C-terminal armadillo repeats are essential and sufficient for association of the plant U-box armadillo E3 ubiquitin ligase SAUL1 with the plasma membrane

Ubiquitination plays important roles in plant growth and development. Whereas ubiquitin-dependent protein degradation and modulation in the cytoplasm and nucleus are well established in plants, ubiquitination events mediated by E3 ubiquitin ligases at the plasma membrane are largely unknown. Here, it is demonstrated that the suppressor of premature senescence and cell death SENESCENCE-ASSOCIATED UBIQUITIN LIGASE 1 (SAUL1), a plant U-box armadillo repeat (PUB-ARM) E3 ubiquitin ligase, localizes at the plasma membrane. Among the members of the PUB-ARM protein family, this localization is unique to SAUL1 and its two closest homologues. A novel armadillo repeat domain was identified at the SAUL1 C-terminus that directs specific association with the plasma membrane and is crucial for SAUL1 function in vivo. The data suggest that a small subgroup of PUB-ARM proteins including SAUL1 have functions at the plasma membrane probably by modifying target proteins by ubiquitination.     

Mitochondrial dynamics and their impact on T cell function

Energy supply is the most prominent function of mitochondria, but in addition, mitochondria are indispensable for a multitude of other important cellular functions including calcium (Ca(2+)) signaling and buffering, the supply of metabolites and the sequestration of apoptotic factors. The efficiency of those functions highly depends on the proper positioning of mitochondria within the cytosol. In lymphocytes, mitochondria preferentially localize into the vicinity (～200nm) of the immune synapse (IS). This localization is regulated by motor-based cytoskeleton-mediated transport, the fusion/fission dynamics of mitochondria, and probably also through tethering with the ER. IS formation also induces the accumulation of CRAC/ORAI1 Ca(2+) channels, the CRAC/ORAI channel activator STIM1, K(+) channels and plasma membrane Ca(2+) ATPase (PMCA) within the IS. Such a large agglomeration of Ca(2+) binding organelles and proteins highlights the IS as a critical cellular compartment for Ca(2+) dependent lymphocyte activation. At the IS, Ca(2+) microdomains generated beneath open CRAC/ORAI channels provide a rapid, robust and reliable mechanism for driving cellular responses in mast cells and T cells. Here, we discuss the relevance of motor-based mitochondrial transport, fusion, fission and tethering for mitochondrial localization in T cells and the importance of subplasmalemmal mitochondria to control local CRAC/ORAI1-dependent Ca(2+) microdomains at the IS for efficient T lymphocyte activation.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).     

Biophysical and biochemical approach to locating an inhibitor binding site on cholesteryl ester transfer protein

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood. To aid in the study of inhibitor interactions with CETP, a torcetrapib-related compound was coupled to different biotin-terminated spacer groups, and the binding of CETP to the streptavidin-bound conjugates was monitored on agarose beads and in a surface plasmon resonance biosensor. CETP binding was poor with a 2.0 nm spacer arm, but efficient with polyethyleneglycol spacers of 3.5 or 4.6 nm. The conjugate based on a 4.6 nm spacer was used for further biosensor experiments. Soluble inhibitor blocked the binding of CETP to the immobilized drug, as did preincubation with a disulfide-containing covalent inhibitor. To provide a first estimate of the binding site for torcetrapib-like inhibitors, CETP was modified with a disulfide-containing agent that modifies Cys-13 of CETP. Mass spectrometry of the modified protein indicated that a single half-molecule of the disulfide was covalently bound to CETP, and peptide mapping after digestion with pepsin confirmed previous reports based on mutagenesis that Cys-13 was the site of modification. Modified CETP was unable to bind to the biosensor-mounted torcetrapib analog, indicating that the binding site on CETP for torcetrapib is in the lipid-binding pocket near the N-terminus of the protein. The crystal structure of CETP shows that the sulfhydryl group of Cys-13 resides at the bottom of this pocket.     

Redox properties of the calcium chelator Fura-2 in mimetic biomembranes

Fura-2 is one of the most commonly used fluorescent dyes to analyze the cytosolic Ca(2+) concentration ([Ca(2+)](i)) of living cells. Fura-2-dependent measurements of [Ca(2+)](i) are susceptible to changes of pH, reactive oxygen species concentration and membrane potential. Fura-2 is often loaded over the lipophilic cell membrane into the cytosol of a cell in its esterified form (Fura-2/AM) which is then cleaved by endogenous esterases. We have analyzed the electrochemical properties of Fura-2/AM and Fura-2 salt by cyclic voltammetry ("three-phase" and "thin-film" electrode methods). Using Fura-2/AM as a redox facilitator, we were able to mimic the transport of various ions across a lipophilic barrier. We show that Fura-2/AM in this biomimetic set-up can be reversibly oxidized in a single electrochemical step. Its redox reaction was highly proton sensitive in buffers with pH< or =6. At physiological pH of around 7.0, the oxidation of Fura-2/AM was coupled to an uptake of mono-anions across the liquid-liquid interface. The voltage-dependence of the redox cycle was sensitive to the free Ca(2+) concentration, either after de-esterification of Fura-2/AM, or when Fura-2 salt was used. The complex between Fura-2 and Ca(2+) ions is ionic (complexation occurs via the dissociated negative groups of Fura forms), while the redox transformations in Fura-2 occurs at the nitrogen atoms of the amino groups. Our results suggest that redox transformations of the Fura-2 forms do not affect the binding ability toward Ca(2+) ions and thus do not interfere with [Ca(2+)](i) measurements.     

A Novel K+ Channel Expressed in Carrot Roots with a Low Susceptibility Toward Metal Ions

Kdc1 is a novel K⁺-channel gene cloned from carrot roots, and which is also present in cultured carrot cells. We investigated the characteristics of the ionic current elicited in Xenopus oocytes coinjected with KDC1 (K⁺-Daucus carota 1) and KAT1 (from Arabidopsis thaliana) RNA. Expressed heteromeric channels displayed inward-rectifying potassium currents whose kinetics, voltage characteristics, and inhibition by metal ions depended on KDC1:KAT1 ratios. At low KDC1:KAT1 ratios, Zn²⁺ inhibition of heteromeric K⁺ current was less pronounced compared to homomeric KAT1 channels, while at higher KDC1:KAT1 ratios, the addition of Zn²⁺ even produced an increase in current. Under the same conditions, the Ni²⁺ inhibition of the current was also reduced, but no current increase was observed. These effects might be explained by the unusual amino acid composition of the KDC1 protein in terms of histidine residues that are absent in the pore region, but abundant (four per subunit) in the proximity of the pore entrance. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals.     

Splinting or surgery for carpal tunnel syndrome? Design of a randomized controlled trial [ISRCTN18853827]

Background  

Carpal tunnel syndrome is a common disorder, which can be treated with surgery or conservative options. However, there is insufficient evidence and no consensus among physicians with regard to the preferred treatment for carpal tunnel syndrome. Therefore, a randomized controlled trial is conducted to compare the short- and long-term efficacy of surgery and splinting in patients with carpal tunnel syndrome. An attempt is also made to avoid the (methodological) limitations encountered in earlier trials on the efficacy of various treatment options for carpal tunnel syndrome.     

Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I).

Waardenburg syndrome type I (WS-I) is an autosomal dominant disorder characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Klein-Waardenburg syndrome (WS-III) is a disorder with many of the same characteristics as WS-I and includes musculoskeletal abnormalities. We have recently reported the identification and characterization of one of the first gene defects, in the human PAX3 gene, which causes WS-I. PAX3 is a DNA-binding protein that contains a structural motif known as the paired domain and is believed to regulate the expression of other genes. In this report we describe two new mutations, in the human PAX3 gene, that are associated with WS. One mutation was found in a family with WS-I, while the other mutation was found in a family with WS-III. Both mutations were in the highly conserved paired domain of the human PAX3 gene and are similar to other mutations that cause WS. The results indicate that mutations in the PAX3 gene can cause both WS-I and WS-III.