The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice

Bone is the major store for Ca(2+) in the body and plays an important role in Ca(2+) homeostasis. During bone formation and resorption Ca(2+) must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca(2+) transport machinery in these bone cells. In this study, we examined the epithelial Ca(2+) channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca(2+) transport genes, calbindin-D(9k) and/or -D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+) ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6(D541A/D541A) mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca(2+) transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice.     

Essential role for CAML in follicular B cell survival and homeostasis

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.     

Conservation agriculture, increased organic carbon in the top-soil macro-aggregates and reduced soil CO2 emissions

Background and aims

Conservation agriculture, the combination of minimal soil movement (zero or reduced tillage), crop residue retention and crop rotation, might have the potential to increase soil organic C content and reduce emissions of CO₂.

Methods

Three management factors were analyzed: (1) tillage (zero tillage (ZT) or conventional tillage (CT)), (2) crop rotation (wheat monoculture (W), maize monoculture (M) and maize-wheat rotation (R)), and (3) residue management (with (+r), or without (?r) crop residues). Samples were taken from the 0–5 and 5–10?cm soil layers and separated in micro-aggregates (< 0.25?mm), small macro-aggregates (0.25 to 1?mm) and large macro-aggregates (1 to 8?mm). The carbon content of each aggregate fraction was determined.

Results

Zero tillage combined with crop rotation and crop residues retention resulted in a higher proportion of macro-aggregates. In the 0–5?cm layer, plots with a crop rotation and monoculture of maize and wheat in ZT+r had the greatest proportion of large stable macro-aggregates (40%) and highest mean weighted diameter (MWD) (1.7?mm). The plots with CT had the largest proportion of micro-aggregates (27%). In the 5–10?cm layer, plots with residue retention in both CT and ZT (maize 1?mm and wheat 1.5?mm) or with monoculture of wheat in plots under ZT without residues (1.4?mm) had the greatest MWD. The 0–10?cm soil layer had a greater proportion of small macroaggregates compared to large macro-aggregates and micro-aggregates. In the 0–10?cm layer of soil with residues retention and maize or wheat, the greatest C content was found in the small and large macro-aggregates. The small macro-aggregates contributed most C to the organic C of the sample. For soil cultivated with maize, the CT treatments had significantly higher CO₂ emissions than the ZT treatments. For soil cultivated with wheat, CTR-r had significantly higher CO₂ emissions than all other treatments.

Conclusion

Reduction in soil disturbance combined with residue retention increased the C retained in the small and large macro-aggregates of the top soil due to greater aggregate stability and reduced the emissions of CO₂ compared with conventional tillage without residues retention and maize monoculture (a cultivation system normally used in the central highlands of Mexico).     

Mechanism and specificity of the human paracaspase MALT1

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.     

Conditional deletion of calcium-modulating cyclophilin ligand causes deafness in mice

Calcium-modulating cyclophilin ligand (Caml) is a ubiquitously expressed cytoplasmic protein that is involved in multiple signaling and developmental pathways. An observation in our laboratory of a protein-protein interaction between Caml and the cytoplasmic region of Cadherin23 led us to speculate that Caml might be important in the inner ear and play a role in the development and/or function of hair cells. To address this question, we generated a mouse line in which Caml expression was eliminated in Atoh1-expressing cells of the inner ear upon administration of tamoxifen. Tamoxifen was administered immediately after birth to neonates to assess the effect of loss of Caml in the inner ear during postnatal development. Hearing in treated animals was tested by auditory brain stem response (ABR) analysis and cochlear pathology was evaluated by light microscopy. Lack of Caml expression in the inner ear leads to severe loss of cochlear hair cells and complete deafness. Elucidating the role of Caml in the inner ear will aid our understanding of the molecular pathways important for auditory development and function.     

alphaB-crystallin competes with Alzheimer's disease beta-amyloid peptide for peptide-peptide interactions and induces oxidation of Abeta-Met35

Alzheimer's disease (AD) is associated with plaque deposition in the brain of AD patients. The major component of the aggregate is a 39-42 long peptide termed beta-amyloid (Abeta). Except for Abeta, plaques contain several other components which co-precipitate together with Abeta. One such component is the small heat shock protein (sHSP) alphaB-crystallin. Instead of preventing the cell from the neurotoxicity of Abeta, alphaB-crystallin induces an increased neurotoxicity. We find - using solution state NMR spectroscopy - that alphaB-crystallin competes efficiently for Abeta monomer-monomer interactions. Interactions between Abeta and alphaB-crystallin involve the hydrophobic core residues 17-21 as well as residues 31-32 of Abeta, and thus the same chemical groups which are important for Abeta aggregation. In the presence of alphaB-crystallin, Met35 in Abeta becomes efficiently oxidized. In order to quantify the redox properties of the different complexes consisting of Abeta/alphaB-crystallin/copper, we suggest an NMR assay which allows to estimate the electrochemical properties indirectly by monitoring the rate of glutathion (GSH) auto-oxidation. The oxidation of the side chain Met35 in Abeta might account for the increased neurotoxicity and the inability of Abeta to form fibrillar structures, which has been observed previously in the presence of alphaB-crystallin [Stege, G.J. et al. (1999) The molecular chaperone alphaB-crystallin enhances amyloid-beta neurotoxicity. Biochem. Biophys. Res. Commun. 262, 152-156.].     

Wind mediated dispersal of freshwater invertebrates in a rock pool metacommunity: differences in dispersal capacities and modes

Current evidence suggests regular overland transport of different freshwater invertebrates by wind, mainly over short distances. Yet, very little is known about the mechanism and scale of this process or about differences in wind dispersal dynamics and capacities among taxa and propagule types. We investigated wind dispersal of freshwater invertebrates in a cluster of temporary rock pools (spatial scale: 9,000 m²) in South Africa. Dispersing propagules and propagule bank fragments (i.e. aggregates of sediments and propagules) were intercepted during 1 month using a combination of windsocks (1.5 m above ground level) and sticky traps (ground level). The potential movement of propagule bank fragments (i.e. aggregates of propagules and sediments) was also simulated by tracking inter-pool movements of differently sized artificial substrate fragments similar to dry propagule bank fragments. We detected differences in the composition of dispersing communities intercepted at different altitudes (ground level and at 1.5 m). Comparison of dispersal distance distributions also revealed significant differences among taxa. Overall, larger propagule types (e.g. adult ostracods and oribatid mites) dominantly travelled near ground level while small resting eggs and cryptobiotic life stages of copepods were most frequently collected at higher altitudes (1.5 m) and dispersed over the longest distances. Finally, not only dispersal of single propagules but also ground level transport of propagule bank fragments was shown to contribute to local dispersal dynamics in temporary aquatic habitats.     

Non-Helicobacter pylori helicobacters detected in the stomach of humans comprise several naturally occurring Helicobacter species in animals

Besides the well-known gastric pathogen Helicobacter pylori , other Helicobacter species with a spiral morphology have been detected in a minority of human patients who have undergone gastroscopy. The very fastidious nature of these non- Helicobacter pylori helicobacters (NHPH) makes their in vitro isolation difficult. These organisms have been designated ' Helicobacter heilmannii '. However, sequencing of several genes detected in NHPH-infected tissues has shown that the ' H. heilmannii ' group comprises at least five different Helicobacter species, all of them known to colonize the stomach of animals. Recent investigations have indicated that Helicobacter suis is the most prevalent NHPH species in human. This species has only recently been isolated in vitro from porcine stomach mucosa. Other NHPH that colonize the human stomach are Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis and ' Candidatus Helicobacter heilmannii'. In numerous case reports of human gastric NHPH infections, no substantial information is available about the species status of the infecting strain, making it difficult to link the species with certain pathologies. This review aims to clarify the complex nomenclature of NHPH species associated with human gastric disease and their possible animal origin. It is proposed to use the term 'gastric NHPH' to designate gastric spirals that are morphologically different from H. pylori when no identification is available at the species level. Species designations should be reserved for those situations in which the species is defined.     

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.