Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base.     

Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity

Interplay of two cytoskeletal systems--microfilaments and microtubules is essential for directional cell movement. To better understand the role of those cytoskeletal systems in polarization of cells, rat fibroblasts were incubated with drugs inhibiting activity of myosin II: blebbistatin and Y-27632. Both drugs led to disappearance of actin-myosin bundles and mature focal cell-matrix adhesions but did not affect polarization and directional motility. The rate of motility even increased after inhibitor treatment. The characteristic feature of inhibitor-treated fibroblasts was collapse of the cytoplasm accompanied by bundling of microtubules that led to transformation of lamellae into long immobile tails. The only exception was the leading anterior lamella which was not transformed into the tail and supported directional movement of the cell. The tail at the cell rear determined the position of anterior lamella and direction of locomotion. Depolymerization of microtubules by colcemid stopped directional locomotion of inhibitor-treated cells. These data show that integrity of the microtubular system provides the basic mechanism of polarization and orientation which is only modified by interactions with actin-myosin system and cell-substrate adhesions. We suggest that the position of bundled tail microtubules and dispersed microtubules in leading lamella determine polarization in cells lacking stress fibers and focal adhesions. Thus, polarization is based on microtubule-dependent mechanisms both in non-contractile and contractile cells. These mechanisms could switch dependent on circumstances as fibroblasts may acquire non-contractile phenotype, not only after direct inhibition of myosin II but also in certain conditions of microenvironment.     

Host Responses to Intestinal Microbial Antigens in Gluten-Sensitive Mice

Background and Aims

Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice.

Methodology/Principal Findings

HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-γ in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota.

Conclusion

Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-γ production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.     

IDH1-Associated Primary Glioblastoma in Young Adults Displays Differential Patterns of Tumour and Vascular Morphology

Glioblastoma is a highly aggressive tumour with marked heterogeneity at the morphological level in both the tumour cells and the associated highly prominent vasculature. As we begin to develop an increased biological insight into the underlying processes driving the disease, fewer attempts have thus far been made to understand these phenotypic differences. We sought to address this by carefully assessing the morphological characteristics of both the tumour cells and the associated vasculature, relating these observations to the IDH1/MGMT status, with a particular focus on the early onset population of young adults who develop primary glioblastoma. 276 primary glioblastoma specimens were classified into their predominant cell morphological type (fibrillary, gemistocytic, giant cell, small cell, oligodendroglial, sarcomatous), and assessed for specific tumour (cellularity, necrosis, palisades) and vascular features (glomeruloid structures, arcades, pericyte proliferation). IDH1 positive glioblastomas were associated with a younger age at diagnosis, better clinical outcome, prominent oligodendroglial and small cell tumour cell morphology, pallisading necrosis and glomeruloid vascular proliferation in the absence of arcade-like structures. These features widen the phenotype of IDH1 mutation-positive primary glioblastoma in young adults and provide correlative evidence for a functional role of mutant IDH1 in the differential nature of neo-angiogenesis in different subtypes of glioblastoma.     

High-performance liquid chromatographic method for the determination of mangiferin, likviritin and dihydroquercetin in rat plasma and urine

The use of reversed-phase high-performance liquid chromatography for the determination of the biologically active plant phenolic compounds mangiferin, likviritin and dihydroquercetin is described. Perchloric acid (35%) was used for deproteinization in the case of mangiferin and likviritin, and acidified methanol for dihydroquercetin. Detection was performed at 254, 275 and 290 nm for mangiferin, likviritin and dihydroquercetin in plasma, and 365, 312 and 290 nm in urine, respectively. The limit of detection was 0.2 μg/ml for plasma and 0.5 μg/ml for urine.     

K(+)-channel blockers do not decrease acetylcholine depolarizations in canine trachealis.

Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10(-6) M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20-30 mV positive to the resting potential, previously shown to be approximately -55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10(-6) M), charybdotoxin (10(-7) M), and glybenclamide (10(-5) M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10(-6) M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K(+)-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K(+)-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.     

Mathematical models for mixing in deep jet bioreactors: analysis

A mathematical model for single and multi step deep-jet bioreactors is presented. A stagewise approach based on macroscopic mechanistic model which divides the reactor into compartments with good quality of mixing and plug flow regions (macromixer), was used. For the mathematical representation of this model a system of differential equations, describing the concentration of tracer in structural elements based on mass balance, and the Runge-Kutta-Fehlberg numerical method of integration, was applied. The mixing time in a 300 dm³ tank was determined by conductivity method with NaCl as tracer.List of Symbols V _g dm³ total volume of liquid - V ₁; V ₆ dm³ volumes of ideally mixed compartments in the vessel - V ₂; V ₇ dm³ volumes of macromixer in the inner circulation flows - V ₃; V ₉ dm³ volumes of liquid phase in the pump - V ₄; V ₈ dm³ volumes of liquid phase in the pipe between the vessel and the pump - V ₅; V ₁₀ dm³ volumes of liquid phase in pipes between the pump and the air input system, including falling jet - F _E; F _E,1; F _E,2 dm³/s the inner volumetric circulation flow rates accross the macromixers - F _E,3; F _E,4 dm³/s exchanges volumetric flow rates between two ideally mixed compartments in the vessel - F _cir; F _1,cir; F _2,cir dm³/s external volumetric circulation flow rates (pumping capacity) - t _A s time interval of puls application - t _AA s time point of impuls application related to the free chosen point of simulation - t _end s end time of simulation - F _qu g²/dm⁶ sum of quadratic error - C _*,* kg/m³ concentration of the tracer in the indicated compartment - C ₀ kg/m³ concentration of the tracer before the injection - C _t kg/m³ concentration of the tracer at the indicated time - C kg/m³ theoretical concentration of full mixed tracer - i index of an arbitrary tank - C _sim kg/m³ calculated concentration of the tracer by numerical integration method     

Daily and circadian rhythms of locomotor activity in the American lobster, Homarus americanus

It is widely accepted that American lobsters, Homarus americanus (Milne-Edwards), are nocturnally active. However, the degree to which this rhythm is expressed by different individuals and the underlying causes of lobster activity rhythms, are poorly understood. In order to address these issues we recorded daily patterns of lobster locomotion using two novel techniques. In the first, reed switch assemblies were used to monitor the distance traveled by freely moving lobsters (n=43), each fitted with a small magnet, as they walked around a 1 m diameter racetrack. The advantages of this technique included: (1) lobsters were freely moving; (2) the system could be deployed in laboratory tanks or in the field and; (3) actual distances moved were measured, not just relative activity. The second technique involved placing individual lobsters (n=10) into custom-designed running wheels. This allowed for continuous monitoring of locomotor activity for extended durations (>45 days) under normal light/dark (L/D) cycles, as well as in constant darkness (D/D) and constant light (L/L).Under ambient light conditions lobsters in the racetracks moved an average of 60.1±6.5 m/day in flow-through seawater tanks. Overall, lobsters were significantly more active at night, moving 4.1±0.4 m/h in the dark vs. 1.0±0.2 m/h in the light. However, many of the lobsters moved as much during the day as during the night.Lobsters in the running wheels moved an average of 36.6±11.7 m/day and 80% expressed clear daily rhythms of activity, with a mean periodicity of 24.0±0.1 h under L/D conditions. Under D/D conditions 90% of the animals expressed free-running circadian rhythms with a mean periodicity of 24.2±0.3 h, indicating that this species possesses endogenous rhythmicity. While the running wheel results show that the nocturnal pattern of locomotor activity for this species is strongly influenced by an endogenous circadian clock, the results from the racetracks show that there is remarkable variability in the extent to which they express this pattern under natural conditions.     

Response of sorghum to a water gradient and potassium variable

Summary A field study utilizing a modified line-source sprinkler system was used to investigate the hydrologic balance of a sorghum crop subjected to water deficit stress. An additional objective was to evaluate the evapotranspiration-yield relationship and to determine if the water use efficiency could be improved through the addition of increased amounts of potassium. Sixteen lysimeters packed with a Greenfield sandy loam and planted to sorghum (Sorghum bicolor) were placed under a moisture gradient irrigation system. Four levels of potassium were used in the experiment (0, 100, 200 or 300 Kg/ha). The results indicated that when the leaching fraction was set at 0.15 near the source, the predicted leaching fraction would go to zero at 10 meters from the source. Although yield was related to evapotranspiration, a better fit to the yield data was obtained when a parameter that included an availability and deficit term was used. The relationship was not linear but instead was described by an exponential fit. This parameter was also highly sensitive to leaf water potential and diffusion resistance. Finally, water use efficiency increased with yield but showed no correlation with potassium application.