Identification of new TRAP markers linked to chlorophyll content, leaf senescence, and cell membrane stability in water-stressed wheat

In order to identify target region amplification polymorphism (TRAP) markers linked to three physiological traits in wheat (Triticum aestivum L.), the segregating F₄ population from the cross between drought-sensitive (Yecora Rojo) and drought-tolerant (Pavon 76) genotypes was made. The parents and 150 F₄ families were evaluated phenotypically for drought tolerance using two irrigation treatments [2.5 and 7.5 m³(H₂O) m^?2(soil)]. Using 40 different TRAP primer combinations tested for polymorphism in parental and F4 family genotypes, the results revealed that quantitative trait locus (QTL) for chlorophyll content was associated with TRAP 5, TRAP 14, and TRAP 20 and explained 18, 16, and 23 % phenotypic variation, respectively. The genetic distance between chlorophyll content QTL and TRAP 5, TRAP 14, and TRAP 20 were 12.3, 19.8, and 13.6 cM, respectively. QTL for flag leaf senescence was associated with TRAP 2, TRAP 3, TRAP 15, and TRAP 16 and explained 33, 27, 28, and 23 % phenotypic variations, respectively. The genetic distance between flag leaf senescence QTL and TRAP 2, TRAP 3, TRAP 15, and TRAP 16 were 9.4, 14.7, 18.1, and 17.3 cM, respectively. QTL for cell membrane stability was associated with TRAP 8, TRAP 9, and TRAP 37 and explained 27, 30, and 24 % phenotypic variation, respectively. The markers TRAP 8, TRAP 9, and TRAP 37 had genetic distances of 17.0, 10.0, and 9.0 cM, respectively. Therefore, these TRAP markers can be used in breeding for drought tolerance in wheat.     

DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells

DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24–72 h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.     

The systematic position of Paraspathidium Noland, 1937 (Ciliophora,Litostomatea?) inferred from primary SSU rRNA gene sequences and predicted secondary rRNA structure

Traditionally the unusual ciliate Paraspathidium has been regarded as a gymnostome haptorid (Litostomatea) based on its morphological features. In order to test this placement, the small-subunit (SSU) rRNA gene was sequenced for two isolates of Paraspathidium apofuscum and phylogenetic trees were constructed. Furthermore, the putative structure of the variable regions 2 and 4 of the SSU rRNA gene were predicted and compared with those of other ciliates. Our analyses of SSU rRNA gene sequences revealed (i) a clear separation of Paraspathidium from the haptorids and indeed the class Litostomatea, rejecting its systematic position based on morphological characters and (ii) an equally clear association with the assemblage comprising the classes Plagiopylea and Prostomatea. Putative secondary structures of the variable regions 2 and 4 of Paraspathidium are similar to those of the plagiopyleans and prostomateans but differ from the hapotrids in Helix 10, Helix E10-1 and Helix E23-5. Taken together, these results support the placement of Paraspathidium close to prostomateans and plagiopyleans, or even as a distinct group possibly at ordinal rank, within the class Plagiopylea.     

Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: Synthesis,biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a–b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a–b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4–5.8% range). In comparison, the percentage NO released was higher (3.1–8.4% range) when these nitrate prodrugs were incubated in the presence of l-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure–activity relationship data acquired showed that compounds having a MeSO₂ COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H₂NSO₂ substituent. Compounds having a MeSO₂ COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED₅₀ = 132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED₅₀ = 118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED₅₀ = 128.7 mg/kg po) but lower than ibuprofen (ED₅₀ = 67.4 mg/kg po).     

Simultaneous Determination of Cd, Pb, Cu, Zn, and Se in Human Blood of Jordanian Smokers by ICP-OES

A total of 73 blood samples (56 from smokers and 17 from nonsmokers) were collected to determine the concentrations of selected heavy metal in the whole blood of smokers and nonsmokers living in and around the city of Amman, Jordan. Analysis of heavy metals in the whole blood samples of various groups took in consideration the number of cigarettes smoked per day. The analysis of blood samples was carried out using inductively coupled plasma optical emission spectrometry. This study aimed to evaluate the blood metal levels in smokers and nonsmokers and to assess the influence of smoking cigarettes on blood metal levels. The results were compared with those from a control group. The results indicated that the average concentrations of cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), and selenium (Se) were 0.0313, 0.344, 2.328, 3.214, and 0.332 mg/L, respectively. Statistical analysis of results indicated that these average concentrations were significantly higher compared with the average concentrations in nonsmokers (P < 0.05). Moreover, the correlations between blood metal and other blood metal levels in smokers, the correlations between blood metal and other blood metal levels in nonsmokers, and the correlations between blood metal concentration in smokers and its concentration in nonsmokers were calculated. The standard reference material (blood serum National Institute of Standards and Technology 1598) and the quality control were used to validate the reliability of the method used for the estimation of heavy metals in blood samples. Results revealed that there was an agreement between the certified values and the measured values.     

TMKP1 is a novel wheat stress responsive MAP kinase phosphatase localized in the nucleus

The regulation of plant signalling responses by Mitogen-Activated Protein Kinases (MAPKs)-mediated protein phosphorylation is well recognized. MAP kinase phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes. We report here the identification and the characterization of TMKP1, the first wheat MKP (Triticum turgidum L. subsp. Durum). Expression profile analyses performed in two durum wheat cultivars showing a marked difference in salt and drought stress tolerance, revealed a differential regulation of TMKP1. Under salt and osmotic stress, TMKP1 is induced in the sensitive wheat variety and repressed in the tolerant one. A recombinant TMKP1 was shown to be an active phosphatase and capable to interact specifically with two wheat MAPKs (TMPK3 and TMPK6). In BY2 tobacco cells transiently expressing GFP::TMKP1, the fusion protein was localized into the nucleus. Interestingly, the deletion of the N-terminal non catalytic domain results in a strong accumulation of the truncated fusion protein in the cytoplasm. In addition, when expressed in BY2 cells, TMPK3 and TMPK6 fused to red fluorescent protein (RFP) were shown to be present predominantly in the nucleus. Surprisingly, when co-expressed with the N-terminal truncated TMKP1 fusion protein; both kinases are excluded from the nuclear compartment and accumulate in the cytoplasm. This strongly suggests that TMKP1 interacts in vivo with TMPK3 and TMPK6 and controls their subcellular localization. Taken together, our results show that the newly isolated wheat MKP might play an active role in modulating the plant cell responses to salt and osmotic stress responses.     

Noninvasive determination of upper airway resistance and flow limitation.

We have shown that a polynomial equation, FP = AP3 + BP2 + CP + D, where F is flow and P is pressure, can accurately determine the presence of inspiratory flow limitation (IFL). This equation requires the invasive measurement of supraglottic pressure. We hypothesized that a modification of the equation that substitutes time for pressure would be accurate for the detection of IFL and allow for the noninvasive measurement of upper airway resistance. The modified equation is Ft = At3 + Bt2 + Ct + D, where F is flow and t is time from the onset of inspiration. To test our hypotheses, data analysis was performed as follows on 440 randomly chosen breaths from 18 subjects. First, we performed linear regression and determined that there is a linear relationship between pressure and time in the upper airway (R2 0.96 +/- 0.05, slope 0.96 +/- 0.06), indicating that time can be a surrogate for pressure. Second, we performed curve fitting and found that polynomial equation accurately predicts the relationship between flow and time in the upper airway (R2 0.93 +/- 0.12, error fit 0.02 +/- 0.08). Third, we performed a sensitivity-specificity analysis comparing the mathematical determination of IFL to manual determination using a pressure-flow loop. Mathematical determination had both high sensitivity (96%) and specificity (99%). Fourth, we calculated the upper airway resistance using the polynomial equation and compared the measurement to the manually determined upper airway resistance (also from a pressure-flow loop) using Bland-Altman analysis. Mean difference between calculated and measured upper airway resistance was 0.0 cmH2O x l(-1) x s(-1) (95% confidence interval -0.2, 0.2) with upper and lower limits of agreement of 2.8 cmH2O x l(-1) x s(-1) and -2.8 cmH2O x l(-1) x s(-1). We conclude that a polynomial equation can be used to model the flow-time relationship, allowing for the objective and accurate determination of upper airway resistance and the presence of IFL.     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

Synthesis and bioevaluation of N-(arylalkyl)-homospermidine conjugates

N1-(Arylalkyl)homospermidines (1c-1f) and terminally piperazine-substituted homospermidine conjugates (2a-2e) were synthesized and evaluated for cytotoxicity in mouse leukemia L1210, alpha-difluoromethylornithine (DFMO)-treated L1210, melanoma B16, spermidine (SPD)-treated B16, and HeLa cell lines. Results demonstrated that homospermidine was a more effective vector than piperazine-substituted homospermidine in ferrying diverse arenes into cells via the polyamine transporter. The leading compound, 9-anthracenemethyl-homospermidine (1a), was shown to induce apoptosis in B16 cells and IL-3 dependent FL5.12A pro-B cells. The novel conjugate 4-biphenylmethyl-homospermidine (1e) could also induce apoptosis. However, it exhibited different effect on the cell cycle of B16 cells compared to 1a.