Directional Ca2+ effect on stimulation of mucin secretion from chicken trachea in vitro

Chicken tracheal mucosa in vitro transported and incorporated radioactive precursors into mucins, which were secreted at a steady rate into the tracheal lumen. Secretion of mucins labelled with ³⁵S and ³H after pulse-labelling of the mucosal layer with Na₂³⁵SO₄ and d-[1-³H]glucosamine as precursors was an energy-dependent process, as it was strongly inhibited by the action of respiratory-chain inhibitors, an uncoupler of oxidative phosphorylation, a metabolic blocker and a temperature shift from 41°C to 5°C. On the other hand, both cholinergic and parasympathomimetic agents considerably increased the secretion of dual-radiolabelled mucins when applied on the submucosal side of the trachea. The effect of Ca²⁺ was directional, since only high submucosal (3.6 or 18mm) or low luminal (zero or 0.18mm) Ca²⁺ massively enhanced the secretion of radiolabelled mucin compared with the mucin output measured under physiological Ca²⁺ conditions (1.8mm). Whereas application of ionophore A23187 on either side of the trachea significantly increased mucin output, its presence in the appropriate tracheal compartment and under appropriate Ca²⁺ conditions further accentuated the output of radiolabelled mucins. Addition of acetylcholine under appropriate conditions also had an additive effect on the Ca²⁺-stimulated secretion of mucins. Ca²⁺ stimulation of mucin secretion appears to be dependent on the metabolic integrity of the mucosal cells. Mucins secreted in response to high submucosal and low luminal [Ca²⁺] appear to consist of a number of different types of glycoproteins, as judged from their ion-exchange-chromatographic behaviour.     

Effect of one-legged exercise on the strength, power and endurance of the contralateral leg. A randomized, controlled study using isometric and concentric isokinetic training.

The purpose of this investigation was to study the effect of one-legged exercise on the strength, power and endurance of the contralateral leg. The performance of the knee extensor and flexor muscle of 20 healthy young adults (10 men and 10 women) was first tested by Cybex II+ and 340 dynamometers. Then 10 subjects were chosen at random to train using one leg three times a week for 7 weeks whilst the other 10 served as controls. During the 8th week, the tests were repeated. Both quadriceps and hamstring muscles of the trained subjects showed a cross-transfer effect from the trained limb to the untrained side. This concerned the strength and power, as well as endurance characteristics of these muscles. The average change in peak torque of the quadriceps muscle was +19% (P less than 0.001) in the trained limb, +11% (P less than 0.01) in the untrained limb and 0% in the control limbs. In hamstring muscles the changes were +14% (P less than 0.01), +5% and -1%, respectively. Concerning muscle endurance (work performed during the last 5 contractions in the 25-repetition test) the corresponding changes were +15% (P less than 0.01), +7% (P less than 0.01), and -1% in quadriceps muscle, and +17% (P less than 0.05), +7%, and -3% in hamstring muscles. The average strength benefit in the untrained limb was +36% (hamstring muscles) and +58% (quadriceps muscle) of that achieved in the trained limb. Untrained hamstring muscle showed better benefits in the endurance parameters than in strength or power parameters, while in the quadriceps muscle this effect was reversed. A positive relationship was observed between the changes (greater improvement in the trained limb resulted in greater improvement in the untrained limb) (hamstring muscles: r = 0.83, P less than 0.001, quadriceps muscle: r = 0.53, P less than 0.001). In endurance parameters, this relationship was almost linear while in the strength and power parameters the results were more in favour of a curvilinear relationship with limited benefit.     

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK)-MEDIATED FEEDBACK LOOPS CONTROL HORMONE SENSING*

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.     

Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry.

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides by using nano-electrospray ionization mass spectrometry (nESI-MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1--3 min. Hence, our data indicated that as little as 3 x 10(-15) mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[(35)S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [(35)S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.     

Early Events following Experimental Infection with Peste-Des-Petits Ruminants Virus Suggest Immune Cell Targeting

Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d’Ivoire ’89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis.     

Murine bladder wall biomechanics following partial bladder obstruction

Evaluation of bladder wall mechanical behavior is important in understanding the functional changes that occur in response to pathologic processes such as partial bladder outlet obstruction (pBOO). In the murine model, the traditional approach of cystometry to describe bladder compliance can prove difficult secondary to small bladder capacity and surgical exposure of the bladder. Here, we explore an alternative technique to characterize murine mechanical properties by applying biaxial mechanical stretch to murine bladders that had undergone pBOO. 5–6 week old female C57/Bl6 mice were ovariectomized and subjected to pBOO via an open surgical urethral ligation and sacrificed after 4 weeks (n=12). Age matched controls (n=6) were also analyzed. Bladders were separated based on phenotype of fibrotic (n=6) or distended (n=6) at the time of harvest. Biaxial testing was performed in modified Kreb's solution at 37 °C. Tissue was preconditioned to 10 cycles and mechanical response was evaluated by comparing axial strain at 50 kPa. The normal murine bladders exhibited anisotropy and were stiffer in the longitudinal direction. All mice showed a loss of anisotropy after 4 weeks of pBOO. The two phenotypes observed after pBOO, fibrotic and distended, exhibited less and more extensibility, respectively. These proof-of-principle data demonstrate that pBOO creates quantifiable changes in the mechanics of the murine bladder that can be effectively quantified with biaxial testing.     

Dietary dependent distribution of acetyl CoA carboxylase between cytoplasm and mitochondria of rat liver

Biotinyl proteins in cytoplasm and mitochondria of rat liver were examined by fluorography and the quantity of acetyl CoA carboxylase was determined after sodium dodecyl sulfate-denatured proteins were incubated with [14C] methyl avidin and separated by polyacrylamide gel electrophoresis. Results show that one-half of the total acetyl CoA carboxylase in liver of fed rats was associated with mitochondria in a relatively inactive form. Fasting shifted the distribution of the enzyme toward the mitochondrial fraction and refeeding previously fasted rats shifted the distribution towards cytoplasm. Thus, acetyl CoA carboxylase can be added to the list of ambiquitous enzymes whose subcellular distribution varies with physiological conditions.