Cutting edge: a key pathogenic role of IL-27 in T cell- mediated hepatitis

The signals driving T cell activation in T cell-mediated fulminant hepatitis are not fully understood. In this study, we identify the cytokine IL-27p28/EBI3 as a major pathogenic factor in the ConA model of T cell-mediated hepatitis. We found an up-regulation of hepatic EBI3 and p28 expression and augmented levels of IL-27 in wild-type mice after ConA administration, suggesting a potential pathogenic role of this cytokine in ConA hepatitis. Consistently, IL-27 EBI3-deficient mice were almost completely protected from ConA-induced liver damage. Such protection was associated with reduced levels of IFN-gamma and its signaling proteins pSTAT-1 and T-bet. Finally, in vivo blockade of IL-27 function using a soluble IL-27 receptor fusion protein led to reduced pSTAT1 levels and suppression of liver injury. Taken together, these data demonstrate a key pathogenic role of IL-27 in T cell-mediated liver injury. Furthermore, in vivo blockade of IL-27 emerges as a novel potential therapy for T cell-mediated hepatitis.     

A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.     

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.     

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.     

LIS1 and spindle orientation in neuroepithelial cells

Asymmetric stem cell division is thought to require precise orientation of the mitotic spindle. However, a recent study in Cell (Yingling et al., 2008) analyzes the role of LIS1 in the developing mouse brain and shows that spindle orientation is more important during early, symmetric progenitor cell divisions than for later asymmetric divisions.     

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.