Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

The alphavbeta6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are common primary hepatic malignancies. Their immunohistological differentiation using specific markers is pivotal for treatment and prognosis. We found alphavbeta6 integrin strongly upregulated in biliary fibrosis, but its expression in primary and secondary liver tumours is unknown. Here, we aimed to evaluate the diagnostic applicability of alphavbeta6 integrin in differentiating primary liver cancers.

Polyunsaturated fatty acid-enriched diets used for the treatment of canine chronic enteropathies decrease the abundance of selected genes of cholesterol homeostasis

Lipids are important for cell function and survival, but abnormal concentrations may lead to various diseases. Cholesterol homeostasis is greatly dependent on the active transport by membrane proteins, whose activities coordinate lipid status with cellular function. Intestinal Niemann-Pick C1-Like 1 protein (NPC1L1) and scavenger receptor B1 (SR-B1) participate in the uptake of extracellular cholesterol, whereas ATP binding cassette A1 (ABCA1) mediates the efflux of excessive intracellular cholesterol. Caveolin-1 binds cholesterol and fatty acids (FA) and participates in cholesterol trafficking. Sterol response element binding protein-2 (SREBP-2) is a sensor that regulates intracellular cholesterol synthesis. Given that cholesterol is a constituent of chylomicrons, whose synthesis is enhanced with an increased FA supply, we tested the hypothesis that feeding polyunsaturated FA (PUFA)-enriched diets in treatment of canine chronic enteropathies alters the mRNA expression of genes involved in cholesterol homeostasis. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we compared the mRNA abundance of NPC1L1, SR-B1, ABCA1, caveolin-1, and SREBP-2 in duodenal mucosal biopsies of dogs with food-responsive diarrhea (FRD; n=14) and inflammatory bowel disease (IBD; n=7) before and after treatment with cholesterol-free PUFA-enriched diets and in healthy controls (n=14). The abundance of caveolin-1, ABCA1, and SREBP-2 were altered by PUFA-enriched diets (P<0.05), whereas that of NPC1L1 and SR-B1 mRNA remained unchanged. The gene expression of caveolin-1, ABCA1, and SREBP-2 was down-regulated (P<0.05) by PUFA-enriched diets in IBD dogs only. Our results suggest that feeding PUFA-enriched diets may alter cholesterol homeostasis in duodenal mucosal cells of dogs suffering from IBD.

Generation and characterization of stable, highly concentrated titanium dioxide nanoparticle aerosols for rodent inhalation studies

The intensive use of nano-sized titanium dioxide (TiO2) particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of TiO2 nanoparticles (NP) with biological systems ideally needs to be investigated using physico-chemically uniform and well-characterized NP. In this article, we describe the reproducible production of TiO2 NP aerosols using spark ignition technology. Because currently no data are available on inhaled NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation studies in rodents. For anticipated in vivo dosimetry analyses, TiO2 NP were radiolabeled with 48V by proton irradiation of the titanium electrodes of the spark generator. The dissolution rate of the 48V label was about 1% within the first day. The highly concentrated, polydisperse TiO2 NP aerosol (3–6 × 106 cm−3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation, and number concentration. Extensive characterization of NP chemical composition, physical structure, morphology, and specific surface area was performed. The originally generated amorphous TiO2 NP were converted into crystalline anatase TiO2 NP by thermal annealing at 950 °C. Both crystalline and amorphous 20-nm TiO2 NP were chain agglomerated/aggregated, consisting of primary particles in the range of 5 nm. Disintegration of the deposited TiO2 NP in lung tissue was not detectable within 24 h.

Highly improved metabolic stability and pharmacokinetics of indium-111-DOTA-gastrin conjugates for targeting of the gastrin receptor

The development of metabolically stable radiolabeled gastrin analogues with suitable pharmacokinetics is a topic of recent research activity. These imaging vectors are of interest because the gastrin/CCK2 receptor is highly overexpressed in different tumors such as medullary thyroid cancer, neuroendocrine tumors, and SCLC. The drawback of current targeting agents is either their metabolic instability or their high kidney uptake. We present the synthesis and in vitro and in vivo evaluation of 11 (111)In-labeled DOTA-conjugated peptides that differ by their spacer between the peptide and the chelate. We introduced uncharged but hydrophilic spacers such as oligoethyleneglycol, serine, and glutamine. The affinity of all radiopeptides was high with IC(50) values between 0.5 and 4.8 nM. The improvement of human serum stability is 500-fold within this series of compounds. In addition the kidney uptake could be lowered distinctly and the tumor-to-kidney ratio improved almost 60-fold if compared with radiotracers having charged spacers such as glutamic acid.

Genome sequence of Desulfovibrio sp. A2, a highly copper resistant, sulfate-reducing bacterium isolated from effluents of a zinc smelter at the Urals

Desulfovibrio sp. A2 is an anaerobic gram-negative sulfate-reducing bacterium with remarkable tolerance to copper. It was isolated from wastewater effluents of a zinc smelter at the Urals. Here, we report the 4.2-Mb draft genome sequence of Desulfovibrio sp. A2 and identify potential copper resistance mechanisms.