Cell-type-specific determination of reactive oxygen species by flow cytometry.

BACKGROUND: Seminal leukocyte-generated reactive oxygen species may have a significant impact on sperm intracellular reactive oxygen species levels, therefore contributing to oxidative damage and consequent functional impairment of spermatozoa. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics. OBJECTIVE: To obtain seminal cell-specific, reactive oxygen species-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying reactive oxygen species overproduction (oxidative burst) from normozoospermic seminal samples. MATERIAL AND METHODS: Ejaculates gained by masturbation were obtained from patients in the framework of andrology consultations. The results published in this paper were generated from samples for which the attending physician requested spermatograms and seminal reactive oxygen species laboratory tests. Routine seminal analyses were performed according to World Health Organization guidelines. Samples were divided into normozoospermic "non-inflamed," and leukocytospermic groups. The semen was stained by 2',7'-dichlorodihydrofluorescein diacetate and the reactive oxygen species-related fluorescence signal and the percentage of reactive oxygen species-positive spermatozoa within the living population were quantified by flow cytometry. RESULTS: Reactive oxygen species-related mean fluorescence intensity was higher in both spermatozoa and leukocytes from leukocytospermic samples than in those from normozoospermic samples. Mean fluorescence intensity in spermatozoa was positively and linearly correlated with mean fluorescence intensity measured in leukocytes in both groups. DISCUSSION: The capacity of spermatozoa to generate reactive oxygen species is at least three log lower than that of granulocytes. The question is whether the reactive oxygen species-producing machinery of spermatozoa is capable of causing autologous oxidative stress or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the reactive oxygen species production of leukocytes may have a significant impact on the overall reactive oxygen species levels measured in spermatozoa. CONCLUSION: Reactive oxygen species-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on reactive oxygen species mean fluorescence intensity measurement.

Preclinical characterization of CPL302-253, a selective inhibitor of PI3Kδ, as the candidate for the inhalatory treatment and prevention of Asthma.

Asthma is a common chronic inflammatory disease. Although effective asthma therapies are available, part of asthmatic population do not respond to these treatment options. In this work we present the result of development of CPL302-253 molecule, a selective PI3Kδ inhibitor. This molecule is intended to be a preclinical candidate for dry powder inhalation in asthma treatment. Studies we performed showed that this molecule is safe and effective PI3Kδ inhibitor that can impact many immune functions. We developed a short, 15-day HDM induced asthma mouse model, in which we showed that CPL302-253 is able to block inflammatory processes leading to asthma development in vivo.

Assessment of the Airway Smooth Muscle Relaxant Effect of Drotaverine.

BACKGROUND: Drotaverine, a type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, blocks the degradation of 3',5'-cyclic adenosine monophosphate. However, published receptor binding data showed that drotaverin also binds to the L-type voltage-operated calcium channel (L-VOCC). Based on these molecular mechanisms of action, a direct and indirect (by blocking the constrictor response) relaxant effect on airway smooth muscle can be predicted, which has not yet been assessed. SUMMARY: Accordingly, drotaverine and reference agents were tested both on the histamine-, methacholine-, or KCl-induced contraction response and on precontracted guinea pig tracheal preparations. It was found that drotaverine not only relaxed the precontracted tracheal preparations but also decreased mediator-induced contraction. These effects of drotaverine were concentration dependent, with a significantly higher potency on the KCl-induced response, than on either the histamine or methacholine induced one. A similar result was noted for nifedipine. The PDE inhibitor, theophylline, also relaxed the precontracted preparations but was ineffective on the mediator-induced contraction in a physiologically relevant concentration range. Moreover, theophylline did not show selectivity and was the least potent relaxant among the 3 tested molecules. Key Message: These results show that drotaverine is a more potent airway smooth muscle relaxant molecule than theophylline. This enhanced potency on relaxation and inhibition of the constrictor response, at least partly, may be explained by the combined L-VOCC blocking and PDE inhibitory potential of drotaverine.

Intercontinental migration pattern and genetic differentiation of arctic-alpine Rhodiola rosea L.: A chloroplast DNA survey.

Our study describes genetic lineages and historical biogeography of Rhodiola rosea a widely distributed arctic-alpine perennial species of the Northern Hemisphere based on sequence analysis of six chloroplast regions. Specimens of 44 localities from the Northern Hemisphere have been sequenced and compared with those available in the GenBank. Our results support the migration of the species into Europe via the Central Asian highland corridor, reaching the European Alpine System (EAS) and also the western European edge, the British Isles. The EAS proved to be an important center of genetic diversity, especially the region of the Eastern Alps and the Dolomites where signs of glacial refugia was observed. Apart from those of the EAS, a common lineage was detected along the Atlantic coast from the British Isles toward Scandinavia as well as Iceland and the eastern parts of North America. Accordingly, the British Isles represent a main link between the northern Atlantic and southern EAS lineages.

Potential L-Type Voltage-Operated Calcium Channel Blocking Effect of Drotaverine on Functional Models.

Drotaverine is considered an inhibitor of cyclic-3',5'-nucleotide-phophodiesterase (PDE) enzymes; however, published receptor binding data also support the potential L-type voltage- operated calcium channel (L-VOCC) blocking effect of drotaverine. Hence, in this work, we focus on the potential L-VOCC blocking effect of drotaverine by using L-VOCC-associated functional in vitro models. Accordingly, drotaverine and reference agents were tested on KCl-induced guinea pig tracheal contraction. Drotaverine, like the L-VOCC blockers nifedipine or diltiazem, inhibited the KCl-induced inward Ca2+- induced contraction in a concentration- dependent fashion. The PDE inhibitor theophylline had no effect on the KCl-evoked contractions, indicating its lack of inhibition on inward Ca2+ flow. Drotaverine was also tested on the L-VOCC-mediated resting Ca2+ refill model. In this model, the extracellular Ca2+ enters the cells to replenish the emptied intracellular Ca2+ stores. Drotaverine and L-VOCC blocker reference molecules inhibited Ca2+ replenishment of Ca2+-depleted preparations detected by agonist-induced contractions in post-Ca2+ replenishment Ca2+-free medium. Theophylline did not modify the Ca2+ store replenishment after contraction. It seems that drotaverine, but not theophylline, inhibits inward Ca2+ flux. The addition of CaCl2 to Ca2+-free medium containing the agonist induced inward Ca2+ flow and subsequent contraction of Ca2+-depleted tracheal preparations. Drotaverine, similar to the L-VOCC blockers, inhibited inward Ca2+ flow and blunted the slope of CaCl2-induced contraction in agonist containing Ca2+-free medium with Ca2+-depleted tracheal preparations. These results show that drotaverine behaves like L-VOCC blockers but, unlike PDE inhibitors using L-VOCC associated in vitro experimental models.

Interaction of SSR161421, a novel specific adenosine A(3) receptor antagonist with adenosine A(3) receptor agonists both in vitro and in vivo.

A novel adenosine A(3) receptor antagonist (SSR161421) was characterized by both receptor binding assays and pharmacological tests. Binding studies on cloned human adenosine receptors showed that SSR161421 has high affinity for adenosine hA(3) receptors (K(i)=0.37 nM) with at least 1000-fold selectivity compared to hA(1), hA(2A) and hA(2B) receptors. The receptor antagonist nature of SSR161421 was determined in a functional study on Chinese hamster ovarian cells (CHO) cells expressing human adenosine A(3) receptors. SSR161421 competitively antagonized the effect of 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) on cAMP production with a pA2 value in a luciferase reporter gene construct. In mice, intravenously administered SSR161421 inhibited the N6-(4-aminobenzyl)-adenosine-5'-N-methyl-uronamide dihydrochloride (AB-MECA) induced increase in plasma histamine levels (ED(50)=2.0mg/kg) and the Cl-IB-MECA evoked plasma extravasation (ID(50)=2.9 mg/kg) and oedema formation (ID(50)=4.6 mg/kg) in mouse ear.

Evaluation of SSR161421, a novel orally active adenosine A3 receptor antagonist on pharmacology models.

The effects of a novel adenosine A(3) receptor antagonist, SSR161421, were examined on both antigen per se and adenosine receptor agonist-increased airway responses in antigen-sensitized guinea pigs. Adenosine (10(-5)M) and AB-MECA [N6-(4-aminobenzyl)-adenosine-5'-N-methyl-uronamide dihydrochloride] (10(-7)M) increased the antigen response up to 61 ± 3.0% and 88 ± 5.2% of maximal contraction, respectively. The agonists of adenosine A(1) and A(2) adenosine receptors NECA [1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-b-d-ribofuranuronamide-5'-N-ethylcarboxamidoadenosine], R-PIA [N(6)-R-phenylisopropyladenosine], and CGS21680 (10(-7)M) were ineffective. In vivo intravenous adenosine (600 µg/kg) and AB-MECA (30 µg/kg) increased the threshold antigen dose-induced bronchoconstriction by 214 ± 13.0% and 220 ± 15.2%, respectively. SSR161421 in vitro (IC(50)=5.9 × 10(-7)M) inhibited the AB-MECA-enhanced antigen-induced airway smooth muscle contractions and also in vivo the bronchoconstriction following either intravenous (ED(50)=0.008 mg/kg) or oral (ED(50)=0.03 mg/kg) administration in sensitized guinea pigs. Antigen itself could evoke tracheal contraction in vitro and bronchoconstriction in vivo in antigen-sensitized guinea pigs. SSR161421 (3 × 10(-6)M) decreased the AUC of the antigen-induced contraction-time curve to 20.8 ± 5.4% from the 100% control level. SSR161421 effectively reversed the antigen-induced bronchoconstriction, plasma leak and cell recruitment with EC(50) values of 0.33 mg/kg p.o., 0.02 mg/kg i.p. and 3 mg/kg i.p., respectively.

Comparative mapping between Medicago sativa and Pisum sativum.

Comparative genome analysis has been performed between alfalfa (Medicago sativa) and pea (Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.

Genetic mapping of the non-nodulation phenotype of the mutant MN-1008 in tetraploid alfalfa (Medicago sativa).

Abstract. Roots of the non-nodulating Medicago sativa mutant MN-1008 neither undergo root-hair curling, cortical cell division nor any of the early molecular events that accompany nodule initiation and development following rhizobial infection or treatment with Nod factor. These observations suggested that the mutation(s) impaired a pivotal function in Nod factor perception or in the signal transduction pathway. In this paper we show that the genetic lesion conditioning the recessive non-nodulation phenotype in the tetraploid alfalfa mutant MN-1008 can be localized to a single region on LG5 of the M. sativa genetic map. This conclusion is based on genetic analyses conducted at the tetraploid level, involving both segregation analysis and genetic mapping of the trait with respect to molecular DNA markers. The genetic mapping of the Nod(-) phenotype was performed in a segregating tetraploid F2 population, taking advantage of the availability of an advanced genetic map for diploid alfalfa. Two tightly linked flanking markers have been identified which will facilitate the physical mapping and cloning of the gene(s) that underlie(s) the non-nodulation phenotype.

Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells.

We have isolated a 483-bp-long full-length cDNA clone encoding a non-symbiotic hemoglobin called Mhb1, the first one found in alfalfa. This non-symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome. The Mhb1 mRNA was found only in the roots of alfalfa plants. The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment. The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture. The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm. A potential link to the nitric oxide signalling pathway is also discussed.

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system.

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf-/Fix-) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6kb genomic region in which mutations resulted in both Inf-/Fix- and K+-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level.

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn 1 and nn 2 )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F1 plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F2 population segregating the nn 1 and nn 2 genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F1 plants.

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.

Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity.

We have shown that a Rhizobium meliloti strain overexpressing nodulation genes excreted high amounts of a family of N-acylated and 6-O-sulfated N-acetyl-beta-1,4-D-glucosamine penta-, tetra-, and trisaccharide Nod factors. Either a C(16:2) or a C(16:3) acyl chain is attached to the nonreducing end subunit, whereas the sulfate group is bound to the reducing glucosamine. One of the tetrasaccharides is identical to the previously described NodRm-1 factor. The two pentasaccharides as well as NodRm-1 were purified and tested for biological activity. In the root hair deformation assay the pentasaccharides show similar activities on the host plants Medicago sativa and Melilotus albus and on the non-host plant Vicia sativa at a dilution of up to 0.01-0.001 microM, in contrast to NodRm-1, which displays a much higher specific activity for Medicago and Melilotus than for Vicia. The active concentration range of the pentasaccharides is more narrow on Medicago than on Melilotus and Vicia. In addition to root hair deformation, the different Nod factors were shown to induce nodule formation on M. sativa. We suggest that the production of a series of active signal molecules with different degrees of specificity might be important in controlling the symbiosis of R. meliloti with several different host plants or under different environmental conditions.

Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for D-glucosamine synthetase.

The nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the D-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS- mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied D-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. melioti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.