Changes in Calcium-Dependent Protein Kinase Activity during in Vitro Tuberization in Potato

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.     

Length dependence of staircase potentiation: interactions with caffeine and dantrolene sodium

In skeletal muscle, there is a length dependence of staircase potentiation for which the mechanism is unclear. In this study we tested the hypothesis that abolition of this length dependence by caffeine is effected by a mechanism independent of enhanced Ca2+ release. To test this hypothesis we have used caffeine, which abolishes length dependence of potentiation, and dantrolene sodium, which inhibits Ca2+ release. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 20 s of repetitive stimulation at 5 Hz were analyzed at optimal length (Lo), Lo - 10%, and Lo + 10%. Potentiation was observed to be length dependent, with an increase in developed tension (DT) of 78 +/- 12, 51 +/- 5, and 34 +/- 9% (mean +/- SEM), at Lo - 10%, Lo, and Lo + 10%, respectively. Caffeine diminished the length dependence of activation and suppressed the length dependence of staircase potentiation, giving increases in DT of 65+/-13, 53 +/- 11, and 45 +/- 12% for Lo - 10%, Lo, and Lo + 10%, respectively. Dantrolene administered after caffeine did not reverse this effect. Dantrolene alone depressed the potentiation response, but did not affect the length dependence of staircase potentiation, with increases in DT of 58 +/- 17, 26 +/- 8, and 18 +/- 7%, respectively. This study confirms that there is a length dependence of staircase potentiation in mammalian skeletal muscle which is suppressed by caffeine. Since dantrolene did not alter this suppression of the length dependence of potentiation by caffeine, it is apparently not directly modulated by Ca2+ availability in the myoplasm.     

Play behavior in immature moor macaques (Macaca maura) and Japanese macaques (Macaca fuscata)

Play is widespread across mammalian taxa, but species strongly vary in the ways they play. In less despotic primate species (i.e., with less steep dominance hierarchies, less severe conflicts, and more reconciliation), play has been described as being more frequent, cooperative, and freely expressed. To study the link between social play and dominance style, we compared play behavior in free-ranging infants, juveniles and subadults of more despotic Japanese macaques (Macaca fuscata, N = 24) and less despotic moor macaques (Macaca maura, N = 17). We found interspecific differences in play behavior that corresponded with the contrasting dominance styles of the study species, largely confirming our predictions. In particular, moor macaques spent a larger proportion of time in solitary and social play than Japanese macaques, while Japanese macaques spent a larger proportion of time in grooming interactions. In moor macaques, play sessions included more players, a larger variety of play behaviors, greater play face rates, a greater proportion of time in contact play, and a higher rate of reciprocal play-biting than in Japanese macaques. Aggressive escalations were not common, but more frequent in Japanese macaques. Finally, a higher frequency of play faces during play sessions predicted the occurrence of more reciprocal play-bites, but not the proportion of time spent in contact play behaviors. Additional studies on other groups and species will allow a better understanding of the link between dominance style and social play.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.     

A prolonged after-effect of intense synaptic activity on acetylcholine in a sympathetic ganglion.

The findings was confirmed that there is a "rebound" increase of stored acetylcholine (ACh) in cat superior cervical ganglia conditioned by prolonged preganglionic stimulation at a frequency high enough to cause initial depletion of the store. Ganglia removed immediately after 60 min of continuous or interrupted stimulation at 50 Hz, with chloralose as anesthetic, contained about 30% more ACh than their unconditioned controls; the rebound rose to about 60% after 15 min of rest and then subsided with an apparent half-time of about 2 h. Tests with hemicholinium, combined with hexamethonium or tubocurarine, showed that rebound ACh was located presynaptically and could be released by nerve impulses; but conditioned ganglia perfused with an eserine-containing medium did not release more ACh than their unconditioned controls, except in circumstances in which the mobilization of ACh from a reserve store appeared to be the rate-limiting process for release. The appearance of rebound ACh during and after conditioning stimulation was suppressed by hexamethonium and by tubocurarine, neither of which has much effect on ACh turnover in ganglia excited at lower frequencies, but not only by atropine, noradrenaline, or phenoxybenzamine. The formation of rebound ACH is thus contingent on the postsynaptic nicotinic response to released ACh, and may represent an augmentation of the transmitter store in structures remote from the release sites.     

BOLD fMRI in the White Matter as a Marker of Aging and Small Vessel Disease

Purpose

Determine whether white matter signal fluctuation on T2* weighted BOLD contrast images are associated with aging and cerebral small vessel disease (SVD).

Methodology

Resting state BOLD data were collected with a 250 ms repetition time (TR) to achieve unaliased, ungated cardiac sampled BOLD (cs-BOLD) images on 11 young adult controls, 10 healthy older adult controls and 7 adults with extensive white matter hyperintensities (WMH) from SVD. Tissue classes (WM and GM) were segmented on T1 images. WMH were identified on FLAIR images in the SVD group. Raw physiological noise (σ_physio) and cardiac pulsatility (i.e. fluctuations at the cardiac frequency) were calculated voxel wise and group differences were tested by ANOVA. It was also possible to calculate σ_physio in 2s TR cardiac aliased whole-brain BOLD (wb-BOLD) data (N = 84) obtained from the International Consortium for Brain Mapping.

Results

CS-BOLD metrics showed an aging and SVD effects (p<0.0005). Covariates such as thermal noise, WM volume and partial volume did not influence the significant aging effect seen on the cardiac pulsatility metric (p<0.017) but did influence the σ_physio (p = 0.184). As a verification of the cs-BOLD findings, the wb-BOLD also showed a linear aging effect of σ_physio in WM. In the SVD adults, cardiac pulsatility and σ_physio were lower in WMH regions compared to normal appearing white matter (NAWM) regions (p<0.0013 and p<0.002, respectively). Cardiac pulsatility was better able to distinguish WMH regions from NAWM than σ_physio as measured by effect size (Cohen’s d 2.2 and 0.88, respectively).

Conclusion

NAWM was found to have graded increases in cardiac pulsations due to age and SVD, independently. Within SVD participants, WMH lesions had reduced physiological noise compared to NAWM. Cardiac pulsatility in resting BOLD data may provide a complementary dynamic measure of WM integrity to add to static FLAIR anatomical images.     

What to Eat: Evidence for Selective Autophagy in Plants

Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process, housekeeping mechanism, and response to environmental stress. In plants, autophagy involves the sequestration of cargo to be degraded, transport to the cell vacuole in a double-membrane bound autophagosome, and subsequent degradation by lytic enzymes. Autophagy has generally been considered to be a non-selective mechanism of degradation. However, studies in yeast and animals have found numerous examples of selective autophagy, with cargo including proteins, protein aggregates, and organelles. Recent work has also provided evidence for several types of selective autophagy in plants. The degradation of protein aggregates was the first selective autophagy described in plants, and, more recently, a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1, which interacts with the autophagy machinery and may function in autophagy of protein aggregates, was described in plants. Other intracellular components have been suggested to be selectively targeted by autophagy in plants, but the current evidence is limited. Here, we discuss recent findings regarding the selective targeting of cell components by autophagy in plants.     

Effects of training on biochemical and functional properties of rodent neonatal heart

This study was undertaken to determine biochemical and functional (in vivo) adaptations of the rodent neonatal heart in response to a training program of endurance running. Ten day-old rats were progressively trained on a treadmill (final intensity, 21 m/min, 30% grade, 1 h/day) until 75 days of age. The training program induced 14, 57, and 24% increases in relative heart mass, skeletal muscle citrate synthase activity, and whole-body maximal O2 uptake, respectively (P less than 0.05). Cardiac myosin (ATPase) and Ca2+-regulated myofibril ATPase were both reduced by approximately 15% in trained vs. sedentary animals (P less than 0.05). In the majority of trained hearts examined, the myosin isozyme profile reflected an estimated 14 +/- 3% shift toward the V3 or low ATPase isozyme. Left ventricular functional indices during submaximal exercise, derived from a fluid-filled indwelling cannula, indicated that the trained animals maintained similar left ventricular (LV) systolic pressure, LV + the time derivative of pressure, and systemic arterial mean blood pressure compared with their sedentary counterparts. These functional parameters were maintained even though the trained animals performed with lower submaximal exercise heart rate. These findings suggest that maximal exercise capacity can be enhanced in neonatal rats even though the biochemical potential for ATP degradation in the cardiac contractile system is lowered. We speculate that the trend to maintain the myosin isozyme pattern further in the direction of the V3 isozyme in the trained neonatal rat heart may reflect a means to economize cross-bridge cycling while maintaining normal levels of ventricle performance at a given submaximal work load.