syndromes (MDS) samples and evaluated its diagnostic and prognostic potential. METHODS: Besides evaluating the four parameters suggested by Ogata, we investigated erythroid precursors and mast cells. We evaluated the six-parameter FCSS in a four-color setting (test cohort: 51 patients; 25 controls), then we implemented it into an eight-color setting and tested it on a validation cohort of patients with MDS (n=31). RESULTS: When we compared MDS cases to non-MDS samples in the test cohort, we detected significant differences regarding not only the four major parameters but also two additional ones, namely CD71 rCV% of erythroid precursors (P=.004) and mast cell percentage (MC%) (P=.001). The utilization of the modified six-parameter FCSS provided high sensitivity and specificity both in the four color (84% and 80%, respectively) and in the eight color (81% and 100%, respectively) setting, with an excellent discriminative power between MDS and non-MDS samples. Furthermore, we found significant difference in event-free survival between the risk groups based on the modified six-parameter FCSS (P=.001). CONCLUSION: We evaluated and validated a single-tube flow cytometric procedure for a simple six-parameter FCSS which has not only high diagnostic but also prognostic power.

Central nervous system, uterus, heart, and leukocyte expression of the LOXL3 gene, encoding a novel lysyl oxidase-like protein.

A BLASTN search using the mouse lor-2 cDNA identified three overlapping ESTs (AI752772, AA852888, and R55706) in the GenBank database. These expressed sequence tags were assembled into a contig of 3121 nucleotides with an open reading frame of 2262 bp. The encoded putative polypeptide of 754 amino acids presented all structural characteristics of the lysyl oxidase (LOX) enzyme family, a copper-binding site with four histidyl residues, the lysyl and tyrosyl residues known to be involved in LOX enzyme in the formation of the quinone cofactor and surrounding sequences, and the cytokine receptor-like domain. In addition, four scavenger receptor cysteine-rich (SRCR) domains were found in the N-terminal region of the protein. The gene encoding this new cDNA, which we have referred to as human lysyl oxidase-like 3 (humanLOXL3), has been mapped to chromosome 2p13.3, overlapping at its 3' end the HtrA2 serine protease gene. The structure of the humanLOXL3 gene was deduced from the BAC clone bac91a19 sequence and contained 14 exons. The expression pattern of this new member of the LOX gene family appears to be different from that of the LOX and LOX-like genes, as the central nervous system, neurons, and also leukocytes expressed humanLOXL3. A BLASTN search of the human EST database indicated the presence of ESTs, corresponding to alternative splice variants of LOXL3, that lacked exon 5 and exon 8. The putative resulting protein retained the region encoding the structural and functional elements of the amine oxidase but the second and fourth SRCR domains were truncated and the potential BMP-1 cleavage site was not present. The presence of domains unrelated to the traditional amine oxidase activity is a strong indication that humanLOXL3 might fulfill other functions in addition to intrinsic enzyme activity.

Characterization of the region encompassing the human lysyl oxidase locus.

A 46,823 bp region of human chromosome 5q23.1 encompassing the seven-exon lysyl oxidase gene was characterized at the primary sequence level. Approximately 17.4% of this region is comprised of repetitive elements. The gene colocalizes with microsatellite marker D5S467. It is flanked by two candidate nuclear matrix association regions (MARs). The 5' MAR centered at position 12,500 is of the AT-rich and curved DNA class. This is followed by a large CpG island containing fifty-seven putative regulatory elements which extend from just upstream of exon 1 to intron 2. The larger 3' MAR, spans position 35,050-39,750 and is characterized by a TG-rich kinked structure that also contains a topoisomerase II binding site. Based on these results model of the transcriptional regulation of the lysy/oxidase gene is presented.

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery.

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.

Long-lasting treatment with adenosine receptor antagonists: effects on hypoxic tolerance and vascular responsiveness.

In electrically driven myocardial preparations obtained from chronically methylxanthine-[aminophylline (APH) and 8-phenyltheophylline (8-PT)] or solvent(DMSO)-treated guinea pigs no differences were found in alteration of mechanical activity under hypoxia and reoxygenation. The vasoconstrictor effects observed after in vitro exposure of pulmonary arterial preparations (excised from either methylxanthine- or solvent-treated guinea pigs) to both noradrenaline and PGF2 alpha were also similar. In methylxanthine-treated vascular tissues, however, nitroglycerin and NO exerted more pronounced vasorelaxant effect than in specimens prepared from solvent-treated guinea pigs.

On the cardioprotective effect of adenosine.

In isovolumically perfused Langendorff heart preparations of guinea pigs adenosine-depending on the experimental protocol-more or less could prevent the hypoxia-induced decrease in myocardial adenosine triphosphate [ATP], creatine phosphate [CP], glycogen and increase in lactate, i.e. showed cardioprotection.

Regulation of purinoceptors in guinea pig pulmonary artery: functional evidences.

In isolated guinea pig pulmonary arteries (precontracted with 1 microM noradrenaline) N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist, exerted a concentration-dependent contraction, whereas 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective A1/A2 receptor agonist, in the presence of DPCPX (a highly selective A1 receptor antagonist), produced a concentration-related rapid relaxation. Pulmonary arteries obtained from guinea pigs treated with aminophylline (APH) or 8-phenyltheophylline (8-PT) for 10 consecutive days, displayed more pronounced contraction in response to CPA compared to those of solvent-treated animals. Relaxant action of NECA was, however, attenuated in arteries prepared from methylxanthine-treated guinea pigs. Opposite changes were found in vascular tissues excised from chronically dipyridamole(DP)-treated guinea pigs.

The effect of chronic diazepam treatment on hypoxia-induced alterations in functional activity of guinea pig myocardium.

The concentration-related sensitization of guinea pig left atrium to adenosine in the presence of diazepam is well established. It was found in our experiments that the cardiodepressive action of hypoxia is significantly enhanced by diazepam in the left atrial myocardium. In atrial preparations obtained from guinea pigs treated with diazepam for 10 days, the hypoxia-induced depression of myocardial contractility was not altered. These results indicate that diazepam-treatment does not impaire the hypoxic tolerance of myocardium.

Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs.

In guinea pig main pulmonary artery precontracted with noradrenaline, adenosine exerted an initial phasic contraction followed by a tonic contraction and a slow relaxation. After selective blockade by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX: 10 nM) of A1 receptors, adenosine only elicited a rapid relaxation. This initial response was characterized by use of adenosine (AR) and its analogues N6-cyclopentyl-adenosine (CPA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), 5'-N-ethyl-carboxamidoadenosine(NECA), N6-2-(4-aminophenyl) ethyl adenosine (APNEA) and 2-p-((carboxyethyl)-phenethylamino)-5'-carboxamidoadenosine (CGS 21 680). The order of potency of the adenosine analogues for purine-induced phasic contraction was CPA > R-PIA > NECA = APNEA > AR > CGS 21 680 suggesting the involvement of activation of A1 type adenosine receptors in the contraction phase. DPCPX antagonized the CPA-induced contraction with a pA2 = 9.27 +/- 0.26, but the Schild plot slope parameter was significantly lower than unity (0.58 +/- 0.09). In contrast, in electrically driven guinea pig atrial myocardium (a tissue reported to possess A1 receptors), the DPCPX-CPA antagonism was purely competitive (pA2 = 8.95 +/- 0.06; slope = 0.93 +/- 0.06). In the presence of 300 nM DPCPX, the rank order of potency for the purine-induced fast relaxation was NECA > CADO = AR > CGS 21 680 = R-PIA > CPA. The NECA- and adenosine-induced relaxation was influenced neither by 300 nM CP 66713 (an antagonist at A2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 microM NG-nitro-L-arginine: L-NOARG).(ABSTRACT TRUNCATED AT 250 WORDS)

[Cardioprotective effect of levcromakalim in isolated guinea pig heart preparations]. / A levcromakalim kardioprotektív hatásának vizsgálata izolált perfundált tengerimalac szíven.

The aim of present work was to study the cardioprotective effect of the potassium channel opener levcromakalim at a low concentration (0.3 microM) which does not depress heart rate and left ventricular developed pressure. For the determination of the protection against 10 or 20 min duration of hypoxia (95% N2 + 5% CO2) in isovolumically-perfused Langendorff heart preparations of guinea-pigs biochemical parameters (ATP, phosphocreatine, lactate and glycogen) were used. Under normoxic (95% O2 + 5% CO2) conditions 0.3 microM levcromakalim has not changed myocardial high energy phosphates, glycogen, lactate and the hypoxia induced decrease in ATP and phosphocreatine. The 10 min hypoxia induced increase in lactate and decrease in glycogen have--slightly but not significantly--been moderated and when the duration of hypoxic perfusion has lasted 20 min, this protection became significant. It is supposed that the preservation of glycogen stores induced by levcromakalim may contribute to the decrease of intracellular acidosis evoked by hypoxia. Therefore--in such experimental condition-, this mechanism may constitute the biochemical basis of cardioprotective effect of levcromokalim.