Adaptation of the Griess reaction for detection of nitrite in human plasma

The determination of nitrite in human plasma or serum has been most frequently used as a marker of nitric oxide (NO) production. In addition, it has recently been suggested that nitrite could act as a vasodilating agent at physiological concentrations by NO delivery. Therefore, nitrite determination in biological fluids is becoming increasingly important. The most frequently used method to measure nitrite is based on the spectrophotometric analysis of the azo dye obtained after reaction with the Griess reagent. This method has some limitations regarding detection limit and sensitivity, thus resulting unsuitable for nitrite detection in plasma. We have identified some drawbacks and modified the original procedure to overcome these problems. By the use of the newly developed method, we measured 221±72 nM nitrite in human plasma from healthy donors.     

Tamoxifen-inducible NaV1.8-CreERT2 recombinase activity in nociceptive neurons of dorsal root ganglia

To explore the function of genes expressed in adult mouse nociceptive neurons, we generated heterozygous knock-in mice expressing the tamoxifen-inducible Cre recombinase construct CreERT2 downstream of the Na(V)1.8 promoter. CreERT2 encodes a Cre recombinase (Cre) fused to a mutant estrogen ligand-binding domain (ERT2) that requires the presence of tamoxifen for activity. We have previously shown that heterozygous Na(V)1.8-Cre mice will delete loxP flanked genes specifically in nociceptive sensory neurons from embryonic day 14. We therefore used the same strategy of homologous recombination and mouse generation, substituting the Cre cassette with CreERT2. No functional Cre recombinase activity was found in CreERT2 mice crossed with reporter mice in the absence of tamoxifen. We found that, as with Na(V)1.8-Cre mice, functional Cre recombinase was present in nociceptive sensory neurons after tamoxifen induction in vivo. However, the percentage of dorsal root ganglion (DRG) neurons expressing functional Cre activity was much reduced (<10% of the number found in the Na(V)1.8-Cre mouse). We also examined Cre recombinase activity in sensory neurons in culture. After treatment with 1 muM tamoxifen for 48 h, 15% of DRG neurons showed Cre activity. Na(V)1.8-CreERT2 animals may thus be useful for single cell studies of the functional consequences of gene ablation in culture, but are unlikely to be useful for behavioral studies.     

Enzymatic production of 18-β-glycyrrhetinic acid from Glycyrrhiza glabra L.

Enzymatic conversion of glycyrrhizinic acid (GL), from root extract of Glycyrrhiza glabra L., into 18--glycyrrhetinic acid (GA) was achieved by using a commercial preparation of -glucuronidase from Aspergillus niger. Two different processes were tested: a batch process and a continuous process. The continuous process was the better one, giving 100% conversion of GL after 5 d and 80% recovery of enzyme activity. The process yielded a product containing 40% GA.     

The mitochondrial calcium uniporter is a multimer that can include a dominant‐negative pore‐forming subunit

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red‐sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino‐acid substitutions in the pore region and indeed MCUb does not form a calcium‐permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant‐negative effect, reducing the [Ca²⁺]_mt increases evoked by agonist stimulation. Accordingly, in vitro co‐expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant‐negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.     

Crowding Activates ClpB and Enhances Its Association with DnaK for Efficient Protein Aggregate Reactivation

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.     

IMPLICATION OF TNF-α CONVERTASE (TACE/ADAM17) IN INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION AND INFLAMMATION IN AN EXPERIMENTAL MODEL OF COLITIS

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-α convertase (TACE; ADAM17). TNF-α plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-α levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-α and NO−x levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-α. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-α release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-α release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

The furoxan system: design of selective nitric oxide (NO) donor inhibitors of COX-2 endowed with anti-aggregatory and vasodilating activities

Several NO donor 3,4-diphenylfuroxan (= 3,4-diphenyl-1,2,5-oxadiazole 2-oxide) derivatives were synthesized and tested for their COX-inhibiting activities. The products were found to be selective COX-2 inhibitors, similar to the structurally related furazans (3,4-diphenyl-1,2,5-oxadiazole), devoid of the NO release property. This behavior was confirmed by a molecular-docking study. The NO-dependent platelet anti-aggregatory and vasodilating activities of the new furoxans 5-7 were studied in vitro. These properties can be modulated by inserting an appropriate spacer between the 4-phenyl group and the furoxan ring, giving rise to new, selective COX-2 furoxan derivatives endowed with anti-aggregatory and vasodilating activities, and with potentially reduced cardiotoxicities.     

New insights into the pathogenesis of vitiligo: imbalance of epidermal cytokines at sites of lesions

Vitiligo is a skin disease that is caused by selective destruction of melanocytes and is characterized by white spots. Melanocytes and keratinocytes seem to exhibit a functional close relationship, mediated at least in part by keratinocyte-derived cytokines, which seem important for survival and activity of melanocytic cells. We wanted to investigate the hypothesis that in vitiligo the expression of epidermal cytokines may be modified compared with normal skin. In 15 patients with active, non-segmental vitiligo, biopsies were obtained from lesional, perilesional and non-lesional skin; normal skin from five healthy donors was also tested. Tissue sections were tested using immunohistochemistry for the expression of keratinocyte-derived cytokines with stimulating activity, such as granulocyte-monocyte colony stimulating factor (GM-CSF), basic fibroblastic growth factor (bFGF), and stem cell factor (SCF) or with inhibiting activity, such as interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) on melanocytes. Cytokine receptors and specific melanocytic markers were also investigated. No melanocyte was identified in lesional skin by means of specific markers or c-kit receptor, whereas in perilesional, non-lesional and healthy skin, melanocytes were found in similar number. In vitiligo skin a significantly lower expression of GM-CSF, bFGF and SCF was found, and a significantly higher expression of IL-6 and TNF-alpha was detected, compared with perilesional, non-lesional and healthy skin. In conclusion, we provided evidence that a significant change of epidermal cytokines exists in vitiligo skin compared with perilesional, non-lesional and healthy skin, suggesting that the cytokine production of epidermal microenvironment may be involved in vitiligo.