Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.     

Myocutaneous flap ischemia: flow dynamics following venous and arterial obstruction.

To further clarify the pathogenesis of the poorer prognosis in skin flaps exposed to venous stasis compared with arterial insufficiency, a microsphere study was conducted in bilateral rectus abdominis island flaps in seven pigs. The relationship between capillary blood flow and arteriovenous (A-V) shunting was studied during progressive 1-hour intervals of arterial insufficiency and venous stasis and during 3 hours of reperfusion. Under controlled conditions, total blood flow was reduced from 100 percent to both 50 and 25 percent by application of an adjustable clamp on the artery supplying one flap and on the vein draining the contralateral flap. The relative distribution between A-V shunt flow and capillary blood flow was different in arterial insufficiency when compared with venous stasis at both the 50 percent and the 25 percent blood flow levels. In the arterial insufficiency flaps, the A-V shunt flow and capillary blood flow shared the total blood flow in the following percentages: 64/36 (at 100 percent total blood flow), 44/56 (at 50 percent total blood flow level), and 22/78 (at 25 percent total blood flow level). In the venous stasis flaps, the A-V shunt flow and the capillary blood flow shared the total blood flow in percentages of 70/30, 66/34, and 55/45, respectively. Hence, in arterial insufficiency flaps, capillary blood flow was spared by a relatively greater decline in A-V shunting compared with venous stasis flaps. Redistribution of capillary blood flow from subcutaneous tissue to muscle was observed, whereas blood flow was equally distributed throughout the length of the flaps at all flow levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycogenosis Type III in the Dog

Enzyme and glycogen structure studies have been carried out on tissues of a glycogenotic dog, the clinical and pathological characteristics of which are reported in the accompanying paper. Liver glucose-6-phosphatase, leukocyte and liver acid maltase, and liver and skeletal muscle glycogen Phosphorylase all appeared largely unaffected. The activity of the muscle and liver debranching enzyme (amylo-l,6-glucosidase), determined by two independent assay methods, was, however, reduced to between 0 and 7 % of normal activity. Glycogen structure studies with Phosphorylase or iodine spectra revealed that the abnormally large amounts of glycogen found in liver and skeletal muscle had abnormally short branches, as would be expected for a deficiency of debranching enzyme. It is thus clear that the dog had suffered from the equivalent of Cori's disease (limit dextrinosis, type III glycogen storage disease). Preliminary data indicate that it may be possible to identify heterozygotes based on a study of the debranching enzyme of leukocytes.     

The Effects of Neuraminidase and Gangliosides on Ovarian LH/hCG Receptors During Rat Development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6–8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (K_D) of about 10⁻⁹ M. The K_D of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10⁻¹⁰ M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptors in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Two new stoloniferous species of Viola (Violaceae) from China

Two new stoloniferous species of Viola (Violaceae) from southern China are described and illustrated. Viola nitida is recognizably different from V. fargesii as the plant is evergreen and glabrous throughout, the leaf blade adaxially nitid, base cuneate or shallowly cordate, and margin serrate. Viola maoershanensis is different from V. diffusa as the leaf blade is serrate, base cordate and not decurrent to petiole, the petiole wingless, the flowers larger, the petals bluish violet or pinkish white, and the lower petal obtuse at the apex. The chromosome numbers of the two new species were counted as 2n = 24 (V. nitida) and 2n = 44 (V. maoershanensis). The taxonomic positions of the two species are discussed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 349–356.     

Cloning and expression of an anti-LDL(-) single-chain variable fragment,and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr^-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr^-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).     

Persistence during Tasks with Horses in Relation to Social Support,General Self-efficacy and Self-esteem in Adolescents

ABSTRACT

In young people, interactions with horses may contribute to the development of social skills and self-efficacy. Our aim was to identify relationships between psychological variables and young people's experience of activities with horses. Participants in this study were 13–15-year-old adolescents (n = 25), given a 4-month intervention consisting of weekly sessions of stable work and horseback riding. We investigated the relationship between psychological variables from survey data with video-recorded behavioral observations during tasks and contact with horses. From the behavioral data, we calculated the frequency per hour of “trying again” when met with a challenge, as a measure of participants' persistence in horse-related tasks. The number of times per hour that that participant initiated contact with the horse was used as a measure of frequency of physical contact (petting). We examined correlations between the change from early to late stages of the intervention in trying again during horse-related tasks and for physical contact and three psychological variables measured prior to the intervention: perceived social support, self-esteem, and general self-efficacy. We detected an association between lower pretest levels of perceived social support and general self- efficacy, and an increase during the intervention in persistence of trying again when met with challenges during tasks with the horse. In a linear regression analysis, controlling for initial levels of persistence during horse-related tasks, self-esteem, and self-efficacy, social support was the only unique predictor of an increase in persistence during the intervention. No association was found between the psychological variables and change in frequency of petting the horse. The results indicate that adolescents with lower levels of perceived social support at the start of the intervention showed greater increases in their persistence in trying again after being met with challenges during horse-related tasks. Since persistence is a central aspect in the development of mastery of tasks, the possible positive social impact of the horse/stable environment should be given more attention in future intervention research.