Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.     

Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses

The chloroplastic drought-induced stress protein of 32 kDa (CDSP32) is a thioredoxin induced by environmental stress conditions. To gain insight into the function of CDSP32, we applied two strategies to analyze its targets. First, using affinity chromatography with an immobilized CDSP32 active site mutant, we identified six plastidic targets of CDSP32. Three of them are involved in photosynthetic processes: ATP-ase gamma-subunit, Rubisco and aldolase. The three others participate in the protection against oxidative damage: two peroxiredoxins, PrxQ and the BAS1 2-Cys peroxiredoxin, and a B-type methionine sulfoxide reductase. Then, we developed a novel strategy to trap targets directly in leaf extracts. The method, based on co-immunoprecipitation using extracts from plants overexpressing Wt CDSP32 or CDSP32 active site mutant, confirmed the interaction in vivo between CDSP32 and the PrxQ and BAS1 peroxiredoxins. We showed that CDSP32 is able to form heterodimeric complexes with PrxQ and that the peroxiredoxin displays CDSP32-dependent peroxidase activity. Under photooxidative stress induced by methyl viologen, plants overexpressing CDSP32 active site mutant exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared with Wt plants and with plants overexpressing Wt CDSP32. We propose that the increased sensitivity results from trapping in planta of the targets involved in the protection against oxidative damage. We conclude that CDSP32, compared with other plant thioredoxins, is a thioredoxin more specifically involved in plastidic responses against oxidative stress.     

Dopaminergic transmission in STOP null mice

Neuroleptics are thought to exert their anti-psychotic effects by counteracting a hyper-dopaminergic transmission. Here, we have examined the dopaminergic status of STOP (stable tubule only polypeptide) null mice, which lack a microtubule-stabilizing protein and which display neuroleptic-sensitive behavioural disorders. Dopamine transmission was investigated using both behavioural analysis and measurements of dopamine efflux in different conditions. Compared to wild-type mice in basal conditions or following mild stress, STOP null mice showed a hyper-locomotor activity, which was erased by neuroleptic treatment, and an increased locomotor reactivity to amphetamine. Such a behavioural profile is indicative of an increased dopaminergic transmission. In STOP null mice, the basal dopamine concentrations, measured by quantitative microdialysis, were normal in both the nucleus accumbens and the striatum. When measured by electrochemical techniques, the dopamine efflux evoked by electrical stimulations mimicking physiological stimuli was dramatically increased in the nucleus accumbens of STOP null mice, apparently due to an increased dopamine release, whereas dopaminergic uptake and auto-inhibition mechanisms were normal. In contrast, dopamine effluxes were slightly diminished in the striatum. Together with previous results, the present study indicates the association in STOP null mice of hippocampal hypo-glutamatergy and of limbic hyper-dopaminergy. Such neurotransmission defects are thought to be central to mental diseases such as schizophrenia.     

Amplification of Nicotiana sylvestris mitochondrial subgenomes is under nuclear control and is associated with phenotypic changes

We have previously shown the presence in a Nicotiana sylvestris protoplast-derived plant of both a nuclear mutation conferring male sterility (ms4) and a mtDNA reorganisation, named U, characterised by the amplification of substoichiometric mtDNA fragments generated by recombination in the parent T mtDNA. Here we show by physical mapping that the recombining repeats are in direct orientation, thus generating two subgenomes both of which are amplified in the U organisation to the detriment of the parent molecule, and are maintained through sexual reproduction. The nuclear ms4 mutation is likely to have play a role in the shift in mitochondrial molecule equilibrium, as higher levels of recombinant fragments were present in protoplast-derived T calli carrying the ms4 allele than in wild type calli or leaves. The MS4 gene could then lead to conflictual situation. However, subgenomic molecules were counter-selected during the regeneration process, suggesting the existence of different selective pressures in differentiated and non-differentiated cells. The U organisation is associated with higher stem height and late flowering, characters that may not be neutral from a selection point of view. The U equilibrium is an unusual example of sudden mtDNA reorganisation, without obvious differences in genetic information and with only a limited phenotypic impact.     

FTIR and UV spectroscopy studies of triplex formation between pyrimidine methoxyethylphosphoramidates alpha-oligodeoxynucleotides and ds DNA targets

The ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes.     

New parameters reducing the interindividual variability of metabolic changes during muscle contraction in humans. A (31)P MRS study with physiological and clinical implications

Interindividual variations in skeletal muscle metabolism make comparative analyses difficult. In this study, we have addressed the issue of capturing the variability of metabolic performance observed during muscle exercise in humans by using an original method of normalization.Metabolic changes induced by various kinds of exercise were investigated using 31P magnetic resonance spectroscopy (MRS) at 4.7 T in 65 normal subjects (23 women and 42 men) and 12 patients with biopsy-proven muscular disorders.Large variations in the extent of PCr breakdown and intracellular acidosis were recorded among subjects and exercise protocols. For all the data pooled, the amplitude of mechanical performance accounts for 50% of these variations. When scaled to the work output, variations of PCr consumption account for 65% of pH changes through a linear relationship. This linear relationship was substantially improved (90%) when both variables were scaled to the square of work output performed (P1 and P2). By capturing most of the initial interindividual variability (90%), P1 vs. P2 relationship represents an ideal standardization procedure, independent of any anthropometric measurements. This relationship also discloses a significant link between the extent of PCr breakdown and intracellular acidosis regardless of exercise protocol. Moreover, changes in the slope of the P1 vs. P2 regression curve, as measured in old subjects and in selected patients, directly reflect alterations of energy production in muscle.     

Optimizing embryogenic callus production and plant regeneration from `Tifton 9' bahiagrass seed explants for genetic manipulation

Bahiagrass (Paspalum notatum Flügge) is a warm season forage grass widely cultivated in southeastern U.S. and South America. The cultivar Tifton 9 has several desirable characteristics such as high forage yield, more vigor at the seedling stage, etc.; but its forage quality is very low. As an initial step for future genetic manipulations to improve its forage characteristics, we have optimized in vitro culture conditions for plant regeneration. In this report, we describe an efficient method for embryogenic callus induction and plant regeneration from bahiagrass (cv. Tifton 9) seed explants, which are readily available and easy to manipulate, compared to other explant sources reported in the literature.Murashige and Skoog (MS) medium containing 30 M dicamba and 5 M 6-benzyladenine (BA) was optimal for callus induction and growth. Out of 9734 seeds cultured, 65.7% germinated and 21.4% produced embryogenic callus on this medium. Shoot formation was best when embryogenic calluses induced in this medium were transferred to MS medium supplemented with 5 M BA and 1 M gibberellic acid with 1640 plantlets formed per gram fresh weight of callus tissue. When transferred to hormone-free SH medium, shoot systems produced well-developed root systems. The resulting plantlets grew normally produced viable seeds when transferred to soil in the greenhouse. Histochemical staining for GUS activity arising from transient expression of the introduced uidA (-glucuronidase) gene indicated that bahiagrass embryogenic callus produced by this method is suitable for gene transfer via biolistic bombardment; and it can serve as a good target tissue for future genetic manipulations to improve the forage quality of bahiagrass (cv. Tifton 9).