Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

The new insight into dynamic crossover in glass forming liquids from the apparent enthalpy analysis

One of the most intriguing phenomena in glass forming systems is the dynamic crossover (T(B)), occurring well above the glass temperature (T(g)). So far, it was estimated mainly from the linearized derivative analysis of the primary relaxation time τ(T) or viscosity η(T) experimental data, originally proposed by Stickel et al. [J. Chem. Phys. 104, 2043 (1996); J. Chem. Phys. 107, 1086 (1997)]. However, this formal procedure is based on the general validity of the Vogel-Fulcher-Tammann equation, which has been strongly questioned recently [T. Hecksher et al. Nature Phys. 4, 737 (2008); P. Lunkenheimer et al. Phys. Rev. E 81, 051504 (2010); J. C. Martinez-Garcia et al. J. Chem. Phys. 134, 024512 (2011)]. We present a qualitatively new way to identify the dynamic crossover based on the apparent enthalpy space (H(a)(') = dlnτ/d(1/T)) analysis via a new plot lnH(a)(') vs. 1∕T supported by the Savitzky-Golay filtering procedure for getting an insight into the noise-distorted high order derivatives. It is shown that depending on the ratio between the "virtual" fragility in the high temperature dynamic domain (m(high)) and the "real" fragility at T(g) (the low temperature dynamic domain, m = m(low)) glass formers can be splitted into two groups related to f < 1 and f > 1, (f = m(high)∕m(low)). The link of this phenomenon to the ratio between the apparent enthalpy and activation energy as well as the behavior of the configurational entropy is indicated.

Microwave-Assisted Bond Forming Reactions

ABSTRACT FOR PART I: PHOSPHA-MICHAEL ADDITIONS TO ACTIVATED INTERNAL ALKENES: STERIC AND ELECTRONIC EFFECTS A method for the phospha-Michael addition of bis(4-methyl)phenyl phosphine oxide to activated internal alkenes has been developed. Michael acceptors including cinnamates, crotonates, chalcones, and internal alkenes containing multiple activating groups were all successfully utilized in this reaction. The reaction was fairly tolerant of electron-donating and electron-withdrawing substituents on the Michael acceptor, and moderate to excellent yields (49-96%) of the adducts were isolated. When steric bulk was increased by a second substituent on the -position of the Michael-acceptor the reaction was suppressed. This was usually overcome by adding a second activating substituent to the -position. ABSTRACT FOR PART II: MICROWAVE-ASSISTED ARYLGOLD BOND FORMATION A microwave-assisted method was developed for the formation of arylgold complexes containing (2-Biphenyl)di-tert-butylphosphine (JohnPhos) as the supporting phosphine ligand. Arylboronic acids with increasingly bulky aromatic groups were screened to determine the steric limitations of the reaction. Arylgold complexes (JohnPhos)Au(p-methoxyphenyl), (JohnPhos)Au(2,4,6-trimethylphenyl), and (JohnPhos)Au(4-bromo-10-anthracene) were all synthesized by microwave irradiation at 70ºC in the presence of Cs2CO3 in either THF or iPrOH. Reactions performed with arylboronic acids containing unhindered ortho positions were carried out in THF. Arylboronic acids with substituents on the ortho position required iPrOH as the reaction solvent. Arylboronic acids with extreme steric hindrance on the ortho position of the aryl substituent, 2,4,6-triisopropylpphenylboronic acid, were unreactive. It was determined that increasing the irradiation temperature increased the formation of side products, therefore to promote conversion to the arylgold complex the duration of the reaction time was increased while maintaining a temperature of 70ºC. Arylgold complexes (JohnPhos)Au(p-methoxyphenyl), (JohnPhos)Au(2,4,6-trimethylphenyl), and (JohnPhos)Au(4-bromo-10-anthracene) were synthesized with moderate yields (40-69%).

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.