1B1, lanes 2 and 3) were both transferred to a PVDF membrane

1B1, lanes 2 and 3) were both transferred to a PVDF membrane

and submitted to Edman degradation. The first 34 amino acid residues from N-terminal sequencing of the reduced protein were determined to be LGPDIVSPPVCGNELLEVGEECDCGTPENCQNE (Fig. 2) and submitted to BLAST. The 10 first amino acids residues of the non-reduced moojenin obtained by Edman degradation showed the same sequence as the reduced moojenin (data not shown). The primary 17-AAG sequence of the reduced moojenin shared a high degree of identity with proteins of the PIIIb subclass of SVMPs, except for a proline (Pro208) where threonine (Thr208) is observed in other known sequences. This sequence begins at the spacer region in other members of the PIIIb subclass of SVMPs (residue 206 – numbering according to Jararhagin), such as the disintegrins Catrocollastatin-C (Calvete et al., 2000) and Jararhagin-C (Usami et al., 1994), suggesting that the moojenin had undergone autolysis. Subclass PIIIb metalloproteinases can undergo proteolysis/autolysis during secretion or in the Natural Product Library venom to generate disintegrin-like and cysteine-rich domains (DC domain) (Fox and Serrano, 2005). However, no proteinase domain released from the DC domain of a PIIIb metalloproteinease has been isolated intact from snake venom, since they are apparently unstable alone (Shimokawa et al., 1997; Moura-da-Silva

et al., 2003; Fox and Serrano, 2005 and Fox and Serrano, 2008). A spacer region, or linker, separates the M from the DC domain and includes a proteolytic site (Moura-da-Silva et al., 2003; Assakura et al., 2003; Muniz et al., 2008), but the cleavage site is not yet known. It has been observed that proteolytic processing occurs in the spacer domain in some members of each of the P classes Galactosylceramidase (Fox and Serrano, 2005). For example, processed PIIIb catrocollastatin-C (Fig. 2) has a spacer region linked to the disintegrin-like domain just as in reduced moojenin (Fox and Serrano, 2005), while native jararhagin-C (Usami et al., 1994) and ALT-C (Souza et al., 2000) contain only the DC domain. Other members of the PIII class undergo autolysis

under non-physiological conditions in vitro ( Takeya et al., 1993); however, the products of this proteolytic processing are not observed by SDS-PAGE under non-reducing conditions, suggesting these domains are connected by disulfide bonds ( Moura-da-Silva et al., 2003). Moreover, under reducing conditions the DC domain was seen without the M domain, since the latter alone is unstable. Other members of the PIII subclass can be manipulated to undergo in vitro autolysis, but the relevance of this processing in vivo is unclear ( Fox and Serrano, 2005). Under physiological conditions, Moojenin probably maintains both native and processed conformations, since the sequence determined for non-reduced moojenin begins at the spacer region.

One reason why we could not identify the relationships

One reason why we could not identify the relationships find more between them may be that the story-comprehension levels did not vary among the participants In fact, they answered the questions about the contents of the Story A and Story B almost perfectly (i.e., they marked 6–8 out of 8 in the questions about the contents of the each story). While the present results suggest mechanisms for phonemic restoration in speech comprehension,

only a limited number of participants were tested. To generalize the results, studies involving a larger number of participants are needed. In addition, assessing the neural activities of brain regions located deeply or frontally was difficult using MEG. Some brain regions involved in phonemic restoration might thus have been missed because of the limitations of MEG. Future studies using other neuroimaging techniques, such as fMRI and PET, would address this limitation. We found brain activations related to phonemic restoration for speech comprehension. The left transverse and superior temporal gyri activated in

response to white-noise stimuli while listening to and understanding the spoken stories, and these brain regions seem to contribute to phonemic restoration for speech comprehension through first processing of speech information. The left inferior frontal gyrus, including Broca’s area, was continuously activated throughout listening to and understanding the spoken stories, and this brain region may contribute

to phonemic restoration for speech comprehension through learn more unconscious sensory repair. These findings may help clarify the neural mechanisms of phonemic restoration and develop innovative treatment methods such as new linguistic training strategies for individuals who suffer from impaired speech comprehension, particularly in noisy environments. Twelve healthy male volunteers (mean (± standard deviation (SD)) age, 26.36±5.54 years) were enrolled in this study. Current smokers, individuals with a history of medical illness such as neurological disease, psychiatric disease, or developmental disorders including reading disabilities, or individuals taking chronic medications or supplements that affect the central nervous system were excluded from the Thymidylate synthase study. All participants had normal hearing and were right-handed according to the Edinburgh handedness inventory (Oldfield, 1971). Normal hearing was ensured by pure tone audiometry and the speech discrimination test. Conventional pure-tone audiometry and speech audiometry were performed using a diagnostic audiometer (AA-78; RION, Tokyo, Japan) in a sound-proof room to assess hearing acuity. In pure-tone audiometry, pure-tone hearing ability was judged normal when all of air-conduction pure-tone thresholds recorded at 7 audiometric frequencies, octave intervals from 125 to 8000 Hz, did not exceed 20 dB hearing level (HL).

, 1999 and Schell and Strick, 1984), which is densely interconnec

, 1999 and Schell and Strick, 1984), which is densely interconnected with the motor cortex (Tanji, 1994). Vim is reciprocally connected with motor cortex and Vop receives considerable input from these cortical motor structures

through the cortico–thalamic Roscovitine molecular weight projection. These connections may explain why increased firing rates in postural tremor are found in both Vim and Vop (Hirai and Jones, 1989, Hua and Lenz, 2005 and Lenz et al., 2002). Spectral analysis showed that coherence and phase for intention ET were more similar to cerebellar tremor than to postural ET (Fig. 4 and Fig. 5). The phase lead was significantly greater for postural ET than for intention ET. Intention ET and cerebellar tremor patients had much lower coherence and SNR than

postural ET subjects. Overall, this physiology seems to confirm the clinical observation that intention ET is similar to cerebellar tremor (Brennan et al., 2002 and Elble and Koller, 1990). The frequency of peak spike power was higher for the postural ET group than for the intention ET or cerebellar tremor groups, which is consistent with the similarities noted above. Clinically, cerebellar tremor is of lower frequency than essential tremor (Elble, 2006 and Findley and Koller, 1987). The lower frequency of intention ET and cerebellar AZD6244 cell line tremor versus postural ET again suggests that intention ET is more like cerebellar tremor than postural ET. Patient 4 was not an exceptional case since there was no bias in the sampling of cell types or predominance of a particular nuclear location. Patient 4 was indistinguishable from other patients in the intention ET group in terms of spontaneous firing rates, and frequency of peak spike power in the tremor range. If a pacemaker of the cerebellum and related systems drives intention ET then a lesion of the cerebellum might further reduce this patient’s tremor and would not increase tremor. In postural ET, lesions of the cerebellum D-malate dehydrogenase or pontine cerebellar connections decrease tremor (Dupuis et al., 1989 and Nagaratnam and Kalasabail, 1997). Therefore, the increase in intention tremor following the cerebellar stroke

in patient 4 is consistent with a mechanism of intention ET related to disruption of the cerebellum rather than to a pacemaker (Destexhe and Sejnowski, 2001, Lenz et al., 1994b and Stein and Oguztoreli, 1976). This difference could be tested by imaging studies of basal- and tremor-evoked activity in patients with either postural ET or intention ET. The analysis of thalamic neuronal activity and of the spike×EMG cross-correlation demonstrates that intention ET is more like cerebellar tremor than like postural ET. Of course, cerebellar tremor is often associated with lesions of the cerebellum or its output pathways (Carrea and Mettler, 1947 and Gilman et al., 1976). Therefore, the present results suggest the intention ET is associated with disruption of the cerebellum, which may be consistent with the histologic changes in Essential Tremor (Louis et al.

In our

study, a gene encoding ARF was up-regulated during

In our

study, a gene encoding ARF was up-regulated during kernel development, suggesting that it also plays a similar role in differentiation during maize embryogenesis. Moreover, many putative protein kinase genes were differentially expressed at various times, which were involved in signaling transduction pathway during maize ear development. For example, homeobox–leucine zipper family protein, a member of the LRR (leucine-rich repeat family protein) subfamily, might be required to increase cell size and the rate of embryonic development. A gene encoding a homeobox–leucine zipper family protein was up-regulated from 15 to 25 DAP, suggesting that this kinase might function in forming organs in the maize embryo. Therefore, we deduced that the accumulation of bZIP transcripts, MADS box-like proteins, and CTLA-4 inhibiton putative laccase resulting VE-822 ic50 from the down-regulation of miR528 might enhance auxin response and, in turn, seed germination in the final stage of seed development (after 22 DAP). However, further work is required to elucidate the functions of these protein kinases. By constructing a small RNA library and characterizing miRNA expression profiles in pooled maize ears at 10, 15, 20, 22, 25 and 30 DAP, at least 21 miRNAs were differentially

expressed. qRT-PCR verification for miR528a and miR167a/miR160b indicated that these miRNAs might be involved in ear development and germination. In addition, functional predictions of target genes indicated that most of

these differentially expressed miRNAs tended to have target genes that were involved in signal transduction and cell communication, particularly those involved in the auxin-signaling pathway. The results of gene expression analysis of candidate germination-associated miRNAs performed by microarray hybridization with a maize genome array demonstrated the differential expression of genes involved in plant hormone signaling pathways. This suggested that phytohormones might play a critical Cell Penetrating Peptide role in the maize ear developmental process. We showed that in combination with other miRNAs, miR528a regulates a putative laccase, a Ring-H2 zinc finger protein and a MADS box-like protein, whereas miR167a and miR160b regulate target genes including ARF (auxin response factor), a member of the B3 transcription factor family that is important for ear germination and physiology. Thus the small RNA transcriptomes and mRNA obtained in this study provide considerable insight into the expression and function of small RNAs in the development of viviparous kernels. This study was supported by grants from the Educational Commission of Sichuan Province (No. 2006J13-039), the Doctoral Program Foundation of Institutions of Higher Education of China (No. 20095103120002), and the National Natural Science Foundation of China (No. 30900901). “
“Rye (Secale cereale L.) is an important cereal crop worldwide.

5 °C) Diffuse reflectance (DR) measurements were performed in di

5 °C). Diffuse reflectance (DR) measurements were performed in diffuse reflection mode with a Shimadzu sampling accessory (DRS8000A). The ground coffee sample was mixed with KBr Cell Cycle inhibitor (100 mg) and then 23 mg of this mixture was placed inside the sample port. Pure KBr was employed as reference material (background spectrum). All spectra were recorded within a range

of 4000–400 cm−1 with a 4 cm−1 resolution and 20 scans, and submitted to background subtraction. The spectra were also truncated to 2500 data points in the range of 3100–600 cm−1, in order to eliminate noise readings present in the upper and lower ends of the spectra. Preliminary tests were performed in order to evaluate the effect of particle size (0.39 mm < D < 0.5 mm; 0.25 mm < D < 0.39 mm; 0.15 mm < D < 0.25 mm; and D < 0.15 mm) and coffee/KBr mass ratio (2, 5, 10, 20, 30, 40 and 50%) on the quality of the obtained spectra. The conditions that provided the best quality spectra (higher intensity and lower noise interference) were D < 0.15 mm and 10% coffee/KBr mass ratio. In order to improve performance of prediction models, the following data pretreatment techniques were evaluated: (0) no additional processing

(raw data), (1) mean centering, (2) normalization, (3) baseline correction, (4) first derivatives www.selleckchem.com/products/AZD6244.html and (5) second derivatives. Mathematical treatments such as mean centering and normalization are commonly applied to data in order to remove

redundant information and enhance sample-to-sample differences ( Wang et al., 2009). Mean centering corresponds to subtraction of the average absorbance value of a given spectrum from each data point. Normalization is calculated by dividing the difference between the response at each data point and the minimum absorbance value by the difference between the maximum and minimum absorbance values. Baseline correction and derivative transformations are usually performed in order to compensate for baseline offset between samples and also to reduce instrument variations ( Esteban-Díez, González-Sáiz, Sáenz-González, & BCKDHB Pizarro, 2007). The statistical software XLSTAT Sensory 2010 (Addinsoft, New York) was employed for all the chemometric calculations. Average spectra obtained for defective and non-defective roasted coffee samples are shown in Fig. 1. A comparative evaluation of these spectra indicates that they are quite similar, although variations in band intensity are perceived, with absorbance values being higher for non-defective and light sour beans and lower for black beans. The two sharp bands at 2920 and 2850 cm−1 have been previously identified in Arabica and Robusta roasted coffee samples (Kemsley et al., 1995) and also on Arabica green coffee samples (Craig et al., 2011 and Craig et al., 2012), in association to asymmetric and symmetric stretching of C–H bonds.

3-A, B, C), the highest response for height under N2 and N0 treat

3-A, B, C), the highest response for height under N2 and N0 treatments (Fig. 3-A), the highest response for leaf area under N2, N1, and N0 treatments (Fig. 3-B), and

the highest response for root surface area under the N1 and N0 treatments (Fig. 3-C). For aboveground biomass, Forestburg had the highest overall response to decreasing N concentration and the worst performance under all treatments (Fig. 3-D). For belowground, Trailblazer had the highest overall response Linsitinib mw to decreasing N concentration (Fig. 3-E, F), but only with the highest response under N0 treatment for belowground biomass (Fig. 3-E). Lowland ecotypes had a lower response than upland ecotypes to decreasing N concentration (Fig. 4). The cultivars responded differently for most agronomic traits when the N deficiency stress was varied. All physiological traits were affected by N deficiency stresses. Only chlorophyll content differed among cultivars (Table S2), with that of Kanlow 1.4% higher than that of all other cultivars (data not shown). A and E were 31% and 23% higher, respectively, FK866 nmr in lowland than in upland ecotypes, but there was no significant difference in these two traits observed across cultivars (Table S2, Fig. 5 and Fig. 6). The N deficiency treatments affected the photosynthetic indices and there was a decrease in A, E, and gs compared with the control.

A similar trend was found with chlorophyll content. All traits showed extreme differences across the four treatments and cultivar-by-treatment interaction. There was no significant ecotype-by-treatment interaction in WUE and chlorophyll content (Table S2). Notably, cultivars performed best under the control condition, followed by moderate stress, and worst under extreme stress (Table 3), suggesting that switchgrass suffered reduced A by an average of 43%, E by 32%, gs by 34%, WUE by 19%, and chlorophyll content by 46% compared with the control ( Table 3). There were highly significant cultivar-by-treatment interactions for all physiological traits (Table S2), meaning that the response to N deficiency stress depended on cultivar. For the six cultivars, A, E, gs, and chlorophyll

content all showed differences across the N2, N1, and N0 treatments ( Fig. 7). For both ecotypes, all of the physiological traits varied across N stress treatments ( Fig. 8). According to Fig. 7, ADAMTS5 accumulation can also be calculated in A, E, gs, and chlorophyll content with increasing stress level for each cultivar (data not shown). For A and E, Kanlow had the lowest overall response and performed best under N2 and N1 treatments, while Pathfinder had the highest overall response to decreasing N level, especially under mild stress ( Fig. 7-A, B). For gs, Trailblazer had the lowest overall response to decreasing N concentration and performed best under N1 and N0 treatments, while Pathfinder had the highest overall response, especially under N1 and N0 treatments ( Fig. 7-C).

4%) and asymptomatic carotid artery stenosis CEA was performed i

4%) and asymptomatic carotid artery stenosis. CEA was performed in 253 patients, whereas 251 patients received endovascular treatment (mainly angioplasty alone). This study excluded high-risk patients, and stents were used selectively, when available, and in only 26% of cases (n = 55). During a median carotid ultrasound follow-up time of 4 years patients undergoing endovascular treatment were found to suffer significantly more often from severe restenosis

(≥70%) or occlusion than patients after CEA [15]. When comparing balloon angioplasty alone to angioplasty and stenting, those patients who were treated with a stent (n = 50) had a significantly lower risk of developing restenosis of ≥70% (adjusted hazard ratio 0.43, 0.19–0.97; p = 0.04). Regarding the clinical complications in patients with a restenosis, the incidence of ipsilateral stroke or transient ischemic attack was significantly

VE-822 in vitro higher in patients with a restenosis ≥70% (cumulative 5-year incidence 22.7% vs. 10.9%, p = 0.04) compared to those with no ISR. Current or past smoking turned out to be independently associated with a higher incidence of restenosis [15]. The Stent-Supported Percutaneous Angioplasty of the Carotid Artery vs. Endarterectomy Trial (SPACE) assessed non-inferiority of CAS to CEA and randomized 1183 patients (CAS n = 605; CEA n = 595) with a symptomatic carotid artery stenosis as assessed with duplex ultrasound (≥50% according MG-132 cost to NASCET criteria, or ≥70% according to ECST criteria) at 35 centres in Austria,

Germany and Switzerland [1]. The type of stent and use of a protection system were chosen at the discretion of the interventionalist. Restenosis during follow-up were observed more frequently in those patients treated with CAS (4.6% vs. 10.7%, p < 0.001) compared to CEA [16]. The majority of the recurrent stenosis occurred within the first 6 months after the initial treatment (CAS n = 28 (51.9%), CEA n = 12 (52.2%)). Furthermore, additional new ISR were observed even after 24 months of follow-up after carotid stenting whereas no new recurrent restenosis was found after CEA beyond 2 years of follow-up. Because a predefined definition of ISR very was not used during the study period and the definition of an ISR depends on the local criteria of each center, a slight overestimation of ISR might be possible [16]. Endarterectomy versus angioplasty in patients with symptomatic severe carotid stenosis (EVA-3S) trial [2] was carried out to demonstrate non-inferiority of CAS compared with CEA and enrolled 527 patients with ≥60% symptomatic carotid stenosis at 30 centres in France. In 507 patients (CAS n = 242, CEA n = 265) serial long-term carotid ultrasound follow-up was performed during a mean follow-up time of 2.1 years [17]. Although the development of a moderate stenosis (≥50–69%) within 3 years was found to differ significantly between the groups with a higher proportion after CAS compared to CEA (12.5% vs.

, 2005 and Frank et al , 2011) Ongoing sequencing efforts reveal

, 2005 and Frank et al., 2011). Ongoing sequencing efforts revealed that the large number of sulfatase genes is indeed a characteristic of the Planctomycetes–Verrucomicrobia–Chlamydia (PVC) superphylum, i.e., Lenthisphaera araneosa ( Thrash et al., 2010), Planctomyces brasiliensis, and Planctomyces maris feature more than 100 and partially even more than 200 sulfatases ( Fig. 1). Sulfatases catalyze

the hydrolytic cleavage of sulfate esters and sulfamates. Three distinct classes of sulfatases have been identified so far. Group I sulfatases (formylglycine-dependent sulfatases) are well-known and widely distributed in eukaryotes and prokaryotes. Group PFT�� supplier II sulfatases (α-ketoglutarate-dependent dioxygenase superfamily alkylsulfatases) and group III sulfatases (Zn2 +-dependent alkyl sulfatases) have been recently discovered and only few examples are known (Müller et al., 2004 and Hagelueken et al., 2006). Substrates range from sulfated proteoglycans and conjugated steroids to smaller aromatic sulfate esters (Ghosh, 2007). Group I sulfatases share a high structural and sequence similarity.

They feature a conserved amino acid signature selleck chemicals including a core pentapeptide (C/S-x-P-x-R), followed by (x(4)-T-G), commonly referred to as sulfatase signature sequence I. The cysteine or serine residue within this signature sequence is posttranslationally modified to a catalytically active formylglycine (FGly). Group I is divided into Cys- and Ser-type sulfatases. Ser-type sulfatases were exclusively found in prokaryotes, while the Cys-type has been detected in both eukaryotes and prokaryotes. Two different pathways for the formylglycine formation were discovered. Formylglycine generating enzymes (FGE) mediate the first mechanism which specifically requires GPX6 a cysteine residue (Dierks

et al., 1999). The second system involves anaerobic sulfatase modifying enzymes (anSME) which are able to convert cysteine or serine in the active site (Berteau et al., 2006). Escherichia coli mutants carrying gene deletions in both described maturation systems still expressed functional sulfatases. Therefore, a third, uncharacterized maturation system seems to exist ( Benjdia et al., 2007). The currently favored mechanism of sulfatase catalysis is a transesterification mechanism, utilizing the hydration of the formylglycine to a geminal diol. In the course of two subsequent nucleophilic attacks, the organic moiety and the sulfate group are released from the initial substrate ( Fig. 2) ( Carlson et al., 2008 and Hanson et al., 2004). It has been suggested that the high number of sulfatases found in Planctomycetes could play a major role in the degradation of sulfated polysaccharides in their environment.

The concentrations of SDs and STs in the test solution were deter

The concentrations of SDs and STs in the test solution were determined by means of gas chromatography–mass spectrometry. The analytical conditions are shown in Table 1. The molecular weight distribution of the test sample was determined by means of gel permeation chromatography. The analytical conditions are shown in Table 2. One milliliter of test sample was

dried under a nitrogen gas purge and the Thiazovivin chemical structure residue was then dissolved in tetrahydrofuran to make 10 mL of tetrahydrofuran solution. The tetrahydrofuran solution was kept at 25 °C for approximately 24 h before use. The Ames test was conducted according to the Organisation for Economic Co-operation and Development (OECD) Guideline for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test [13], as follows: 1) Chemical treatment and colony counting A pre-incubation method in the presence or absence of S9 mix was used [14]. Triplicate plates were used for each dose. S. typhimurium strains TA100, TA1535, TA98, and TA1537 and E. coli strain WP2uvrA were used as the bacterial tester strains. The test solution was diluted with acetone to prepare the test doses. The maximum concentration of the test doses was 10% (w/v). The test sample formulation

was mixed with the bacterial culture in the presence or absence of S9 mix and pre-incubated GDC-0068 cost at 37 °C for 20 minutes. Soft agar was added to the mixture, which was then poured onto a minimal glucose agar plate (Tesmedia AN; Oriental Yeast Co., Tokyo, Japan). Triplicate plates were used for each dose. The final concentration of S9 in the top agar layer was 2%. After incubation at 37 °C for 48 h, the number of revertant colonies was counted

by using a colony counter system (CA-11D; System Sciences, Tokyo, Japan). Precipitation of the test sample and inhibition of bacterial growth were also checked macroscopically. To confirm the reproducibility of the test results, two independent tests were conducted. 2) Evaluation of results The Ames test was considered positive when the number of revertant colonies was increased to two or more times that of the negative control and when the response was dose-related or reproducible, or both. All other cases were considered negative. No statistical methods were used. The in vitro chromosomal aberration test was conducted according to OECD Guideline for Orotidine 5′-phosphate decarboxylase the Testing of Chemicals, No. 473, In Vitro Mammalian Chromosome Aberration Test [15], as follows: 1) Chemical treatment, slide preparation, and assessment The procedure reported by Ishidate and Odashima [16] was followed. CHL/IU cells were pre-cultured in 10% (v/v) heat-inactivated newborn calf serum/minimum essential medium in CO2 incubator (MCO-18AIC, SANYO Electric, Osaka, Japan), which was set at 37 °C and an atmosphere of 5% CO2 under a humid condition. Duplicate dishes were used for each dose. The test solution was diluted with acetone to prepare the test doses. The maximum concentration of the test dose was 50% (w/v).

4 μg/ml ptaquiloside (Pt), 4 4 μg/ml ptaquiloside + 0 1 mM seleni

4 μg/ml ptaquiloside (Pt), 4.4 μg/ml ptaquiloside + 0.1 mM selenium (co-incubation) (PtSe) and 0.1 mM selenium (Se). All treatments were incubated for 1 h at 37 °C in a humidified atmosphere with 5% CO2. Following treatment, the cells were washed and re-suspended in complete RPMI medium and then prepared for the detection and quantification of the proteins metallothionein 1 and 2 (Mt1 and Mt2) and free zinc (Zn2+) as an indicator of their activities. Following in vitro treatment of cultures of non-adherent splenic cells, the cells were adjusted to 1 × 106 cells/50 μl and incubated with 0.5 μl Mouse BD Fc

Block™ (clone 2.4G2, BD Pharmingen) for 5 min (to block the Fc-mediated adherence of antibodies) prior to staining with specific antibodies. These cells were then stained (simultaneously) for surface antigens MK-2206 mouse (CD3 and NK1.1) for 30 min at 4 °C in the dark. The cells were then washed in 2 ml PBS, fixed and permeabilized with a Cytofix/Cytoperm Plus Kit (BD Biosciences) following the manufacturer’s protocol. During the permeabilization step, the cells were stained intracellularly with the primary antibody (anti-metallothionein that cross reacts with

Mt1 and Mt2, clone UC1MT, PS341 Abcam) for 30 min at 4 °C in the dark, then washed and stained with the secondary antibody (FITC-labeled goat polyclonal anti-mouse IgG, Abcam). Finally, the cells were washed free of unbound antibody and then resuspended in PBS for flow cytometry using a FACSCalibur™ flow cytometer equipped with Cell Quest Pro® software (Becton Dickinson [BD] Immunocytometry System). A total of 100,000 target cells were collected by flow cytometry, and the results were expressed as mean fluorescence intensity (MFI). Data analyses were performed with FlowJo 7.6.4® software (Tree Star Inc., Ashland, KY). The free intracellular zinc concentration in NK cells was measured

using the method proposed by Haase et al. (2006), with Dichloromethane dehalogenase modifications. Following the in vitro treatments outlined above, the non-adherent cells were adjusted for 1 × 106 cells/well. FluoZin™-3 AM ester, dissolved in Pluronic® F-127 (1:1) (Molecular Probes), was then added to the cultures at a final concentration of 1 μM and the cells were incubated at 37 °C in a humidified atmosphere at 5% CO2 for 30 min. The cells were then washed in PBS (5 min, 2000 rpm) and incubated with 0.5 μl Mouse BD Fc Block for 5 min (to block the Fc-mediated adherence of antibodies) prior to staining with specific antibodies. The cells were then stained (simultaneously) for surface antigens (CD3 and NK1.1) for 30 min at room temperature in the dark. Finally, the cells were washed free of unbound antibody and resuspended in PBS for flow cytometry using a FACSCalibur™ flow cytometer equipped with Cell Quest Pro® software (Becton Dickinson [BD] Immunocytometry System).