Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 M) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion. (Mol Cell Biochem 156: 79-85, 1996)     

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (g _j ), we compared the effects of 2mm 1-heptanol and 20 μm 2-(methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A₂C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (r_ss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6±1.1% (mean ±sem,n=5) and 9.8±0.6% (n=5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2mm heptanol uncoupled cell pairs completely (n=6), whereas 20 μm A₂C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n=5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased r_ss by 14.9±3.0% (n=5), i.e., decreased cholesterol domain fluidity, whereas A₂C had no effect on r_ss (−0.4±6.7%,n=5). Following an increase of cellular “cholesterol” content (by loading the cells with DHE), 2mm heptanol did not uncouple cell pairs completely:g _j decreased by 80±20% (range 41–95%,n=5). The decrease ing _j was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of non-junctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreasesg _j by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.     

Gamma Oscillations as a Neural Signature of Shifting Times in Narrative Language

Verbs and other temporal expressions allow speakers to specify the location of events in time, as well as to move back and forth in time, shifting in a narrative between past, present and future. The referential flexibility of temporal expressions is well understood in linguistics but its neurocognitive bases remain unknown. We aimed at obtaining a neural signature of shifting times in narrative language. We recorded and analyzed event-related brain potentials (ERPs) and oscillatory responses to the adverb ‘now’ and to the second main verb in Punctual (‘An hour ago the boy stole a candy and now he peeled the fruit’) and Iterative (‘The entire afternoon the boy stole candy and now he peeled the fruit’) contexts. ‘An hour ago’ introduces a time frame that lies entirely in the past, ‘now’ shifts the narrative to the present, and ‘peeled’ shifts it back to the past. These two referential shifts in Punctual contexts are expected to leave very similar traces on neural responses. In contrast, ‘The entire afternoon’ specifies a time frame that may encompass past, present and future, such that both ‘now’ and ‘peeled’ are consistent with it. Here, no time shift is required. We found no difference in ERPs between Punctual and Iterative contexts either at ‘now’ or at the second verb. However, reference shifts modulated oscillatory signals. ‘Now’ and the second verb in Punctual contexts resulted in similar responses: an increase in gamma power with a left-anterior distribution. Gamma bursts were absent in Iterative contexts. We propose that gamma oscillations here reflect the binding of temporal variables to the values allowed by constraints introduced by temporal expressions in discourse.     

Scab resistance in ‘Geneva’ apple is conditioned by a resistance gene cluster with complex genetic control

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most severe diseases of apple worldwide. It is the most studied plant–pathogen interaction involving a woody species using modern genetic, genomic, proteomic and bioinformatic approaches in both species. Although ‘Geneva’ apple was recognized long ago as a potential source of resistance to scab, this resistance has not been characterized previously. Differential interactions between various monoconidial isolates of V. inaequalis and six segregating F1 and F2 populations indicate the presence of at least five loci governing the resistance in ‘Geneva’. The 17 chromosomes of apple were screened using genotyping‐by‐sequencing, as well as single marker mapping, to position loci controlling the V. inaequalis resistance on linkage group 4. Next, we fine mapped a 5‐cM region containing five loci conferring both dominant and recessive scab resistance to the distal end of the linkage group. This region corresponds to 2.2 Mbp (from 20.3 to 22.5 Mbp) on the physical map of ‘Golden Delicious’ containing nine candidate nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) resistance genes. This study increases our understanding of the complex genetic basis of apple scab resistance conferred by ‘Geneva’, as well as the gene‐for‐gene (GfG) relationships between the effector genes in the pathogen and resistance genes in the host.     

Hand position on the bunch and source–sink ratio influence the banana fruit susceptibility to crown rot disease

The postharvest development of crown rot of bananas depends notably on the fruit susceptibility to this disease at harvest. It has been shown that fruit susceptibility to crown rot is variable and it was suggested that this depends on environmental preharvest factors. However, little is known about the preharvest factors influencing this susceptibility. The aim of this work was to evaluate the extent to which fruit filling characteristics during growth and the fruit development stage influence the banana susceptibility to crown rot. This involved evaluating the influence of (a) the fruit position at different levels of the banana bunch (hands) and (b) changing the source–sink ratio (So–Si ratio), on the fruit susceptibility to crown rot. The fruit susceptibility was determined by measuring the internal necrotic surface (INS) after artificial inoculation of Colletotrichum musae. A linear correlation (r = −0.95) was found between the hand position on the bunch and the INS. The So–Si ratio was found to influence the pomological characteristics of the fruits and their susceptibility to crown rot. Fruits of bunches from which six hands were removed (two hands remaining on the bunch) proved to be significantly less susceptible to crown rot (INS = 138.3 mm ²) than those from bunches with eight hands (INS = 237.9 mm ²). The banana susceptibility to crown rot is thus likely to be influenced by the fruit development stage and filling characteristics. The present results highlight the importance of standardising hand sampling on a bunch when testing fruit susceptibility to crown rot. They also show that hand removal in the field has advantages in the context of integrated pest management, making it possible to reduce fruit susceptibility to crown rot while increasing fruit size.     

HSPB1, HSPB6, HSPB7 and HSPB8 protect against RhoA GTPase-induced remodeling in tachypaced atrial myocytes

Background

We previously demonstrated the small heat shock protein, HSPB1, to prevent tachycardia remodeling in in vitro and in vivo models for Atrial Fibrillation (AF). To gain insight into its mechanism of action, we examined the protective effect of all 10 members of the HSPB family on tachycardia remodeling. Furthermore, modulating effects of HSPB on RhoA GTPase activity and F-actin stress fiber formation were examined, as this pathway was found of prime importance in tachycardia remodeling events and the initiation of AF.

Methods and Results

Tachypacing (4 Hz) of HL-1 atrial myocytes significantly and progressively reduced the amplitude of Ca²⁺ transients (CaT). In addition to HSPB1, also overexpression of HSPB6, HSPB7 and HSPB8 protected against tachypacing-induced CaT reduction. The protective effect was independent of HSPB1. Moreover, tachypacing induced RhoA GTPase activity and caused F-actin stress fiber formation. The ROCK inhibitor Y27632 significantly prevented tachypacing-induced F-actin formation and CaT reductions, showing that RhoA activation is required for remodeling. Although all protective HSPB members prevented the formation of F-actin stress fibers, their mode of action differs. Whilst HSPB1, HSPB6 and HSPB7 acted via direct prevention of F-actin formation, HSPB8-protection was mediated via inhibition of RhoA GTPase activity.

Conclusion

Overexpression of HSPB1, as well as HSPB6, HSPB7 and HSPB8 independently protect against tachycardia remodeling by attenuation of the RhoA GTPase pathway at different levels. The cardioprotective role for multiple HSPB members indicate a possible therapeutic benefit of compounds able to boost the expression of single or multiple members of the HSPB family.     

Metabolism of C2 compounds inAcetobacter aceti

The presence of isocitrate lyase and malate synthase was detected in cell-free extracts ofAcetobacter aceti, grown in a mineral medium with acetate as sole carbon source. The presence of these enzymes explains the ability of this strain to grow with ethanol or acetate as sole carbon source, which is an important characteristic in Frateur's classification system forAcetobacter. In addition to isocitrate lyase and malate synthase, these cell-free extracts were found to contain glyoxylate carboligase, tartronicsemialdehyde reductase and glycerate kinase. The induction of these enzymes during growth on acetate is thought to be caused by the very high activity of isocitrate lyase, which may lead to an accumulation of glyoxylate. The importance of this pathway in cells growing with acetate as sole carbon source for the synthesis of their carbohydrate components is discussed. The presence of the enzymes from the pathway from glyoxylate to 3-phosphoglycerate explains the ability of this strain to grow with ethyleneglycol and glycollate as sole carbon source.     

Gait acts as a gate for reflexes from the foot

During human gait, electrical stimulation of the foot elicits facilitatory P2 (medium latency) responses in TA (tibialis anterior) at the onset of the swing phase, while the same stimuli cause suppressive responses at the end of swing phase, along with facilitatory responses in antagonists. This phenomenon is called phase-dependent reflex reversal. The suppressive responses can be evoked from a variety of skin sites in the leg and from stimulation of some muscles such as rectus femoris (RF). This paper reviews the data on reflex reversal and adds new data on this topic, using a split-belt paradigm. So far, the reflex reversal in TA could only be studied for the onset and end phases of the step cycle, simply because suppression can only be demonstrated when there is background activity. Normally there are only 2 TA bursts in the step cycle, whereas TA is normally silent during most of the stance phase. To know what happens in the stance phase, one needs to have a means to evoke some background activity during the stance phase. For this purpose, new experiments were carried out in which subjects were asked to walk on a treadmill with a split-belt. When the subject was walking with unequal leg speeds, the walking pattern was adapted to a gait pattern resembling limping. The TA then remained active throughout most of the stance phase of the slow-moving leg, which was used as the primary support. This activity was a result of coactivation of agonistic and antagonistic leg muscles in the supporting leg, and represented one of the ways to stabilize the body. Electrical stimulation was given to a cutaneous nerve (sural) at the ankle at twice the perception threshold. Nine of the 12 subjects showed increased TA activity during stance phase while walking on split-belts, and 5 of them showed pronounced suppressions during the first part of stance when stimuli were given on the slow side. It was concluded that a TA suppressive pathway remains open throughout most of the stance phase in the majority of subjects. The suggestion was made that the TA suppression increases loading of the ankle plantar flexors during the loading phase of stance.