For visualization of EGFP expression, spores of pHxk1-EGFP transformed strains were inoculated in MM and grown for 10 h at 28 °C. These cultures were used directly for microscopic observation. Fluorescence and light microscopy were performed using a Nikon Eclipse 80i fluorescence microscope and images were captured under control of nis-elements ar software. Previous work showed that H. jecorina hexokinase-negative strains were not able to grow on MM containing d-fructose as the sole carbon source (Hartl & Seiboth 2005).

To test the utility of the two polyols d-mannitol and d-sorbitol as osmotic stabilizer and as a selective carbon source we tested the growth of H. jecorina TU-6H and the parent strain on MM agar plates with different carbon sources. Growth tests showed that TU-6H strains were Wortmannin chemical structure not able to grow on MM containing 10 g L−1d-mannitol and d-sorbitol, whereas the parent strain was able to use both polyols as sole carbon source. This indicates that, in H. jecorina, the two polyols d-mannitol and d-sorbitol are catabolized via d-fructose as intermediate (Elorza & Arst, 1971; Solomon et al., 2007). To demonstrate the utility of this transformation system, H. jecorina TU-6H was transformed

with the reporter plasmid pHxk1-EGFP. For determination of the optimal selective carbon source and osmotic stabilizer, we compared the effect of 1 M d-sorbitol and 1 M d-mannitol in the regeneration medium on transformation efficiency. Results from three independent parallel transformation experiments showed that d-mannitol leads to higher transformation efficiencies than d-sorbitol. Using d-mannitol, approximately 500–1000 colonies μg−1

Natural Product Library mouse plasmid DNA were obtained, whereas using d-sorbitol, only 100–200 colonies were found. Sodium butyrate In control transformation assays with the EGFP expression, plasmid pIG1783 alone or without plasmid DNA, no colonies were found on the selective plates. As shown in Fig. 1a, transformed colonies of variable sizes were seen on selective plates. After purification of the transformants, their growth on different carbon sources was compared with growth on the parental strains (Fig. 1b). The growth of the transformants was fully restored on MM with d-mannitol or d-glucose as carbon source. The presence of the reporter plasmid pHxk1-EGFP in d-mannitol-utilizing transformants was confirmed by the presence of both hxk1 and egfp in the isolated gDNA of different transformants as detected by PCR (Fig. 2a). Three randomly picked transformants were further analyzed by Southern blot to determine the pattern of integration. Figure 2b shows that pHxk1-EGFP integrated into the genome of all analyzed transformants at the hxk1 locus; in all cases, the plasmid integrated next to the promoter region of hxk1. Some of the transformants showed additional hybridizing bands, most likely due to tandem integration or insertion of the plasmid by ectopic integration as well as targeted integration.

The fixation point was a red (R255 G0 B0) square (0.67 × 0.67°); the directional cue was a red (R255 G0 B0) arrow (0.67 × 0.67°); targets were white (R255 G255 B255) figure 8s (0.62 × 1°); discrimination symbols were white (R255 G255 B255) Es or 3s (0.62 × 1°);

distractors were white (R255 G255 B255) 2s or 5s (0.62 × 1°). Targets were located at the four corners of an imaginary square, each 5.4° diagonally from the central fixation point. Each block of trials started with a check of the calibration quality and, if required, a two-dimensional 13-point re-calibration procedure covering the display area. At the beginning and end of each recording, a sequence of reflexive saccades was recorded to provide data for post hoc assessment and adjustment of the calibration if required. Stimuli were presented using PsychoPy, an open-source experimental control selleck kinase inhibitor software package (Peirce, 2007, 2008). All participants attended two testing sessions. At the first session, after a 6-m visual acuity test with the Snellen wall chart (each subject was required to have visual PI3K Inhibitor Library cell line acuity of no worse than 6/12 corrected in their best eye), each participant’s vision was checked whilst they were seated in front of the computer screen with the chin supported

by the chinrest of the recording column. At a viewing distance of 600 mm, some participants’ own corrective lenses were not suitable. A range of corrective lenses of various strengths was then tried until the best possible acuity at 600 mm was achieved. Vision was then tested again with an array of symbols at

the size and contrast actually used in the experiments. The actual test and recording started after calibration of the eye movement recording system. At the first session, subjects first performed two blocks of the saccade task ‘without discrimination’, and then two blocks of the saccade task ‘with Venetoclax purchase discrimination’. The saccade task ‘without discrimination’ was always performed at the start of the first session, while participants were not yet aware of the potential relevance of the symbol-changes. Another two blocks of the task ‘with discrimination’ were performed at the second session, 1 week after the first session. In the task ‘with discrimination’, each trial was followed by a visual prompt asking the participant whether E or 3 had appeared. Participants responded E or 3 with a right or left manual button press, respectively. Participants were explicitly told to guess if unsure of the answer. They were also told that on some trials there would be no discrimination symbol, and to push one of the two buttons at random when they thought no discrimination symbol had appeared. In No-change and Distractor trials there was no discrimination symbol, but subjects were not told about the different symbol-change conditions or the likelihood of a discrimination symbol occurring.

The results showed that the paddy soil profile harbored diverse bacterial communities and experienced depth-related changes in community structure and carbon source utilization. The bacterial communities and functions might be shaped by the soil edaphic characteristics along the soil profile. “
“HAS University of Applied Sciences, Venlo, The Netherlands Pseudomonas fluorescens SS101 produces the cyclic lipopeptide massetolide with diverse functions in antimicrobial activity, motility, and biofilm formation. To understand how massetolide biosynthesis is genetically regulated in SS101, c. 8000 random plasposon mutants were

screened for reduced or loss of massetolide production. Of a total of 58 putative mutants, 45 had a mutation

in one Androgen Receptor Antagonist cell line PLX4032 mouse of the three massetolide biosynthesis genes massA, massB, or massC. For five mutants, the insertions were located in the known regulatory genes gacS, gacA, and clpP. For the remaining eight mutants, insertions were located in clpA, encoding the ClpP chaperone, in phgdh, encoding D-3-phosphoglycerate dehydrogenase, in the heat shock protein-encoding dnaK, or in the transmembrane regulatory gene prtR. Genetic, chemical, and phenotypic analyses showed that phgdh, dnaK, and prtR are indeed involved in the regulation of massetolide biosynthesis, most likely by transcriptional repression of the LuxR-type regulator genes massAR and massBCR. In addition to their role in massetolide biosynthesis, dnaK and prtR were found to affect siderophore and extracellular protease(s) production, respectively. The identification of new regulatory genes substantially extended insights into the signal transduction pathways of lipopeptide biosynthesis

in P. fluorescens and into regulation of other traits that may contribute to its life-style in the rhizosphere. “
“The two-component system (TCS), consisting of a response regulator (RR) and a cognate histidine kinase (HK), responds to extra-/intercellular cues and triggers adaptive changes. The RR, RavR, has been reported to act as a positive virulence regulator and a c-di-GMP hydrolase in Xanthomonas campestris Methocarbamol pv. campestris (Xcc). Here, we identified the cognate HK, RavA, that regulate RavR phosphorylation levels and bacterial pathogenesis. Deletion of ravA, a putative HK gene flanking ravR, dramatically attenuated Xcc virulence. Phenotypes of the double mutant ΔravR/ΔravA were similar to those of ΔravR, suggesting that RavR is a downstream component of RavA signaling. RavA interacts with RavR and positively influences the phosphorylated RavR levels. In vitro analysis suggests that RavR is a bifunctional enzyme involved in c-di-GMP synthesis and degradation.

“
“Within the phylum Bacteroidetes, the gyrB gene, encoding for the B subunit of the DNA gyrase, has been used as a phylogenetic marker for several genera closely related to Flavobacterium. Dabrafenib price The phylogenies of the complete 16S rRNA gene and the gyrB gene were compared for 33 Antarctic Flavobacterium isolates and 23 type strains from closely related Flavobacterium species. gyrB gene sequences provided

a higher discriminatory power to distinguish between different Flavobacterium groups than 16S rRNA gene sequences. The gyrB gene is therefore a promising molecular marker for elucidating the phylogenetic relationships among Flavobacterium species and should be evaluated for all the other type strains of described Flavobacterium species. Combining the phylogeny of both genes, the new Antarctic Flavobacterium strains constitute 15 Flavobacterium groups, including at least 13 potentially new species together with one group of isolates probably belonging to the species Flavobacterium micromati and one group close to Flavobacterium gelidilacus. Heterotrophic bacterial communities in Antarctica are highly diverse in aquatic (Bowman et al., 2000; Van Trappen et al., 2002) as well as in terrestrial (Aislabie et al., 2006; Babalola et al., 2009) habitats. A genus that has been isolated

often from these environments is Flavobacterium (Brambilla et al., 2001; Humphry et al., 2001; Van Trappen et al., 2002), and several novel Flavobacterium species were described from Antarctic habitats (Flavobacterium gelidilacus, Flavobacterium gillisiae, Flavobacterium hibernum, OSI-906 supplier Flavobacterium micromati, Flavobacterium psychrolimnae, Flavobacterium xanthum) or other cold environments (Flavobacterium xinjangense and Flavobacterium omnivorum). Other Flavobacterium species have been mainly isolated from freshwater fish (Flavobacterium ADAMTS5 branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum), temperate freshwater (Flavobacterium aquatile, Flavobacterium flevense, Flavobacterium saccharophilum) and from soil (Flavobacterium johnsoniae, Flavobacterium pectinovorum). Most Flavobacterium species are psychrotolerant and as they are able to hydrolyse several carbohydrates and biomacromolecules

such as gelatine, casein and starch, they might be of biotechnological importance (Bernardet & Bowman, 2006). The family Flavobacteriaceae (phylum Bacteroidetes) as well as the genus Flavobacterium have been revised and added to repeatedly over the years (Vandamme et al., 1994; Bernardet et al., 1996, 2002). Flavobacterium was created in 1923 for all bacteria that formed yellow- or orange-pigmented colonies and weakly produced acid from carbohydrates (Bergey et al., 1923). This broadly defined and taxonomically heterogeneous group was further refined using phenotypic characteristics (Holmes et al., 1984) and the determination of guanine plus cytosine (G+C) content (Reichenbach, 1989). The introduction of the 16S rRNA gene oligonucleotide catalogue (Paster et al.

, 2006) is a competing software package for reverse complementary 16S sequence detection and orientation. The software operates by Selumetinib supplier matching short oligonucleotide sequences at highly conserved positions along the gene and offers a user-friendly interface combined with an impressive processing speed. In order to compare the detection efficiency and reliability of this tool, we processed the bacterial and archaeal full-length, V1-V3 and V1-V2 datasets. The detection efficiency of orientationchecker decreased with decreasing sequence length, showing detection of 100%, 95% and 2% for the full-length,

V1-V3 and V1-V2 datasets, respectively. Although the performance on full-length sequences was somewhat similar to that of v-revcomp, orientationchecker failed to detect the correct orientation of 124 full-length sequences and incorrectly assigned 10 as being reverse complementary. The lack of detection selleck products increased by 5% on V1-V3 sequences when compared with v-revcomp and the tool almost completely failed to detect the shorter V1-V2 sequences. In conclusion, v-revcomp demonstrated superior performance, especially on shorter sequences, and features a more reliable mechanism by screening multiple conserved regions at once. Furthermore, HMMs will be more flexible in detecting deviant sequences than a simple pattern matching using oligonucleotide

sequences. In addition, the command-line nature of v-revcomp facilitates incorporation into automated software pipelines (e.g. Barker et al., 2010; Caporaso et al., 2010), which makes it especially suited to screen HTS datasets. In order to assess the status of reverse complementary sequences in public data repositories, we ran v-revcomp on the 1 113 159 bacterial and 58 487 archaeal 16S sequences of a minimum length of 500 bp that were available in GenBank as of 1 July 2010. The 16S status was determined by screening the GenBank definition line for various synonyms for this gene; therefore, 16S sequences including parts of up- or downstream regions of the gene (e.g.

promoter region, intergenic spacer) were coextracted. A total of 1 158 546 sequences (i.e. 98.9%) were reported by v-revcomp to be in the correct orientation, 9067 (0.8%) in the reverse orientation, 185 (0.02%) were flagged as uncertain and 3848 (0.3%) did not show any HMM detection at all such that no decision ALOX15 was obtained (Fig. 1b). The following reasons accounted for the failure to detect any HMM in the 3848 sequences. In 3437 cases (89.3%), only a very small segment was actually identified as the 16S, whereas most of the sequence information comprised either the intergenic spacer region downstream of the gene (3421 cases) or regions, such as promoters, upstream of the gene (16 cases). In 220 cases (5.7%), the sequences showed only partial, poor or no match to any entry in GenBank as assessed through blast, and are therefore likely to be artefacts created during PCR amplification, sequencing or data processing. In 26 cases (0.

[10-12] College freshmen living in dormitories are at particularly high risk for developing meningococcal disease.[13] Because of this, students from overseas who are planning to live in college dormitories

are usually required to provide proof of meningococcal immunization in the United States and other countries such as the United Kingdom. Many Taiwanese students preparing to study in the United States are required Selleckchem CH5424802 to have the vaccination, which is not a routine immunization in Taiwan.[14] In addition, the vaccine is available only at 12 Centers for Disease Control contracted hospitals due to the scarceness of the vaccine in Taiwan. However, receiving vaccination without learning about the disease is not enough to assure prevention and patient-level factors may influence immunization coverage. Furthermore, educating patients about the Enzalutamide risk of contracting the disease and the importance of the vaccine

should be an essential part of the physician–patient discussion about vaccination. Thus far, few studies have investigated the awareness and attitudes toward meningococcal disease among high-risk students. We designed a study to survey the knowledge, attitudes, and behaviors about the disease among Taiwanese students planning to study in the United States. A cross-sectional questionnaire survey on Taiwanese college students planning to study in the United States was conducted in National Taiwan University Hospital in Taipei, a medical center-based travel medicine clinic, from January 2009 to December 2010. The questionnaire and consent forms were distributed to all college-age nonmedical students from different universities planning to study in the United

States. All study procedures were approved by the ethical committee of Idelalisib chemical structure the National Taiwan University Hospital. A self-administered, single-choice questionnaire surveyed the background information, attitudes toward, and knowledge about meningococcal disease. The questionnaire was based upon personal practice experiences and designed after a careful literature review. Excluding background information, the questionnaire included two questions about attitudes, five questions about general knowledge of the disease, four questions on preventive or postexposure management, and two questions on individual preventive practices. Five experts tested the content validity, while the face validity was tested by five college students. Data management and statistical analyses were performed using SPSS 11.0 software. Frequency distributions were used to describe the demographic data. Stepwise logistic regression analysis determined the relative values of the variables related to positive attitudes on receiving vaccines and willingness to perform individual preventive practices. A p-value less than 0.05 was considered statistically significant.

TLR2 and TLR4 genes increased 6.54-fold and 5.28-fold, respectively, in the healthy control group. However, the expression of IL10 (2.90-fold) 3-h poststimulation was less compared than that seen in the stimulated tuberculosis group (8.74-fold). We compared the gene expression levels in the two groups. The results showed that two of the seven genes examined (TLR2 and IL10) were differentially

expressed in both the stimulated tuberculosis subjects and the stimulated healthy control subjects (P≤0.05 by t-test). Although TLR2 showed increased expression in Sotrastaurin both stimulated groups, it had a greater fold increase in the stimulated control group (6.54-fold) over the stimulated tuberculosis group (2.64-fold). This may indicate that TLR2 plays a larger role in regulation in healthy animals. In contrast, IL10 expression in stimulated tuberculosis animals (8.74-fold) was greater than that seen in the stimulated control group (2.90-fold). Thus, TLR2 may

play a key role in the response of MDMs from healthy cattle to M. bovis stimulation, while IL10 may play a similar key role during M. bovis stimulation of MDMs from tuberculosis cattle. The CPE and the relationship between M. bovis and MDMs cells were observed directly by microscopy (Fig. 2) and Ziehl–Neelsen stain (Fig. 3). The present findings learn more demonstrate that the CPE could be seen under microscopy after 3 h of stimulation, and it became

more severe over time. Necrosis and detachment Protein kinase N1 of cells were caused by the intrusion and adherence of bacteria. At 3 h, the medium incubated with cells was almost clear and intracellular fast-acid bacteria were seldom seen. However, by 10 h, the medium became unclear due to cellular debris and dead cell granules and bacteria could be observed inside the cytoplasm and the nucleus of some cells. Twenty-four hours after stimulation, the massive cellular death made microscopy images obscure and only a small number of cells survived. The M. bovis used in this study was a virulent strain, triggering a strong interaction and quickly leading to massive cellular death. Based on the observations made by microscopy and fast-acid stain, there are no obvious differences in CPE of M. bovis between MDMs from tuberculosis and healthy control cattle. The growth and survival status of intracellular M. bovis were assessed by bacterial CFU in the MDMs from tuberculosis and healthy control cattle (Fig. 4). Our data indicate that at 3 h, the CFU of intracellular survival of M. bovis is very low and difficult to measure in several subjects (less than three bacterial clones on a 7H10 agar plate). This result is consistent with the previous observation, by microscopy and fast-acid stain, that M. bovis grows poorly in cells after 3 h of infection. This study also shows that 10 h after stimulation, CFUs of M.

hep-druginteractions.org) GPP 8.3.1 We recommend starting ART in HIV-positive patients with

KS. 1A   We recommend starting ART in HIV-positive patients with non-Hodgkin lymphoma (NHL). 1B   We suggest starting ART in HIV-positive patients with cervical cancer. 1C   We recommend starting ART in HIV-positive patients who are commencing radiotherapy or chemotherapy for cervical cancer. 1D 8.3.2 We suggest starting ART in HIV-positive patients with non-AIDS-defining malignancies (NADMs). 2C   We recommend starting ART in HIV-positive Ku-0059436 ic50 patients who are commencing immunosuppressive radiotherapy or chemotherapy for NADMs. 1C 8.3.3 We recommend that potential pharmacokinetic interactions between ARVs and systemic anticancer therapy be checked before administration (with tools such as: http://www.hiv-druginteractions.org). GPP   We suggest avoiding ritonavir-boosted ART in HIV-positive PI3K Inhibitor Library mouse patients who are to receive cytotoxic chemotherapy agents that are metabolized by the cytochrome P450 (CYP450) enzyme system. 2C   We recommend against the use of ATV in HIV-positive patients who are to receive irinotecan. 1C   We suggest avoiding ARV agents in HIV-positive patients who are to receive cytotoxic chemotherapy agents that have overlapping toxicities. 2C 8.4.2 We recommend patients with symptomatic HIV-associated NC disorders start ART irrespective

of CD4 lymphocyte count. 1C 8.4.3 We recommend patients with HIV-associated NC disorders start standard combination ART regimens. 1C 8.4.4 In patients with ongoing or worsening NC impairment despite ART we

recommend the following best practice management: GPP • Reassessment for confounding conditions. • Assessment of cerebrospinal fluid (CSF) HIV RNA, CSF HIV genotropism and genotyping of CSF HIV RNA. • In subjects with detectable CSF HIV RNA, modifications Etofibrate to ART should be based on plasma and CSF genotypic and genotropism results. 8.5.1 We recommend patients with HIVAN start ART immediately irrespective of CD4 cell count. 1C   We recommend patients with end-stage kidney disease who are suitable candidates for renal transplantation start ART irrespective of CD4 cell count. 1C 8.5.2 We recommend against the use of ARV drugs that are potentially nephrotoxic, in patients with stages 3–5 chronic kidney disease (CKD) if acceptable alternative ARV agents are available. GPP   We recommend dose adjustment of renally cleared ARV drugs in patients with reduced renal function. GPP 8.6.4 We suggest avoiding: ABC, FPV/r and LPV/r in patients with a high cardiovascular disease (CVD) risk, if acceptable alternative ARV drugs are available. 2C 8.7.2 We recommend therapy-naïve HIV-positive women who are not pregnant start ART according to the same indicators as in men (see Section 4: When to Start) 1A 8.7.

Relative to saline controls, rats in the 7-day but not the 1-day abstinence group had higher levels of DARPP-32 phosphorylated at the protein kinase A site in the insular cortex. These results demonstrate incubation of drug seeking following extended access to nicotine self-administration and suggest that enhanced protein kinase A signaling in the insular cortex via phosphorylation of DARPP-32 at Thr34 is associated with this effect. “
“We used knock-in mice that express green fluorescent protein (GFP)-labeled

embryonic-type acetylcholine receptors to investigate postsynaptic responses to denervation of fast-twitch and slow-twitch muscle fibers, and to visualize the integration of newly synthesized GFP-labeled embryonic-type receptors into adult synapses. The embryonic-type receptors GSK3 inhibitor are transiently expressed and incorporated into the denervated endplates. They replaced synaptic adult-type receptors in a directed fashion, starting from the endplate’s periphery and proceeding to its

central regions. The progress of embryonic-type receptor expression with respect to transcriptional control is a transient, short-term activation mechanism. The less pronounced increase in the expression levels of the GFP-labeled receptors revealed a differential shift in the integration and degradation processes that constitute the INK 128 in vivo dynamic equilibrium of the synaptic receptor pool. Therefore, we were able to model the changes in the total receptor load of the neuromuscular endplate following denervation as a function of the abundance of available receptors and the initial

receptor load of the endplate. “
“Inattention and impulsivity are the most prominent clinical features of attention deficit hyperactivity disorder (ADHD) in adulthood. Oxalosuccinic acid Structural and functional neuroimaging studies of subjects with ADHD have demonstrated abnormalities in several brain areas, including fronto-striatal and fronto-cerebellar networks. Mostly, these studies were based on volumetric measurements and have been conducted in children. We investigated white matter (WM) integrity and correlation with measures of attention and impulsivity in adult patients with ADHD adopting diffusion tensor imaging (DTI). N = 37 (21 males) never-medicated adult patients with ADHD combined subtype and N = 34 (16 males) healthy controls were investigated. ADHD diagnosis (DSM-IV) was assessed with clinical interviews and rating scales, subjects also underwent a large neuropsychological test battery including tests of attention and impulsivity. DTI was acquired, and group differences of fractional anisotropy (FA) and mean diffusivity (MD) as well as correlation analyses with measures of attentional performance and impulsivity were calculated using voxel-based analyses.

The DSR system presumably forms intracellular sulfite that is oxidized by an enzyme system consisting of Sat, Apr, and Qmo proteins (Rodriguez et al., 2011). The electron acceptors, cytochrome c, and menaquinone (Fig. 1) are ultimately oxidized

by the photosynthetic reaction center. In cultures of Cba. tepidum that contain both sulfide and thiosulfate, sulfide is oxidized preferentially while sulfur globules are formed (Chan et al., 2008; Azai et al., 2009; Holkenbrink et al., 2011). Following sulfide depletion, thiosulfate and sulfur globules are oxidized to sulfate. The molecular mechanism of ABT-199 chemical structure this phenomenon is poorly understood. Sulfide possibly inhibits thiosulfate oxidation either by substrate competition between sulfide and thiosulfate (the SOX system oxidizes sulfide in vitro; Ogawa et al., 2010) or by saturation of the electron acceptor pool. Regulation of sulfur metabolism genes in GSB is poorly described, but it is known that SoxA is induced by thiosulfate in Chlorobaculum thiosulfatiphilum (Verté et al. 2002). http://www.selleckchem.com/products/azd4547.html In the purple sulfur bacterium, Allochromatium vinosum, sox and dsr genes are expressed at a low constitutive level in the absence of reduced sulfur substrates and are induced by thiosulfate and sulfide, respectively (Grimm et al., 2010, 2011). Chlorobaculum tepidum TLS was grown under incandescent illumination in CL medium (Frigaard et al., 2004). For experiments comparing early and late exponential growth phase, wild-type

cells were grown at 45 °C in 1-L flasks under a light intensity of 200 μmol photons m−2 s−1. For experiments comparing wild type and the dsrM mutant (Holkenbrink et al., 2011), cells were grown at 42 °C in 15-mL

tubes under a light intensity of 50 μmol photons m−2 s−1 and harvested in the late exponential growth phase. Cells were harvested by centrifugation and stored at −20 °C prior to analysis. Bacteriochlorophyll c was determined by extracting the cell pellet with acetone : methanol (7 : 2 by vol) (Frigaard et al., 1997). Sulfide was measured using the colorimetric methylene blue method (Cline 1969). Sulfate Oxymatrine and thiosulfate were measured by ion chromatography (Dionex, Hvidovre, Denmark) using a carbonate buffer as eluent. Samples for analysis of elemental sulfur were dissolved in methanol and analyzed as S8 by HPLC using a Sykam pump (S1100), UV–VIS detector (Sykam S3200), Zorbax ODS-column (125 × 4 mm, 5 μm; Knauer, Berlin, Germany) and methanol as the eluent at a flow rate of 1 mL min−1. Elemental sulfur was detected at 265 nm. Cell pellets were thawed and extracted in acetone : methanol (7 : 2 by vol) to remove pigments. The colorless cell pellets were solubilized in an SDS-containing buffer (Laemmli, 1970) supplemented with a complete protease inhibitor cocktail (Roche, Hvidovre, Denmark) at 95 °C for 3–5 min and cleared by centrifugation. Prior to digestion, proteins were reduced with dithiothreitol (1 mM) and alkylated with iodoacetamide (5 mM).