The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs.

Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded - whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.

Forces shaping the Drosophila wing.

How genes encode the three-dimensional shape of tissues is a fascinating problem in biology. Pioneering genetic studies in the fruit fly Drosophila have identified key genes that control the generation of force patterns in the developing wing. Shortrange force patterns generated by planar polarised myosins can promote boundary formation and tissue elongation during the larval wing disc stage. Long-range force patterns are also crucial to shaping the wing during the pupal stage. We review the different ways in which both local and global force patterns can be generated, such as: patterned acto-myosin contractility, patterned anchorage to the extracellular matrix, and patterned tissue growth. In all cases, the balance between force, mass, and resistance explains how the resulting mechanical response produces particular tissue forms-a point underscored by the ability of computer simulations of tissue mechanics to reproduce such morphogenetic events.

The effects of a high-intensity free-weight back-squat exercise protocol on postural stability in resistance-trained males.

The purpose of this study was to investigate the effects of a high-intensity free-weight back-squat exercise on postural stability characteristics in resistance-trained males. Eighteen college-aged (mean ± SD: age = 22.9 ± 2.9 years; height = 175.8 ± 6.4 cm; mass = 86.3 ± 9.3 kg), resistance-trained males performed postural stability testing before and after completing five sets of eight repetitions of back-squat exercises at 80% of one-repetition maximum. A commercial balance testing device was used to assess sway index at pre- and at 0, 5, 10, 15 and 20 min post-exercise. Each balance assessment consisted of four, 20-s static stance conditions: eyes-open firm surface, eyes-closed firm surface, eyes-open soft surface and eyes-closed soft surface. Sway index was greater (P = 0.001-0.020) at Post 0 than at all other time points. No differences (P > 0.05) were observed between any other time phases. Sway index was greater (P < 0.001) for eyes-closed soft surface than all other conditions. These findings revealed sway index for all conditions significantly increased following completion of the back-squat; however, sway index recovered within 5 min of exercise. Higher sway index values as a result of neuromuscular fatigue induced by a back-squat exercise may have performance and injury risk consequences to subsequent activities that rely on postural stability. However, these findings suggest balance impairments may recover in ~5 min following high-intensity lower body resistance exercise.

Sex comparisons of strength and coactivation following ten weeks of deadlift training.

OBJECTIVES: To examine the effects of a ten week deadlift training program on peak torque and agonist-antagonist coactivation. METHODS: Fifty-four untrained subjects (mean age=23 years) participated in this investigation, and were randomly assigned to a training (males, n=17; females, n=17) or control (males, n=9; females, n=11) group. The subjects in the training group performed deadlifts twice per week. Isometric peak torque for the leg extensors and flexors and surface electromyographic (EMG) amplitude for the superficial quadriceps and biceps femoris muscles were assessed. RESULTS: Deadlift training increased leg extension peak torque for the males (P=.008, Cohen's d=0.43) and females (P=.003, d=0.48). Leg flexion peak torque improved for the females (P=.001, d=0.45). Increased EMG amplitude for the superficial quadriceps femoris muscles when they served as agonists was demonstrated for the females (P=.010, d=0.40), but not the males (P=.059, d=0.20). For both sexes, the effect sizes for the decline in biceps femoris coactivation were large. CONCLUSION: Deadlift training elicited improvements in strength and agonist-antagonist coactivation in untrained subjects, and particularly, novice females.

Effects of neuromuscular fatigue on the electromechanical delay of the leg extensors and flexors in young and old men.

PURPOSE: The purpose of this study was to investigate the effects of a fatigue-inducing bout of submaximal, intermittent isometric contractions on the electromechanical delay (EMD) of the leg extensors and flexors in young and old men. METHODS: Twenty young (mean ± SD: age = 25 ± 2.8 years) and sixteen old (age = 70.8 ± 3.8) recreationally active men performed maximal voluntary contractions (MVCs) followed by a fatigue-inducing protocol consisting of intermittent isometric contractions of the leg extensors or flexors using a 0.6 duty cycle (6 s contraction, 4 s relaxation) at 60 % of MVC until volitional fatigue. MVCs were again performed at 0, 7, 15, and 30 min post fatigue. A three-way mixed factorial ANOVA was used to analyze the EMD data. RESULTS: There was a two-way muscle × time interaction (P = 0.039) where the EMD of the leg flexors was greater (P = 0.001-0.034) compared with baseline at all post fatigue time periods, but was only greater at immediately post fatigue for the extensors (P = 0.001). A significant two-way interaction for muscle × age (P = 0.009) revealed that the EMD was greater (P = 0.003) for the extensors for the old compared with the young men, but not different for the flexors (P = 0.506). CONCLUSIONS: These findings showed differential fatigue-induced EMD recovery patterns between the leg extensors and flexors with the flexors being slower to recover and also that age-related increases of EMD are muscle group specific. The sustained increased EMD of the flexors during recovery may have important injury and performance implications in a variety of populations and settings.

Influence of acute eccentric exercise on the H:Q ratio.

The purpose of the present study was to examine the effects of an acute bout of eccentric exercise on maximal isokinetic concentric peak torque (PT) of the leg flexors and extensors and the hamstrings-to-quadriceps (H:Q) strength ratio. Sixteen male (mean±SD: age=20.9±2 years; stature=177.0±4.4 cm; mass=76.8±10.0 kg) volunteers performed maximal, concentric isokinetic leg extension and flexion muscle actions at 60°·sec - 1 before and after (24-72 h) a bout of eccentric exercise. The eccentric exercise protocol consisted of 4 sets of 10 repetitions for the leg press, leg extension, and leg curl exercises at 120% of the concentric one repetition maximum (1-RM). The results indicated that the acute eccentric exercise protocol resulted in a significant (P<0.05) decrease in isokinetic leg flexion (13-19%) and leg extension (11-16%) PT 24-72 h post-exercise. However, the H:Q ratios were unaltered by the eccentric exercise protocol. These findings suggest that an acute bout of eccentric exercise utilizing both multi - and single - joint dynamic constant external resistance (DCER) exercises results in similar decreases in maximal isokinetic strength of the leg flexors and extensors, but does not alter the H:Q ratio.

The relationship between passive stiffness and evoked twitch properties: the influence of muscle CSA normalization.

Passive stiffness measurements are often used as a clinical tool to examine a muscle's passive lengthening characteristics. The purpose of this study was to examine the relationship between passive stiffness and evoked twitch properties prior to and following normalization of passive stiffness to muscle cross-sectional area (CSA). Ten healthy volunteers (mean ± SD age = 23 ± 3 year) performed passive range of motion, evoked twitch, and muscle CSA assessments of the plantar flexor muscles. Passive stiffness was determined from the slope of the final 5° of the angle-torque curve. Peak twitch torque (PTT) and rate of torque development (RTD) were determined via transcutaneous electrical stimulation, and muscle CSA was assessed using a peripheral quantitative computed tomography scanner. Pearson product moment correlation coefficients (r) were used to assess the relationships between passive stiffness and PTT and RTD and normalized passive stiffness (passive stiffness ⋅ muscle CSA(-1)) and PTT and RTD. Significant positive relationships were observed between passive stiffness and PTT (P = 0.003, r = 0.828) and RTD (P = 0.003, r = 0.825). There were no significant relationships between normalized passive stiffness and PTT (P = 0.290, r = 0.372) or RTD (P = 0.353, r = 0.329) demonstrating that stiffness did not account for a significant portion of the variance in twitch properties. Passive stiffness was largely influenced by the amount of muscle tissue in this study. Future studies that examine muscle stiffness and its relationship with performance measures, among different populations, and following various interventions may consider normalizing stiffness measurements to muscle CSA.

Temperature alters lipopolysaccharide-induced cytokine secretion by RAW 264.7 cells.

Local and systemic temperature change is associated with the immune response to infection, but the role of temperature remains poorly understood. To study the effect of temperature on macrophage activation by lipopolysaccharide (LPS), RAW 264.7 cells were incubated with LPS at different temperatures and secretion of three cytokines was measured. Incubation at 31 degrees C increased tumour necrosis factor (TNF) secretion when compared with 37 degrees C, while cells exposed at 39 degrees C secreted less TNF. Interleukin-6 (IL-6) secretion was less at 31 degrees C than at 37 degrees C and remained unchanged at 39 degrees C. Interleukin-10 secretion was depressed on either side of 37 degrees C. Only IL-6 secretion was sensitive to preincubation temperature effects. The kinetics of cytokine secretion and steady-state mRNA analysis indicated potentially different mechanisms of temperature regulation for TNF and IL-6.

Identification of genes induced by growth hormone in rat liver using cDNA arrays.

Growth hormone (GH) is a pleiotropic cytokine that acts upon its target cells to regulate their growth, differentiation and metabolism. GH is thought to act by altering gene expression in target cells, but few GH-regulated genes are known. In this study, we used cDNA array analysis to identify genes rapidly induced in the liver of GH-deficient dwarf rats following a single systemic injection of GH. Eight genes were found to upregulate their mRNA expression within 1-3 hours of GH administration, results which were confirmed by northern analysis. The identity of these genes suggests GH may influence a diversity of cellular processes. A role for GH in regulating cytokine and growth factor signalling is suggested by upregulation of mRNAs encoding three signal transducers: a subunit of the receptor for IL-6-type cytokines (gp130), STAT3 (signal transducer and activator of transcription) and p38MAPK (mitogen activated protein kinase). Two genes involved in DNA repair and cell cycle control, APEN (apurinic endonuclease) and GADD45 (growth arrest and DNA damage 45) were upregulated. Other induced genes include those encoding a lactate transporter (MCT-1), an extracellular matrix remodelling enzyme, MTI-MMP (membrane type 1 matrix metalloproteinase) and an acute phase protein (fibrinogen beta). In summary, this work is the first to apply cDNA arrays to the study of peptide hormone action in vivo and has identified 8 novel GH target genes.

Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues.

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.

Lessons from the front: implementing outcomes projects.

These issues are representative of the challenges facing those wishing to implement outcomes evaluations in the dramatically different settings of today's behavioral healthcare. To maintain acceptable levels of credibility, difficult issues such as these need to be addressed openly and assertively. We remain optimistic about the value of measuring the effects of behavioral healthcare interventions. Such measurement facilitates the development of communication across service providers and creates accountability where once there was none. When standards of quality research are applied, outcomes management offers great potential benefit to consumers, providers, and behavioral healthcare organizations.

Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein.

The gene for C4-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter(-1) of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.

Biosynthesis of fatty acids by lactating human breast epithelial cells: an evaluation of the contribution to the overall composition of human milk fat.

The objective of this study was to characterize the fatty acid biosynthetic pathway of the lactating human breast. Mixed cell populations, obtained by centrifugation of human milk, were enriched in breast epithelial cells by a selective adsorption procedure. Confirmation of the identity of the breast epithelial cells was obtained immunohistochemically. These viable breast epithelial cells incorporated radioactively labeled acetate predominantly into fatty acids with less than 16C atoms. The presence of the two key enzymes characteristic of the medium-chain fatty acid biosynthetic pathway of nonruminants, fatty acid synthetase, and thioesterase II, was demonstrated both qualitatively, by immunohistochemistry, and quantitatively, by enzyme assay. The results indicate that the lipogenic system of the human breast is qualitatively very similar to that of rats, mice, and rabbits, which also secrete milk fats containing medium-chain fatty acids. Quantitatively, however, the mammary fatty acid biosynthetic pathway appears to be less active in humans than in these other species.

Thioesterase II, a new marker enzyme for human cells of breast epithelial origin.

Metabolic, enzymologic, and immunohistochemical techniques have been used to show that a human cell line of breast epithelial origin synthesized medium chain fatty acids via the ubiquitous fatty acid synthetase and a mammary-specific chain-terminating enzyme, thioesterase II. Previous studies in our laboratory with rodents indicated that thioesterase II is expressed exclusively in mammary epithelial cells, an observation consistent with the physiologic role of the enzyme in milk fat synthesis. Results of the present study suggest that the enzyme exhibits a similar cell specificity in its expression in humans and that the specificity is maintained in normal and neoplastic tissues. Thus thioesterase II was detected immunohistochemically in normal human breast epithelia derived from both lactating and nonlactating breast tissue, in cultured cells derived from both primary breast epithelial tumors and from a metastatic tumor of breast origin, and in several human breast epithelial cell lines; the enzyme could not be detected in HeLa cells, in a colon carcinoma, or in a mammary myoepithelial cell line. These findings raise the possibility that thioesterase II may be of use as a diagnostic tool to identify human tumors of breast epithelial origin.

Localization of thioesterase II, the chain-length regulatory enzyme of milk fatty acid synthesis, in rat mammary gland epithelial cells.

Two approaches were used to establish the intercellular distribution of fatty acid synthetase and thioesterase II in the lactating rat mammary gland. Thioesterase II is the chain-length regulatory enzyme in the biosynthesis of the medium-chain fatty acids characteristic of milk fat. Using immunohistochemical techniques, immunoreactive fatty acid synthetase was found in both mammary adipocytes and epithelial cells; in contrast, immunoreactive epithelial cells were isolated from lactating rat mammary glands after digestion with collagenase and thermolysin, and their lipogenic activity was studied using isotopically labelled acetate. Consistent with the immunohistochemical data, adipocytes synthesized exclusively long-chain fatty acids whereas epithelial cells synthesized predominantly chain fatty acids. The results indicate that the capacity for synthesis of medium-chain fatty acids is a unique property of the epithelial cell component of the mammary gland.