Aromatic L-amino acid decarboxylase (AADC) enzymes catalyze the synthesis of biogenic amines, including the neurotransmitters serotonin and dopamine, throughout the animal kingdom. These neurotransmitters typically perform important functions in both the nervous system and other tissues, as illustrated by the debilitating conditions that arise from their deficiency. Studying the regulation and evolution of AADC genes is therefore desirable to further our understanding of how nervous systems function and evolve.C. elegans, the bas-1 gene is required for both serotonin and dopamine synthesis, and maps genetically near two AADC-homologous sequences. We show by transformation rescue and sequencing of mutant alleles that bas-1 encodes an AADC enzyme. Expression of a reporter construct in transgenics suggests that the bas-1 gene is expressed, as expected, in identified serotonergic and dopaminergic neurons. The bas-1 gene is one of six AADC-like sequences in the C. elegans genome, including a duplicate that is immediately downstream of the bas-1 gene. Some of the six AADC genes are quite similar to known serotonin- and dopamine-synthetic AADC's from other organisms whereas others are divergent, suggesting previously unidentified functions. In comparing the AADC genes of C. elegans with those of the congeneric C. briggsae, we find only four orthologous AADC genes in C. briggsae. Two C. elegans AADC genes – those most similar to bas-1 – are missing from C. briggsae. Phylogenetic analysis indicates that one or both of these bas-1-like genes were present in the common ancestor of C. elegans and C. briggsae, and were retained in the C. elegans line, but lost in the C. briggsae line. Further analysis of the two bas-1-like genes in C. elegans suggests that they are unlikely to encode functional enzymes, and may be expressed pseudogenes.In the nematode bas-1 gene of C. elegans encodes a serotonin- and dopamine-synthetic AADC enzyme. Two C. elegans AADC-homologous genes that are closely related to bas-1 are missing from the congeneric C. briggsae; one or more these genes was present in the common ancestor of C. elegans and C. briggsae. Despite their persistence in C. elegans, evidence suggests the bas-1-like genes do not encode functional AADC proteins. The presence of the genes in C. elegans raises questions about how many 'predicted genes' in sequenced genomes are functional, and how duplicate genes are retained or lost during evolution. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.The  Drosophila, a single gene encodes the serotonin- and dopamine-synthetic AADC  mutant is serotonin- and dopamine-deficient, and displays several behavioral abnormalities  The C. elegans and C. briggsae bas-1 genes have identical genomic structure which differs from that of C05D2.3 and F12A10.3, which are more similar to one another , we found that current splicing predictions for the gene were incorrect. We sequenced six F12A10.3 clones and found two slightly different splicing patterns, both different from Genefinder and Intronerator predictions. The two types of clones differed only in whether a final intron was removed or not. We found 4 clones with 9 exons, and 2 clones with 8 exons. The failure of the gene prediction programs in this case is likely to be due to their preference for creating functional transcripts. All F12A10.3 cDNAs instead appear to be non-functional: they have frameshifts relative to AADC-homologous reading frames. The first frameshift occurs in the second exon and quickly leads to a premature stop codon. At best F12A10.3 transcripts would result in a 158 amino acid protein that could not function as an AADC. F12A10.3 therefore appears to be an expressed pseudogene. DNA microarray experiments and representation in cDNA sequencing projects suggest that F12A10.3, like C05D2.3, is likely expressed at a low level  comparing the bas-1 orthologs and bas-1-like genes. We also calculated these values for the other AADC ortholog pairs from C. elegans and C. briggsae (Table A/KS < 1 indicates purifying (negative) selection, KA/KS = 1 indicates no selection (as in true pseudogenes), and KA/KS < 1 indicates Darwinian (positive) selection. KA/KS for 8179 C. elegans and C. briggsae ortholog pairs had a mean value of 0.06, indicating most genes are under purifying selection [bas-1 genes are under purifying selection (KA/KS = 0.039), but the bas-1-like genes appear to be under reduced selective pressure; the average KA/KS for comparisons with bas-1-like genes was 0.148, more than three times the value of the bas-1 ortholog comparison. The proportion of observed to potential non-synonymous substitutions (pN) among the bas-1-like gene comparisons was similarly much higher than for the bas-1 orthologs.In order to assess whether the election . We founbas-1 orthologs  was near mutational saturation (pS > 0.75); KA/KS cannot be calculated when pS > 0.75. Thus, a value could not be calculated either for full-length tdc-1 alignments, or using a tdc-1 core sequence. We were able to calculate a value by aligning the N- and C-terminals sequence of the tdc-1 orthologs , corresponds to the genetically-defined eficient , and neibas-1 gene is expressed in at least two alternatively spliced forms, one of which appears to be less common and contains a small additional 27 nucleotide exon. The short segment of protein encoded by the additional exon, and the surrounding region are not found in other AADC proteins, suggesting a novel function for this region of the AADC protein. In other organisms, the serotonin- and dopamine-synthetic AADC genes have alternative splicing that result in tissue-specific protein isoforms. Currently we have no indication that bas-1 is expressed in any cells other than serotonergic and dopaminergic neurons, and no information about the functional significance of this alternative splicing.The in vitro experiments [C. elegans AADCs by phosphorylation remains speculation.AADC has received somewhat less attention with respect to the regulation of serotonin and dopamine synthesis than the specific, rate-limiting synthetic enzymes tryptophan hydroxylase and tyrosine hydroxylase . This iseriments , althougeriments . Our exaC. elegans with those of other organisms in order to guess about their possible functions. This is particularly relevant because all bas-1 mutants retain weak, residual serotonin immunoreactivity ([tdc-1 [Drosophila (G30446), an uncharacterized AADC homolog, is likely to encode the fly's octopamine-synthetic tyrosine decarboxylase. It has long been known that a separate gene encoded this enzymatic activity in flies, since the activity is still detectable in Ddc deletion mutants [tdc-1 protein could be reflected in the much slower rate of amino acid substitution seen in its C. elegans & briggsae orthologs than in the bas-1 orthologs which encode more 'promiscuous' enzymes.We compared the protein sequences of other predicted AADCs in ctivity . As with tdc-1, C. elegans ZK829.2 and its C. briggsae ortholog have also evolved more slowly than the bas-1 orthologs. A recent analysis of eukaryotic AADC sequences that includes the C. elegans ZK829.2 and its C. briggsae ortholog as the only nematode representatives clearly demonstrates that AADC genes can evolve at very different rates, and that a constant "molecular clock" cannot be assumed in phylogenetic analyses [Whether  elegans , this obanalyses .Finally, since the C09G9.4 predicted protein is so highly divergent from the typical AADC, and lacks a critical lysine residue that binds the PLP cofactor, it is unlikely to be an AADC enzyme. It has a similar level of divergence from genuine AADCs as do other group II PLP-dependent enzymes such as cysteine sulfinic acid decarboxylase, to which it has little or no similarity. Whatever the function of a C09G9.4-encoded protein, it appears to represent a new PLP-DC-related protein; sequencing of more genomes may yet reveal additional members.bas-1 in C. elegans, the genes C05D2.3 and F12A10.3, are missing from C. briggsae. Furthermore, phylogenetic analysis indicates the two extra genes did not arise in the C. elegans line, but were present (or their commmon ancestor was present) in the species that gave rise to both the C. elegans and C. briggsae lines. Finally, careful examination of the cDNAs and predicted protein sequences of C05D2.3 and F12A10.3 reveals that neither is likely to be functional as an AADC: the former lacks critical amino acids and the latter can encode only a truncated protein. Both are expressed, based on the presence of cDNAs, but probably at a very low level, which is not above background in microarray experiments. It is possible that the duplicate genes are functionally 'lost' in C. elegans as well.We found that the closest relatives of C05D2.4/C. elegans. Taking a random sampling of predicted genes and generating transgenics with reporter fusion constructs (in order to determine a pattern of expression), Mounsey and colleagues [C. elegans may be pseudogenes. In fact, careful inspection of recently duplicated genes showed that many were actually pseudogenes, like we found to be the case for F12A10.3. Overall, close inspection of predicted genes revealed at least 4% were pseudogenes.The features of C05D2.3 and F12A10.3 raise a number of interesting questions about the fate of duplicate genes, and the true nature of many 'predicted genes' in lleagues  found thC. elegans if they lack a function? C. briggsae and C. elegans may have diverged 80 – 110 million years ago [bas-1-like gene or genes were likely present in the common ancestor of C. elegans and C. briggsae, then there seems to have been ample time for loss in the C. elegans line. Under a simple model of gene loss following duplication, only a few million generations would be the mean time to fix a null allele of the gene duplicate [Caenorhabditis, a million generations could be completed in 10,000 years or less. This seems to suggest that the downstream duplicate of bas-1 (ancestor of C05D2.3) may have continued to function for a considerable time after the duplication, perhaps by gene conversion which might have continued until sufficient divergence from bas-1 [bas-1, but has not accumulated stop codons and frameshifts expected for a pseudogene. Walsh [So, why are C05D2.3 and F12A10.3 still present in ears ago ,38. Sincuplicate . In Caenom bas-1 . Loss ofe. Walsh  has propbas-1::GFP construct is expressed well without such sequences in cis.C05D2.3 and F12A10. 3 seem to have been retained longer than expected. Lynch and Force  proposedbas-1-like genes C05D2.3 and F12A10.3 are under relaxed selection relative to bas-1 and other AADCs. It should be noted that precise quantitative comparisons cannot be made with the results presented in the C. briggsae whole genome analysis [A/KS; however our calculations indicate that bas-1 and the other AADC's, like most genes in the Caenorhabditis genomes, are under strong purifying selection. Even if both C05D2.3 and F12A10.3 are now pseudogenes, some significant period of time during which they functioned and were under purifying selection could act to obscure this fact in an analysis of KA/KS. Even if C05D2.3 has acquired a new, adaptive function, such a new function might result from changes in only a few sites in the protein, and so again this could be obscured by a majority of sites under purifying selection. With the sequencing of three related Caenorhabditis species it will be interesting to learn of the fates of bas-1 and the bas-1-like genes in other lines.Our analysis of synonymous vs. non-synonymous substitutions indicates that the analysis , since wbas-1 gene encodes a serotonin- and dopamine-synthetic AADC enzyme in C. elegans. The C. elegans genome possesses five other AADC-homologous genes, two of which are closely related to bas-1. These bas-1-like genes are missing, however, from the congeneric C. briggsae, and evidence suggests that, despite their persistence in C. elegans, the genes do not encode functional AADC proteins. Since one or more of the bas-1-like genes was likely present in the common ancestor of C. elegans and C. briggsae- which may have diverged over 80 million years ago – it is unclear why the bas-1-like genes have been retained in the C. elegans line. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.The Caenorhabditis elegans was performed as described by Brenner [C. elegans genetics conforms to the conventions set forth by Horvitz et al. [him-5(e1490)V; MT7988: bas-1(ad446)III; MT7990: bas-1(n2948)III; MT8002: bas-1(n3008)III; LC7: bas-1(pa4)III; LC33: bas-1(tm351)III. The him-5(e1490) strain generates approximately 30% males by increased X chromosome non-disjunction [Routine culturing of  Brenner . Nomenclz et al. . Strainsjunction , but is bas-1 mutant strains  and then PCR-amplified small segments of C05D2.4 and C05D2.3 protein-coding sequence. Five pairs of primers were used to survey the C05D2.4 gene  and 4 pairs for C05D2.3 . Bands of the predicted size were excised from 2% agarose gels and purified with GeneClean (Bio101/Qbiogene), then sequenced. All mutations were confirmed by sequencing both strands for two independent PCR reactions. All PCR and sequencing primers were designed using the program Primer3  from four imer3 . Primerrol-6(su1006) [bas-1 mutant worms. Progeny of injectees that express the rol-6 dominant plasmid have the easy-to-identify Roller phenotype which results in worms with a helically-twisted body along the anterior-posterior axis. Roller transgenic worms typically carry co-injected DNAs. Transgenic Roller progeny were isolated and propagated; rescue of bas-1 mutants was scored by staining with serotonin antiserum as described previously[All DNAs for microinjection were purified with Qiagen tip20 columns. Experimental DNAs were co-injected with the dominant marker plasmid pRF4 containing the mutant gene (su1006)  into N2 reviously,67.bas-1 mutants. Clones were analyzed by restriction digests. We also made C05D2XN derivatives in which either C05D2.4 and C05D2.3 was mutated to introduce a premature stop in coding sequence. Clones were sequenced to determine the nature of the introduced mutation. Clone pCL6991 had a 4 bp deletion in the second exon of C05D2.4; pCL7991 had a 2 bp insertion in the first exon of C05D2.3; each causing a frameshift mutation. In the clone pCL7003, derived from the rescuing plasmid pCL3001, almost the entire C05D2.3 coding sequence is deleted. The bas-1 GFP reporter construct (within transgenics) was kindly provided by Ian Hope, and consists of a PCR-generated fragment of C05D2 with 4595 bp upstream of the predicted translation start site and 403 bp protein coding region to make an in-frame protein fusion in the 2nd exon. The construct was created by a multi-site recombination reaction with the C05D2 fragment, PCR-generated GFP, and vector using the Invitrogen Gateway cloning system  as continuation of a project to determine expression patterns for C. elegans genes through reporter gene technology [The rescuing plasmid C05D2XN contains a 15.8 kbp genomic DNA insert (XhoI to NheI) derived from the cosmid C05D2, and contains both C05D2.4 and C05D2.3 predicted genes. A number of deletions of C05D2XN were made to test for rescue of chnology .bas-1 mRNA.) Worms were isolated from six 100 mm NGM plates, washed several times with M9 buffer, and pooled to form a ~100 μl pellet of worms. The pellet was mixed with 175 μl RNA lysis buffer , frozen and ground to a fine powder with a pestle and mortar cooled with liquid nitrogen. The powder was recovered in a fresh microfuge tube, mixed with 350 μl SV RNA dilution buffer and centrifuged to remove debris. The cleared lysate was transferred to a new tube and precipitated with 200 μl 95% ethanol and applied to the SV spin column assembly. The remaining RNA purification was performed exactly as described for the SV System "RNA Purification by Centrifugation." Purified RNA was eluted from the spin column with 100 μl nuclease-free water. RNA was converted to cDNA in a 80 μl reaction containing 800 Units of M-MLV Reverse Transcriptase (Life Technologies/BRL), 16 μl 5X RT buffer, 25 mM dNTPs, 80 Units RNAsin (Promega) 2.0 μg random hexamer primers (Life Tech), and 50 μl purified RNA (from above). Bas-1(C05D2.4) and C05D2.3 cDNAs were amplified by PCR from him-5 cDNA produced as described above, typically using Failsafe PCR mixes (Epicentre Technologies). Conditions for PCR were 94°C (1 min.), 50°C (1 min.), 72°C (3 min.) for 40 cycles, then 72°C (10 min.). Primers used to amplify C05D2.4 cDNAs were SL1-B + C05D2-L and SL1-B + C05D2-B; a partial C05D2.3 cDNA was amplified with primers C05D2-M and -N. Spliced leader primer sequences were as follows: SL1-B: AAAGGATCCTTTAATTACCCAAGTTTGAG; SL2-B: AAAGGATCCTTTTAACCCAGTTACTCAAG. Appropriately-sized bands were isolated with GeneClean or Ultrafree-DA (Millipore), then cloned directly into the pCRII-TOPO vector using the TOPO-TA Cloning kit (Invitrogen). From seven of our RT-PCR derived clones we sequenced, we found two different splice variants different form the Genefinder prediction (see results).We isolated RNA for RT-PCR from mixed stage CB1490 worms. . DNA we received from the ORFeome project is purified from a pool of transformants derived from ligation and transformation of their original RT-PCR, thereby allowing the isolation of internal splice variants from the mix. We transformed with this DNA and isolated several clones. We sequenced two ORFeome clones completely; six more clones were partially sequenced. Interestingly, each of the 8 clones appeared to have at least one mutation (compared to known genomic sequence). Since we found one splice variant containing the 27 bp microexon from among the eight clones that we sequenced, we analyzed 29 additional ORFeome project-derived clones by PCR from single isolated bacterial colonies. Amplification of the region between primers C05D2-T and -L allowed us to distinguish between clones with or without the 27 bp microexon based on product size (464 bp versus 437 bp). Seven of 29 clones were the larger size, so likely contained the 27 bp microexon. None of the ORFeome clones analyzed by sequence or PCR appeared to use the alternative exon 3 splice acceptor noted above. Finally, the two 'YK' cDNA clones from the C. elegans EST project that we examined are from a 'full-length' capped library, and each has an SL1 leader and a poly-A tail. We found that each bas-1 'YK' clone, however, contained a different internal deletion . Each of the deletions was adjacent to a short repeated sequence.We also obtained cDNA clones from the ORFeome project  and the C. elegans genomic and predicted cDNA sequences were retrieved from ACeDB, WormBase, and/or GenBank. Blast searches and Blast2 comparisons were performed using the NCBI Blast server. C. briggsae genomic sequence was searched using TBLASTN of the 7/12/02 shotgun assembly . Assembly and consensus sequence determination of our own cDNA sequences was done using the program SeqMan . Some information about ORFeome clones was retrieved from WorfDB . Phosphv.1.1 . Multipociates, ). Some aociates, . EstimatGojobori .AADC – aromatic amino acid decarboxylaseDDC – dopa decarboxylaseHisDC – histidine decarboxylaseGAD – glutamic acid decarboxylaseTrpDC – tryptophan decarboxylasePLP – pyridoxal 5'-phosphateNSM – neurosecretory motoneuronHSN – hermaphrodite-specific neuronPDE – postdeirid sensory neuronADE – anterior deirid sensory neuronRN – ray sensory neuronCEPD, CEPV – dorsal or ventral cephalic sensory neuronADF – amphid sensory neuron, dual cilia, designation FAIM – ring interneuron, designation MRIH – ring interneuron (unpaired), designation HCP – posterior daughter of designation C cell, male-specific ventral cord motoneuronEH prepared deletion and expression constructs, cloned and sequenced bas-1 cDNAs and mutant alleles, participated in anti-serotonin staining and genetics, and drafted the manuscript. CL conceived and directed the study, generated transgenics by microinjection, performed sequence and phylogenetic analyses, and completed the manuscript. Both authors read and approved the final manuscript.


A prominent and influential hypothesis of vision suggests the existence of two separate visual systems within the brain, one creating our perception of the world and another guiding our actions within it. The induced Roelofs effect has been described as providing strong evidence for this perception/action dissociation: When a small visual target is surrounded by a large frame positioned so that the frame's center is offset from the observer's midline, the perceived location of the target is shifted in the direction opposite the frame's offset. In spite of this perceptual mislocalization, however, the observer can accurately guide movements to the target location. Thus, perception is prone to the illusion while actions seem immune. Here we demonstrate that the Roelofs illusion is caused by a frame-induced transient distortion of the observer's apparent midline. We further demonstrate that actions guided to targets within this same distorted egocentric reference frame are fully expected to be accurate, since the errors of target localization will exactly cancel the errors of motor guidance. These findings provide a mechanistic explanation for the various perceptual and motor effects of the induced Roelofs illusion without requiring the existence of separate neural systems for perception and action. Given this, the behavioral dissociation that accompanies the Roelofs effect cannot be considered evidence of a dissociation of perception and action. This indicates a general need to re-evaluate the broad class of evidence purported to support this hypothesized dissociation. Paul Dassonville and Jagdeep Bala challenge a prominent hypothesis that proposes the existence of two separate visual systems within the brain, one creating perception and the other guiding action Several anatomical, neurophysiological, clinical, and behavioral investigations of human subjects and nonhuman primates have provided evidence for two separate and dissociable cortical systems for visual processing. One of these systems—the ventral stream—resides in a swath of cortex that extends in an anteroventral direction from primary visual cortex to the temporal lobe. The second system resides in a dorsal stream that roughly extends from primary visual cortex to the parietal lobe. These ventral and dorsal systems were originally thought to be dedicated to the visual processing required to determine an object's identity and location, respectively . HoweverMuch of the evidence for separate perception and action systems has come from behavioral studies of normal subjects. The general logic of these behavioral paradigms e.g.,  is as foAs a specific example of this type of evidence, Although these findings are compatible with the hypothesis of two separate visual systems that are differentially affected by Roelofs illusion, it is important to consider the possibility of an alternative explanation. While it is true that both perceptual and sensorimotor measures within the task of We first sought to replicate the findings of A second group of subjects was instructed to make open-loop saccadic eye movements to the target location. Saccades performed immediately after the frame and target were extinguished showed no significant effect of frame position  provided informed consent to participate and were compensated with either course credit or a small monetary payment, as per a protocol approved by the University of Oregon Committee for the Protection of Human Subjects/Institutional Review Board.Subjects were placed in a completely darkened room and presented with a visual display that was back-projected  onto a screen measuring 128 × 96 cm, positioned 122 cm from the eyes. Visual targets were small  red spots, located −4°, −2°, 0°, 2°, or 4° from the subject's midline, at eye level. During experimental trials, targets were presented within a large red unfilled frame  that was either centered with respect to the subject's midline or shifted 5° left or right of the midline. All visual images were presented on a black background, with the high contrast of the Barco Cine7 projector preventing subjects from seeing the edges of the screen.Head and binocular eye positions were monitored at 250 Hz with an eye tracker  that allowed head-free measurement of gaze (precision = 0.01°); however, target placement was such that head movements contributed to only a small fraction of the total gaze displacement on any trial. To start each session (and as necessary throughout each session), eye-tracker calibration was performed using a 3 × 3 grid of targets spaced 13.5° apart in the horizontal dimension and 10.5° apart in the vertical dimension. For each subject, the average fixation error across the nine calibration targets was required to be 1° or less before beginning the subsequent practice and experimental trials; thus, absolute tracking errors within this calibration field were at most 1° in magnitude and were typically only 0.5°. In addition, subjects began each experimental trial by directing the eyes to a fixation point and pressing the space bar of a keyboard to indicate readiness. Upon this signal, the eye-tracker computer performed an adjustment of the calibration to correct for any drift that had occurred since the onset of the previous trial; that is, the calibration was adjusted so that the signal of eye position matched the known location of the fixation point. In experiments requiring eye movement responses, the gaze signals from the two eyes were averaged to yield a single representation of gaze direction as the dependent variable.Each experiment was preceded by a set of practice trials  in which subjects performed the appropriate task (see below) in the absence of the large Roelofs-inducing rectangular frame. Feedback was provided at the end of each practice trial to assist subjects in improving their performance. For all experiments, feedback included the illumination of a small circle at the target location. For the experiment in which subjects provided a button press to report the perceived target identity, a computer-generated voice also provided auditory feedback to indicate the correct target identity. For all experiments in which subjects indicated the target position with a saccadic eye movement, feedback following each practice trial also included the illumination of a small square to indicate the final gaze position; subjects were instructed to use the feedback in an attempt to minimize the distance between the target and final gaze positions. No feedback concerning response accuracy was ever provided during the experimental trials.To test the perceptual effects of Roelofs illusion see A, subjecSimilar trials were used to test the sensorimotor effects of Roelofs illusion see A, with tTo test the effects of Roelofs illusion on remembered visual space see A, subjecTo test the effects of Roelofs illusion on the apparent midline see A, subjecTo determine whether the delayed sensorimotor Roelofs effect was modulated by the presence of the frame during the imposed delay period see A, all trTo test the effects of Roelofs illusion on saccades directed toward targets defined allocentrically see A, stimulIn each experiment, the signed magnitudes of localization errors  were analyzed with a full-factorial analysis of variance, in a 5 (target location) × 3 (frame position) × 2 (response delay) design see , or a 4 Figure S1(A) Time line of task events for immediate (black) and delayed (gray) perceptual judgments of target location. Note that in this and all other experiments, feedback was presented only during practice trials, and the frame was presented only during experimental trials.(B) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of five target locations.(C) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of ten subjects.(579 KB TIF).Click here for additional data file.Figure S2(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses.(B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of five target locations.(C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of ten subjects.(1.1 MB TIF).Click here for additional data file.Figure S3(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward remembered reference-array locations.(B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference-array locations, for each of five target locations. The nonlinear effect of target location has been addressed elsewhere .(C) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference array locations, for each of ten subjects.(2.5 MB TIF).Click here for additional data file.Figure S4(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward the apparent midline.(B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward the apparent midline, for each of ten subjects.(856 KB TIF).Click here for additional data file.Figure S5(A) Time line of task events for delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period  or was present throughout the delay .(B) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period , or was present throughout the delay , for each of five target locations.(C) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period  or was present throughout the delay , for each of ten subjects.(4.7 MB TIF).Click here for additional data file.Figure S6(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses to targets defined allocentrically.(B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of five target locations.(C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of ten subjects.(1.8 MB TIF).Click here for additional data file.


The present study aimed to investigate the relationship between dexamethasone suppression test, personality disorder, stressful life events and depression.Fifty patients  aged 41.0 ± 11.4 years, suffering from Major Depression according to DSM-IV criteria entered the study.Diagnosis was obtained with the aid of the SCAN v 2.0 and the IPDE. Psychometric assessment included the HDRS, HAS, the Newcastle Scale (version 1965 and 1971), the Diagnostic Melancholia Scale, the Personality Deviance Scale and the GAF scale. The 1 mg DST was used.Included MANOVA, ANOVA with LSD post hoc test and chi-square test.Sixteen (32%) patients were non-suppressors. Eight patients without Personality Disorder (PD) (23.5%), and 5 of those with PD of cluster B (50%) were non-suppressors. Atypical patients were the subtype with the highest rate of non-suppression (42.85%). No difference between suppressors and non-suppressors was detected in any of the scales.The results of the current study suggest that pathological DST is not a core feature of major depression. They also suggest that there are more than one subtypes of depression, concerning the response to stress. It seems that the majority of depressed patients (50%) does not experience high levels of stress either in terms of self reported experience or neuroendocrine function. The rest of patients however, either experience high levels of stress, or manifest its somatic analogue (DST non-suppression) or have a very low threshold of stress tolerance, which makes them to behave in a hostile way. Life events and environmental stressful factors may relate to the development of depression -4. HowevThe Dexamethasone Suppression Test (DST)  is the mThe present study aimed to investigate the relationship between dexamethasone suppression test, personality disorder (PD), stressful life events and clinical manifestations of major depression. The hypothesis to test was that subtypes of depression could be identified on the basis of the presence of personality disorder , the presence of abnormal DST results and/or hypercortisolemia (which both constitute an idiosyncratic neuroendocrine response to stress) and the presence or not of stressful life events .The presence or not of Personality Disorder, and the response to the DST are both characteristics of the patient. Life events reflect the impact of the environment on the patient. So, life events provoke responses from the side of the patient, which are largely determined by Personality and DST response. Thus, four groups of patients can be identified and studied, according to the combination of the co-existence of DST non-suppression and personality disorder.Fifty (50) major depressive patients  aged 41.0 ± 11.4 (range 21–60) years ,10, tookrd department of psychiatry, Aristotle University of Thessaloniki, Greece. They constituted consecutive cases that fulfilled the inclusion criteria and no systemic bias exists.Patients were in- or outpatients of the 3The SCAN v 2.0  was usedSeventeen patients (34%) suffered from a personality disorder (PD). Ten of them (20%) had a cluster B PD. Concerning depressive subtypes, 5 (out of 16) melancholics (26.32%), 7 (out of 14) atypicals (50%), 9 (out of 32) patients with somatic syndrome (28.13%), and 3 (out of 9) 'undifferentiated' patients (33.33%), fulfilled criteria for PD . No patient suffered from a paranoid, schizotypal, antisocial, dissocial, narcissistic, and avoidant PD, although individual criteria were met. No criteria belonging to the schizotypal or antisocial PDs were met.No patient fulfilled criteria for catatonic or psychotic features or for seasonal affective disorder. No patient fulfilled criteria for another DSM-IV axis-I disorder, excepting generalized anxiety disorder (N = 10) and panic disorder (N = 7). Another 5 patients had both generalized anxiety disorder and panic disorder .The present study did not include a normal controls group, since the aim of the study was to compare depressive subtypes between each other.Laboratory Testing included blood and biochemical testing, test for pregnancy, T3, T4, TSH, B12 and folic acid.The Psychometric Assessment included the Hamilton Depression Rating Scale (HDRS), the Hamilton Anxiety Scale (HAS), the 1965 and 1971 Newcastle Depression Diagnostic Scale (1965 and 1971-NDDS) and the Diagnostic Melancholia Scale (DMS) [le (DMS)  and the le (DMS) . An attele (DMS) . This waThe PDS consists from the following subscales:Extrapunitive Scale (ES) which consists of 1. HT: Hostile Thoughts and 2. DO: Denigratory Attitudes Toward other People. All these scales and subscales are scored in such a way that high scores denote lack of the characteristic.a. Intropunitive Scale (IS), which consists of 1. LSC: Lack of Self-Confidence and DEP: Overdependency on Others. All these scales and subscales are scored in such a way that high scores denote presence of the characteristic.b. Dominance Scale (DS) which consists of 1. MIN: Domineering Social Attitude and 2. HA: Uninhibited Hostile Acts. The MIN is scored in such a way that high scores denotes presence of the characteristic, while HA has opposite properties.c. a. age of onset b. presence of a recent suicide attempt c. history of such attempts d. The questionnaire of Holmes  was usedDexamethasone Suppression Test (DST) protocol demands the administration of 1 mg dexamethasone per os at 23.00 of the first day, and determination of cortisol serum levels simultaneously and the next day at 16.00 and 23.00. Cortisol levels expressed in μg/dl were measured with Luminance Immunoassay . Non-suppression cut-off level: 5 μg/dl.The 1 mg Multiple Analysis of Variance (MANOVA) was performed with DST (suppression vs. non suppression) and Personality Disorder (present vs. absent) as factors. The dependent variables list included: Age, Age of Onset, Number of previous episodes, Number of DSM-IV Criteria, Number of atypical features, Number of melancholic features, GAF, NDDS 1965, NDDS 1971, Endogenous axis of DMS, Reactive axis of DMS, Number of stressful life events, HDRS-17, HDRS-21, HDRS Depressive index, HDRS Anxiety index, HDRS Sleep index, HDRS non-specific index, HAS, HAS Somatic subscale, HAS Psychic subscale, PDS-Hostile Thoughts Scale, PDS-Denigratory Attitude Scale, PDS-Extrapunitive Scale, PDS-Low Self Confidence Scale, PDS-Overdependency by others Scale, PDS-Intropunitive Scale, PDS-Domineering Social Attitude Scale, PDS-Uninhibited Hostile Acts Scale and PDS-Dominance Scale.Afterwards, Analysis of Variance (ANOVA) with Least Significance Difference (LSD) test as post-hoc test was performed.st degree relatives, and Family history of depression in 2nd degree relatives.Finally, Chi-square test was performed. PD and DST were independently placed in cross-tabulation with the presence or absence of Recent Suicide Attempt, History of Suicide Attempt, Generalized Anxiety or Panic Disorder, Melancholic Features, Atypical Features, Somatic Syndrome, 'Undifferentiated' symptomatology, Full and sustained remission, With Relapsing circumscribed episodes, Chronic Depression without full remission, Presence of Stressful life events, Family history of any mental disorder, Family history of depression in 1Women were twice as many as men (70% versus 30%), which is not uncommon  and reflSixteen out of 50 depressed patients (32%) were DST non-suppressors (NS).Eight out of 17 (47.05%) depressed patients with PD were also NS.When the patients with a coexistent personality disorder (PD) were excluded, then 8 out of 33 (24.24%) patients left, were NS.When only cluster b PDs were excluded, the respected percentage of NS climbs to 27.5% (11 out of 40).Fifty percent of Cluster b PD patients were NS (5 S and 5 NS).Six out of 14 (42.85%) atypical patients were NS, and this percentage makes this subtype the one with the highest NS percentage.No one of Chi-square tests revealed any significant findings (at p > 0.01).MANOVA results were significant both for Personality Disorder p < 0.001) and for DST (P < 0.001)  in depressed patients is 2.5–3 times higher in comparison to the general population. Half (47.05%) of these PD patients were also DST non-suppressors (NS). Atypical patients was the depressive subtype with the highest frequency of both personality psychopathology and DST NS.Figure DST results seem to be a severity marker rather than directly related to symptomatology. In patients without PD, DST NS , and were characterized by the absence of atypical features. One could say that they represent a more 'formal' group of depressed patients. The rest of patients were equally distributed in the three groups . Groups B and C may represent two distinct types of vulnerability to stress , while group D seems to represent a more severe form of depression, with an 'autonomous' hostility independent from the environment. This severe type could be considered to be the product of the accumulation of both vulnerabilities that characterize groups B and C, with the addition of a very low threshold for the tolerance of stress.Nearly 4–10% of normal persons are reported to be DST-NS. The reason for this is unknown, however it has been suggested that it is due to an underlying mood disorder or family history of affective disorder. Another explanation suggests that DST reflects in fact the degree of psychological pressure or discomfort of the subject and not a specific vulnerability or characteristic of depression. It seems that non-suppression is gradually increasing along a continuum, which has mourning outpatients on the one pole (13% NS) and severe psychotic melancholic inpatients with psychotic features and suicidal ideation on the opposite one (64% NS) . In thisDST NS and hypercortisolemia may constitute two separate entities. For example, a patient may have baseline cortisol equal to 6 μg/dl, second cortisol value equal to 2.5 μg/dl and third cortisol value equal to 5.5 μg/dl and thus is classified as NS, but is not hypercorisolemic. On the contrary, a patient with baseline cortisol value equal to 10 μg/dl, second value equal to 4 μg/dl and third also equal to 4 μg/dl, is classified as NS, but is hypercorisolaimic.Kirschbaum et al  reportedIn contrast to what is widely accepted, NS is appeared to be closer to the atypical subtype. There are no direct reports in the international literature on this matter. However, the results of the study of Kocsis et al , in esseRothschild et al  related Although the study sample of the current study is relatively small, the results suggest that there are more than one subtypes of depression, concerning the response to stress. The majority of depressed patients (50%) seems not to experience high levels of stress both in terms of self reported experience and neuroendocrine function. The rest of patients however, experience high levels of stress, either internally or have the somatic analogue of it (DST non-suppression) or have a very low threshold of stress tolerance, which makes them to behave in a hostile way.The authors declare that they have no competing interests.


Multiple sclerosis (MS) can be an unpredictable disease. It develops when the body's immune system attacks healthy nerve cells and disrupts normal nerve signaling. Patients experience a wide range of symptoms—including tingling, paralysis, pain, fatigue, and blurred vision—that can appear independently or in combination, sporadically or persistently. Although symptoms appear in no particular order, flare-ups are common in the majority of patients.MS flare-ups are commonly treated with beta-interferon. Adverse effects are not uncommon, and, more importantly, a sizable proportion of patients show a reduced response, or no response at all. Given the variability of the disease and treatment response, being able to predict how a particular patient is likely to respond to interferon would help doctors decide how close to monitor the patient or even whether to consider alternative treatments. In a new study, Sergio Baranzini et al. describe a computational model that can predict a patient's therapeutic response to interferon based on their gene expression profiles.Immune cells typically secrete interferons to fend off viruses and other pathogens. Interferons stem viral infection by inhibiting cell division in neighboring cells—thus preventing the virus from reproducing—and triggering pathways that kill the infected cells. It's thought that interferon therapy may relieve symptoms associated with MS by correcting imbalances in the immune system that lead to disease. Interferon therapy produces changes in the gene expression profile of targeted cells—that is, it inhibits or activates certain genes—which in turn alters the cells' activity.Blood samples were taken from 52 patients with relapsing-remitting MS , and their RNA was isolated from a class of immune cells called peripheral blood mononuclear cells. After patients started interferon therapy, blood was taken at specific time points over the course of two years. Baranzini et al. measured the expression level of 70 genes—including a number involved in interferon interactions and immune regulation—at each time point.The authors used statistical analyses to search for gene expression profiles that were associated with patients' therapeutic outcomes. They looked for patterns in analyses of single genes, gene pairs, and gene triplets, and found their model's predictive accuracy increased with gene number. They also looked for genes that showed different expression patterns over the two years based on patient response, time passed, and patient response over time. These analyses identified genes that increased activity independently of clinical response (interferon can activate genes that have no effect on disease), as well as genes that were associated with a good or poor response. Some of these genes were also the best predictors of patient response before therapy was started.This approach can predict the probability of a good or poor clinical response with up to 86% accuracy. Baranzini et al. offer hypotheses to explain how the observed gene activity might produce the differential responses to therapy—for example, a poor response may stem from downstream signaling events rather than from problems with drug metabolism. But the authors caution that the mechanisms connecting these genetic signatures to specific outcomes—and the mechanisms that produce a positive interferon response—have yet to be established. For now, these patterns should be thought of as markers. Still, these results suggest that doctors could one day tailor MS patients' treatments to their molecular profile, and perhaps take some of the uncertainty out of this capricious disease.


Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper Under conditions of oxidative stress, methionine auxotrophy in  As a consequence of living in an aerobic world, organisms face the challenge of maintaining a favorable cellular redox status. Oxidative stress is caused by imbalances between the production and disposal of reactive oxygen species, which can damage proteins, lipids, and nucleic acids. Bacteria encounter reactive oxygen intermediates that are generated as byproducts of aerobic metabolism or during challenge by the immune systems of hosts. Thus, understanding the effects of oxidative stress on the cell, as well as elucidating cellular defense mechanisms, is of considerable interest.Escherichia coli have been reported that collectively suggest a link between oxidative stress, methionine limitation, and the enzyme that catalyzes the final step in methionine biosynthesis, cobalamin-independent methionine synthase (MetE). In E. coli, the sulfur of methionine comes from cysteine, which in turn obtains the sulfur group from sulfate via the sulfate assimilation pathway  were sensitive to oxidative challenge and had an oxygen-dependent growth requirement for the sulfur-containing amino acids cysteine and methionine (trxA grxA (thioredoxin 1 and glutaredoxin 1) and grxA grxB grxC gor  strains of E. coli, cells required cysteine or methionine for growth, presumably because of oxidative inactivation of 3′-phosphoadenylylsulfate (PAPS) reductase -independent methionine synthase (the metE gene product) or B12-dependent methionine synthase (the metH gene product). Both enzymes employ a catalytic zinc to transfer a methyl group from methyltetrahydrofolate to L-homocysteine to form methionine  use only the B12-independent isoform  reversibly oxidizes MetE at a rate that correlates with enzyme inactivation. GSSG was found to specifically glutathionylate MetE and induce a conformational change. In vivo thiol-trapping experiments provide direct evidence that MetE is readily oxidized in cells experiencing a variety of oxidative stress conditions. Moreover, oxidation of MetE is associated with a cellular dependence upon methionine for growth, suggesting that oxidant-mediated inactivation of MetE may have a profound impact on the stressed cell.In this study, we investigated the effects of oxidative stress on MetE and methionine availability within the cell. Transient oxidative stress conditions induced by diamide were found to elicit a methionine auxotrophy in E. coli. We investigated the duration of growth lags induced by oxidative stress in cells growing in glucose-minimal medium containing or lacking methionine. In the experiment shown in E. coli cultures logarithmically growing in glucose-minimal medium with and without methionine experienced a lag in growth upon challenge with diamide  was used to induce oxidative stress. Addition of methionine to the medium significantly decreased the growth lag produced by H2O2 treatment  as loss of activity (0.07 min−1).The methionine auxotrophy observed under oxidative stress conditions could reflect an increased demand for methionine or a decreased rate of de novo synthesis due to inactivation of MetE. To distinguish between these possibilities, we assayed the activity of the purified enzyme upon challenge with GSSG . Nearly While the rate of thiol oxidation could be measured with DTNB, the absolute number of cysteines oxidized was ambiguous. MetE contains seven cysteines, yet DTNB titration of the fully reduced enzyme gave values between 7.2 and 9.0 cysteines for different protein preparations, and oxidation of MetE led to an average decrease of 1.8 cysteines per mol. Thus it was important to use independent means to determine the number of cysteines oxidized by GSSG.Oxidation of MetE by GSSG would be expected to proceed by initial formation of a mixed disulfide between MetE and glutathione .An inter- or intramolecular disulfide bond could then be generated by attack on the mixed disulfide by a second cysteine residue. These two possibilities can be distinguished by using mass spectrometry to determine the molecular mass of the oxidized protein. The reduced protein was found to be 84,530 ± 6 Da , whereas the mass of the protein oxidized by GSSG was 84,835 ± 8 Da. The mass difference of 305 ± 10 Da between oxidized and reduced MetE is consistent with the addition of a single glutathione adduct . Formation of an intramolecular disulfide bond would only decrease the mass by 2 Da, which would not be detected by mass spectrometry in such a large protein.To obtain evidence for stoichiometric glutathionylation of MetE, the oxidized protein was reduced with DTT to release the glutathione adduct. The DTT and GSH were separated from the protein using a Microcon concentrator. The filtrate was assayed for GSH using fluorescamine, which reacts with primary amines to form a fluorescent product. The reduced protein did not release GSH (0.0 mol GSH per mol protein) while oxidized protein released 0.9 mol GSH per mol of protein. Thus both mass spectrometry and the fluorescamine assay were consistent with glutathionylation of MetE by GSSG.In order to determine the site of oxidation and verify that an intramolecular disulfide was not being formed concomitant with oxidation, we performed disulfide mapping experiments based on the method of This approach allows for relatively straightforward mapping of disulfide bonds. The acidic pH employed minimizes the possibility of disulfide bond scrambling. Furthermore, the ability to cleave N-terminally to each cyanylated cysteine allows cleavage between closely spaced, even adjacent, cysteines. These factors provide a significant advantage over traditional methods of peptide mapping, particularly for MetE, which contains cysteines spaced only two residues apart at the active site (cysteines 643 and 645). Moreover, for large proteins, most proteolytic and chemical cleavage methods have the potential to generate numerous peptides, whereas this approach produces a more limited number.I, and β-elimination at cysteine 560 produced a small amount of product H. Identification of these side products provides additional evidence that cysteines 560 and 726 were reduced and available for cyanylation. Disulfide mapping of a mutant MetE, MetEC>A1–4, in which cysteines 323, 353, 516, and 560 were changed to alanines, further confirmed the mass assignments. Only peaks corresponding to cleavage next to the three remaining cysteines  were observed  need to be taken into account. Based on the equilibrium constant for GSSG oxidation, MetE activity at equilibrium was calculated for typical cellular glutathione concentrations  at varioE is –0.252 V at pH 7.2  of 1, 2, or 5 mM, and the calculated [GSH]2/[GSSG], [GSH] and [GSSG] can be determined at each cellular potential. The ratio of [GSH]/[GSSG] may then be used to calculate the relative amount of MetE activity expected at equilibrium bywhere se in pH ), R is 8mix is 1.4.)where KE. coli growing under normal conditions (–250 mV to –280 mV /[GSSG] ratio of less than 1.4 would be required to accumulate that much oxidized MetE . Moreover, in vitro oxidation of MetE by GSSG is rather slow, further suggesting that glutathionylation of MetE via thiol-disulfide exchange is not the primary mechanism functioning in vivo. Rather, the in vitro and in vivo data for MetE are consistent with initial formation of a reactive intermediate followed by glutathionylation of cysteine 645, resulting in enzyme inactivation.The ratios of [GSH] to [GSSG] in wild-type (DHB4) and ectively . Though trxA strain. Since E. coli lacking trxA do not experience a substantial amount of cytosolic disulfide bond formation (2O2) accumulate in the trxA strain, since thioredoxin appears to be required to regenerate the reduced thiol status of these proteins. Our preliminary in vitro experiments indicate that glutathionylated MetE is inefficiently reduced by thioredoxin (data not shown), which is not unexpected since glutaredoxins are generally more specific for glutathione-protein adducts  results in irreversible modification, whereas oxidation in the presence of GSH (as would be expected in vivo) yielded reversible glutathionylation  or sulfonic (–SO3) acid adducts (data not shown). Thus, glutathionylation of MetE following the formation of a reactive intermediate may serve the dual purpose of modulating enzyme activity while protecting the active site from more extensive damage. Reduction by cellular thioreductases could then readily reactivate the enzyme.Protein glutathionylation has been suggested to play a protective role in oxidative stress. Studies with purified eukaryotic proteins have shown that incubation with oxidants  and Grx3 (grxC) are structurally distinct from Grx2 (grxB), yet all three proteins are capable of reducing glutathionylated PAPS reductase . This places the sulfhydryl in direct contact with the solvent, where it is accessible to oxidation and reduction. Glutathionylation of cysteine 645 at the entrance to the active site may sterically prevent substrate binding, leading to the observed inactivation of the enzyme. Addition of the glutathione adduct also appears to correlate with a weakened binding of the catalytic zinc to the enzyme (data not shown). The active site of MetE is located in a cleft formed between two domains . Addition of the glutathione tripeptide to cysteine 645 may provide enough bulk to push the two domains apart, giving rise to the observed conformational change and increased hydrodynamic radius. CD spectroscopy indicates that oxidation does not result in gross unfolding of MetE  as well as wild-type cells challenged with diamide or H2O2. Our results further show that oxidation of MetE is associated with a methionine limitation imposed by oxidative stress. Hence this study provides insight into a previously unknown, but important aspect of the E. coli cellular response to oxidative stress.In conclusion, we have established that MetE, a highly expressed protein in 6S)-5-methyltetrahydropteroyltriglutamate was synthesized from pteroyltriglutamate , as described previously , ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions . Iodoacetate and iodoacetamide were purchased from Fluka . (eviously . L-Homocolactone . PolycloE. coli K-12 strain DHB4 and isogenic E. coli K-12 strains WP843 (trxA gor) and WP778 (trxB gor) (E. coli K-12 strain GW2531 (metE) . Wild-type rxB gor)  were obt1 (metE) . The Met1 (metE)  using Pf1 (metE) , specify600 ∼1). Cultures were incubated at 37 °C with shaking at 250 rpm for approximately 30 h before harvesting. The proteins were purified as described previously  (2O2 concentrations were determined similarly (ɛ240 = 43.6 M−1cm−1)  medium  supplemeM−1cm−1) . H2O2 coM−1cm−1) .−1cm−1 using the Endelhoch method -5-methyltetrahydropteroyltriglutamate, and enzyme in a total volume of 400 μl. After a 30-s incubation at 37 °C, the reaction was quenched by addition of 100 μl of 5 N hydrochloric acid/60% formic acid, followed by heating at 88 °C for 10 min. After cooling on ice, the reaction was centrifuged at 14,000g for 5 min at 4 °C to remove precipitated protein. The amount of tetrahydrofolate product (converted to methenyltetrahydrofolate by formic acid) was quantitated by its absorbance at 350 nm. Excess GSSG used to oxidize the enzyme did not significantly alter activity during the 30-s assay.The molar absorption coefficient for MetE at 280 nm was determined to be 157,000 ± 1000 Mh method . The speh method , but omig for 30 min at 4 °C. The supernatant was removed, and the pellet was dissolved in 100 mM potassium phosphate buffer (pH 7.3) containing 0.25 mM DTNB, 6 M guanidinium chloride, and 1 mM EDTA. Complete dissolution of the pellet required vortexing for 15–20 min. Reduced cysteines were quantitated by measuring the absorbance at 412 nm after subtraction of a blank containing DTNB and buffer but no protein. The extent of oxidation was determined using the fraction of reduced cysteines at the indicated time compared to the value at time zero (taken as 100%).MetE (50 μM) in 100 mM Tris chloride (pH 7.2) was equilibrated to 37 °C, and GSSG was added  to initiate the oxidation reaction. The GSSG concentration was established by assay with glutathione reductase . At timePurified MetE was desalted into 100 mM Tris chloride (pH 7.2) to remove DTT from the storage buffer. The reduced protein (50 μM) was then incubated with 10 mM GSSG for 90 min at 37 °C. Samples were concentrated using Microcon concentrators , and GSSG was removed using Bio-Gel P-6 columns  equilibrated with 100 mM Tris chloride (pH 7.2). Reduction and reactivation of GSSG-oxidized MetE was accomplished by incubation of the oxidized enzyme with 2 mM reductant at 37 °C. For preparation of samples of diamide-oxidized MetE to be used as standards for in vivo thiol-trapping assays, diamide (1 mM) was incubated with MetE (10 μM) in 100 mM Tris chloride (pH 7.2) at 37 °C. After 45 min, diamide was removed by gel filtration using Bio-Gel P-6 columns.g for 5 min at room temperature. Aliquots (100 μl) of the filtrate were added to 1.7 ml of 200 mM borate buffer (pH 9.0) containing 1 mM p-(hydroxymercuri)benzoic acid. While vortexing, 0.6 ml of 1 mg/ml fluorescamine (in acetone) was added. The fluorescence emission of the samples was measured at 485 nm with excitation at 390 nm. A range of GSH concentrations (0–125 mM) was assayed to generate a standard curve. GSH concentrations were determined by DTNB titration (Reduced and GSSG-oxidized MetE preparations were exchanged into 50 mM potassium phosphate (pH 7.2) using Bio-Gel P-6 columns. Proteins were then washed three times with the same buffer using Centricon concentrators. MetE (50 μM) was incubated with 2 mM DTT in 50 mM potassium phosphate (pH 7.2) in a volume of 200 μl for 1 h at 37 °C. Each solution was then loaded into a Microcon concentrator and centrifuged at 12,000itration . The stoCDAP dissolved in 200 μl of 100 mM citrate buffer (pH 3) containing 6 M guanidine hydrochloride was added to 100 μl of MetE in 100 mM Tris chloride (pH 7.2) and incubated for 15–20 min at room temperature. The stock concentrations were adjusted so that CDAP was approximately 25-fold in excess over the total thiol concentration. CDAP was removed from the solution by gel filtration, using Bio-Gel P-6 columns equilibrated with 6 M guanidine hydrochloride solution (brought to pH ∼7–8 with potassium hydroxide). Concentrated ammonium hydroxide was added , and the reaction was incubated at room temperature for approximately 75 min. Samples were then lyophilized to remove the ammonia and stored at –80 °C. Prior to LC-MS analysis, DTT was added  and allowed to incubate at room temperature for 30 min. Solutions were then diluted into 6 M guanidine hydrochloride solution (adjusted to pH 3 with acetic acid) and characterized by LC-MS.Samples (250–500 pmol) were injected onto a Vydac  250 mm × 2.1 mm C4 column (214MS52) using an analytical HPLC Surveyor from ThermoFinnigan . The column was equilibrated with 97% solvent A (water with 0.1% v/v acetic acid and 0.02% v/v TFA) and 3% solvent B (acetonitrile with 0.1% v/v acetic acid and 0.02% v/v TFA), and samples were eluted at a flow rate of 0.25 ml/min with a three-step linear gradient: 3% B for 15 min, 3% to 30% B in 9 min, and 30% to 65% B in 35 min. For LC-MS of the holo-protein, samples were desalted using a two-step linear gradient: 20% B for 10 min, and 20% to 70% B in 25 min. Fractionation was monitored by the UV chromatogram recorded at 280 nm. The eluent was diverted to waste for the first 15 min, and then directly infused into the electrospray ionization source of a ThermoFinnigan LCQ mass spectrometer. Mass spectral data were obtained in the positive mode, and ESI spectra were deconvoluted using the BioMass software provided by ThermoFinnigan.Oxidation of MetE by diamide was monitored at 37 °C using a Hi-Tech Scientific  SF-61DX2 stopped-flow spectrophotometer equipped with a xenon light source for single wavelength detection. Diamide (1 mM) and MetE (100 μM) in 100 mM Tris chloride (pH 7.2) were mixed, and reduction of diamide was monitored at 325 nm.MetE (20 pmol) was injected onto an Alltech  macrosphere size exclusion HPLC column (250 mm × 4.6 mm) with a 300-Å pore size (7-μm particle size) and run at 0.5 ml/min with 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride. For GroEL interaction experiments, GroEL (2 μM) was incubated with MetE (1 μM) in 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride for 30 min at 37 °C before analysis by size exclusion chromatography.g at 4 °C and then diluted to 0.4 mg/ml. Far UV CD spectra were recorded using a JASCO  J-810 spectropolarimeter and 0.1 cm cuvettes. Absorbance spectra of the same sample were recorded on a JASCO V-550 UV/Vis spectrophotometer to determine the precise protein concentration. The data were then normalized to a protein concentration of 4.5 μM MetE.Reduced and oxidized MetE were buffer exchanged into 10 mM potassium phosphate buffer (plus 500 μM DTT for the reduced protein) (pH 7.2). The final filtrates were collected and used as a blank. Samples were centrifuged for 1 h at 14,000Aliquots containing 2.5 mg/ml reduced or oxidized MetE in 50 mM potassium phosphate (pH 7.2) were digested with 0.02% (w/v) trypsin at room temperature. At indicated times, 5-μl samples were quenched with 10 μg of 1-tosylamido-1-lysyl chloromethylketone, and then analyzed by SDS-PAGE (10% gel).350 directly correlates to the amount of product in the activity assay, relative MetE activity was calculated by:MetE (10 μM) was incubated with varying concentrations of GSH and GSSG in 100 mM Tris chloride (pH 7.2) at 37 °C. GSH  and GSSG350(x) is the absorbance at 350 nm for sample x, A350(oxid) is for completely oxidized MetE , and A350(red) is for completely reduced MetE . The equilibrium constant, Kmix, was then determined by plotting relative MetE activity versus the [GSH]/[GSSG] ratio.where AE. coli cultures used for in vivo thiol-trapping experiments were grown aerobically at 37 °C. E. coli wild-type (DHB4) and trxA gor (WP843) strains were grown in glucose-minimal MOPS medium supplemented with 10 μM thiamine and all amino acids except methionine  was transformed into electrocompetent trxB gor (strain WP778) cells by electroporation. An overnight culture of WP778/pJG816 in LB medium containing 20 μg/ml kanamycin, 50 μg/ml ampicillin, and 4 mM DTT was diluted to an OD600 of approximately 0.02 into the same medium (without kanamycin). Care was taken to ensure that an overnight culture from the same inoculum failed to grow when DTT was not included in the medium. The 50-ml culture was grown to an OD600 of approximately 0.4, and then filtered into a prewarmed sterile filter flask. The cells were washed twice with prewarmed sterile LB medium to remove residual DTT, and resuspended in 50 ml of prewarmed LB medium. At the indicated time points following DTT removal, samples were taken for alkylation as described.For in vivo thiol-trapping experiments performed on the E. coli were grown in glucose-minimal MOPS medium without methionine as in the growth experiments described above, except that diamide  was added at an OD600 of approximately 0.4. Aliquots were removed at intervals following diamide addition for treatment with alkylating agents.To assay the oxidation status of MetE in diamide-treated cells, wild-type g) for 30 min at 4 °C, washed with cold 10% TCA (once) followed by ethanol (twice), and then dried by incubating approximately 20 min in a 37-°C incubator. The pellet could then be stored at –80 °C prior to secondary alkylation with iodoacetic acid. Pellets were dissolved in 50 μl of alkylation buffer containing DTT , and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate  and incubating 2 min at room temperature. For some samples, excess DTT was added to quench the reaction; however, this was found to not be necessary. Alkylated proteins were then frozen in liquid nitrogen and stored at –80 °C prior to analysis by isoelectric focusing.In order to trap oxidized thiols in vivo, 1 ml of culture was added to 250 μl of 0.5 M iodoacetamide in 100 mM Tris chloride (pH 7.2) that had been pre-equilibrated at 37 °C. Samples were incubated 2 min at 37 °C, quenched by the addition of 140 μl of 100% (w/v) ice-cold TCA, and incubated on ice for at least 30 min. Precipitated proteins were pelleted by centrifugation  at 4 °C for 30 min and then washed with cold 10% TCA (once) and either ether (3 times) or 1 M Tris chloride (pH 7.2) followed by water. Pellets were then alkylated with iodoacetate as described for the in vivo culture samples.For the reduced and oxidized MetE standards, 25 μl of 0.5 M iodoacetamide was added to 100 μl of 10 μM MetE in 100 mM Tris chloride (pH 7.2) and incubated for 1 min at 37 °C. The reaction was quenched by addition of 125 μl of 20% (w/v) ice-cold TCA and incubation on ice for at least 30 min. The proteins were pelleted by centrifugation , 50 mM DTT, and 0.15% SDS. Care was taken not to increase the solution temperature above 30 °C to avoid breaking down urea, which could then carbamylate proteins. Aliquots of the sample buffer were snap-frozen in liquid nitrogen and stored at –20 °C.An Xcell Surelock Mini-cell gel apparatus from Invitrogen was assembled with the freshly prepared gel, and degassed cathode  and anode  solutions were added. Samples were diluted at least 10-fold into sample buffer and loaded. Power was applied for 1 h at 100 V followed by 1 h at 200 V and finally 30 min at 500 V. Gels were then stained as described previously  or blottGels were electroblotted onto PVDF membrane  according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE  and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody  followed by direct detection using a Molecular Dynamics  Storm phosphorimager.http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are AhpC (P26427), glutaredoxin 1 (P00277), glutaredoxin 2 (P39811), glutaredoxin 3 (P37687), glutathione reductase (P06715), GroEL (P06139), iron superoxide dismutases (P09157), manganese superoxide dismutase (P00448), MetA (P07623), MetE  (P82610), MetE (E. coli) (P25665), MetE (T. maritima) (Q9X112), MetH (P13009), OxyR (P11721), PAPS reductase (P17854), thioredoxin 1 (P00274), thioredoxin reductase (P09625), and Tpx (P37901).The Swiss-Prot (


Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B.Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3–4°C higher over wild type. All mutants showed 2-fold higher thermal stability at 55°C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities.We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity. Acinetobacter calcoaceticus has great potential for application as a constituent of an electron mediator-type glucose sensor. The conventionally utilized enzyme for glucose measurement, glucose oxidase (GOD), inherently utilizes oxygen as its electron acceptor during the oxidation of glucose. In contrast, PQQGDH-B is completely independent of oxygen, resulting in improved accuracy and rapidity during glucose measurement. However, because the substrate specificity and stability of PQQGDH-B remain somewhat inferior to those of GOD, we have been engaged in the improvement of these enzymatic properties through protein engineering to further increase the application of PQQGDH-B in glucose monitoring systems [Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from  systems -17.The subunit structure of PQQGDH-B was determined to be homodimeric with no activity observed in its monomeric form . CorrectIn this paper, we report on our rational designing of hydrophobic interaction in the dimer interface to increase the stability of PQQGDH-B. We identified protein regions at the dimer interface where potential novel hydrophobic interactions could be introduced by amino acid substitution, thereby increasing the stability.Among 19 amino acid residues located at the dimer interface, 8 residues were predicted not to be involved in the formation of hydrogen bonds, electrostatic interactions, or hydrophobic interactions Fig. , no inteThe activity and stability of each of the three constructed double mutant enzymes were then analyzed. All three mutant enzymes showed slightly higher thermal stability than wild-type PQQGDH-B upon incubation for 10 min at various temperatures Fig. . The temInvestigation of the kinetic parameters of the wild-type and mutant enzymes shows that the mutations did not significantly affect the overall kinetic properties of PQQGDH-B Table . The spePQQGDH-B is a 6-blade β-propeller protein, with each blade consisting of a 4-stranded anti-parallel β-sheet (W-motif) . The strBased on the predicted structure of the mutant enzymes shown in Figure The addition of hydrophobic interactions in other enzymes has been reported to result in thermostability increases of 2 to 10°C -24, compWe improved PQQGDH-B's thermal stability by increasing the dimer interface interaction through rational design based on its quaternary structure. We demonstrated that this can be achieved by selecting residues located at the dimer interface that do not contribute to the intersubunit interaction and substituting them to hydrophobic ones via site-directed mutagenesis. In each case tested, the enzyme's thermal stability was increased without decreasing its catalytic activity. This rational design approach will provide relevant information for future designs by combining with other mutant PQQGDH-Bs with narrowed substrate specificity and improved catalytic efficiency.KpnI was obtained from TOYOBO  and HindIII from New England BioLabs .Glucose, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), and magnesium chloride were obtained from Kanto Kagaku , 3-(N-morpholino) propane sulfonate (MOPS) from Dojin , and pyrroloquinoline quinone from Mitsubishi Gas Chemical Company . All other regents were of analytical grade. KpnI-HindIII fragment containing the PQQGDH-B gene was transferred from pGB to pKF18k and mutagenesis was carried out with the Mutan-Express Km kit (Takara) according to the manufacturer's instructions with the oligonucleotides Asn340Phe (5'-GGTGGGACAAAGAATTTACCAGTCC-3'), Tyr418Phe (5'-CGGTACAGCGTCATCAAAAGTAGTGC-3'), Tyr418Ile (5'-CGGTACAGCGTCATCAATAGTAGTGC-3'), and Thr416Val/Thr417Val (5'-CAGCGTCATCATAAACAACGCTATAAGTTGGATC-3'). The mutations (underlined) were confirmed by automated DNA sequencing . The mutated genes were digested with KpnI and HindIII and were replaced into pGB to construct expression vectors containing mutated PQQGDH-B. Numbering of the amino acid positions starts from the first residue of the signal peptide (24 residues).The structural gene for wild-type PQQGDH-B was previously amplified by polymerase chain reaction (PCR) and inserted into the expression vector pTrc99A (Pharmacia) to create pGB . A 1.2-kEscherichia coli and the enzymes purified as previously reported [2 at room temperature (25°C) to produce the holoenzyme, GDH activity was measured by using 0.6 mM PMS and 0.06 mM DCIP. The enzyme activity was determined by measuring the decrease in absorbance of DCIP at 600 nm.The PQQGDH-B genes were expressed in reported ,16. FollThe thermal stability of wild-type and mutant PQQGDH-B was determined with 0.075 μg/mL protein, as previously reported . ThermalThree-dimensional and quaternary structures were predicted using Molecular Operating Environment (MOE) . By using the available PDB data of the wild-type PQQGDH-B, 1QBI , we madeST carried out the site-directed mutagenesis of PQQGDH-B as well as the preparation and characterization of the resulting proteins. SI carried out the 3D modeling and participated in the design of the study. SF participated in interpretation of the results and in drafting the manuscript. KS conceived of the study, participated in its design and coordination, as well as in drafting the manuscript. All authors read and approved the final manuscript.


Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied.B. bassiana extracts was measured using intradermal skin testing.IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana.Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana. Metarhizium anisopliae, Verticillium lecanii, and Beauveria bassiana are being used as biocontrol agents for a number of crop, livestock, and human nuisance pests [B. bassiana have been licensed for commercial use against whiteflies, aphids, thrips, and numerous other insect and arthropod pests. B. bassiana fungal formulations are being spread onto a range of vegetables, melons, tree fruits and nuts, as well as organic crops. As alternatives to chemical pesticides these agents are natural occurring and are considered to be non-pathogenic to humans, although a few cases of B. bassiana mediated tissue infections have been reported [Microorganisms are currently under intensive study for use as biopesticides -3. Severce pests -7. Straireported ,9.Alternaria, Aspergillus, and Cladosporium genera [Airborne mold spores are widespread, and many have been identified as inhalant allergens eliciting type I hypersensitive reactions in atopic individuals -14. Commm genera -19. The m genera ,20-22, aBeauveria bassiana crude extracts contain numerous allergens capable of being recognized by human serum IgEs. The allergens were proteinaceous in nature, and immunoblot inhibition experiments revealed the presence of shared epitopes between Beauveria and several other common fungal moulds. Potential Beauveria-specific allergens were also identified, including a strongly reactive ~35-kDa protein band. Intradermal skin testing using B. bassiana extracts resulted in allergenic reactions in several individuals, including some who have had occupational exposure to the fungus.In the present study, we demonstrate Beauveria bassiana (ATCC 90517) was grown on Sabouraud dextrose + 0.5–1% yeast extract or Potato dextrose (PD) media on either agar plates or in liquid broth. Plates were incubated at 26°C for 10–12 days and conidia were harvested by flooding the plate with sterile dH2O containing 0.01% Tween-20. Liquid cultures were inoculated with conidia harvested from plates at 0.5–1 × 105 conidia/ml.Alternaria alternata, Aspergillus fumigatus, Candida albicans, Cladosporium herbarum, Epicoccum purpurascens, and Penicillium notatum were acquired from Greer Laboratories inc., . Extracts were resuspended in TE  to a final concentration of 2 mg/ml. Beauveria bassiana was grown in Sabouraud's broth containing 1% yeast extract with aeration at 25°C for 3–5 d. Cellular mass was harvested by centrifugation  and freeze-dried. Cells were resuspended in TE containing 0.1% phenylmethylsulfonyl fluoride (PMSF) and homogenized using a bead-beater apparatus.B. bassiana were subjected to three successive precipitations before use in Western blots.Crude extracts of B. bassiana extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation , and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF.Homogenized Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged  in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate.Beauveria extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation . The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use.The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate . Saturated ammonium sulfate (120 ml) was added dropwise to the Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis  using standard protocols. Gels were stained with Gelcode blue stain reagent  and subsequently de-stained with dH20.Protein samples were separated under reducing conditions using 10% Bis-tris polyacrylamide gels (Invitrogen Mops system) and transferred to polyvinylidene-fluoride (PVDF) membranes (Invitrogen) as described. Immunoblot experiments were performed using individual and pooled human sera as the primary antibody solution as indicated. Typically, sera were diluted 1:5 with Tris-HCl buffered saline (TBS) containing 5% dry milk + 0.1% Tween-20. IgE-specific reactivity was visualized using a horseradish peroxidase (HRP) conjugated goat anti-human IgE  secondary antibody . Membranes were washed with TBS containing 0.1% Tween-20 and bands were visualized using the Immuno-Star HRP detection system .B. bassiana crude extracts was treated with Proteinase K  following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution  for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H  and peptide: N-Glycosidase F  according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer  at 100°C for 10 min prior to the addition of the EndoH  and PNGaseF reaction buffers (50 mM sodium phosphate pH 7.5) and enzymes (5 μl), respectively. Reactions were incubated at 37°C for 2 h before being analyzed by SDS-PAGE and Western blotting.The ammonium sulfate fraction of B. bassiana proteins were competed with proteins of other fungal extracts. SDS-PAGE resolved B. bassiana proteins were electroblotted to PVDF membranes as described above. Membranes were blocked with TBS containing 5% dry milk + 0.1% Tween-20 and strips were incubated with pooled human sera (1:5 v/v in same buffer) containing 100–500 μg of the indicated fungal crude protein extract.IgE binding to Alternaria tenius, Aspergillus fumigatus, Cephalosporium (Acremonium strictum), Curvularia spp. Bipolaris, Epicoccum nigram, Fusarium spp., Helminthosporium sativum, Hormodendrum horde, Penicillium . Extracts were tested using a 1:10 dilution of the 20,000 PNU/ml stock solution, and skin sensitivity was recorded on a relative scale from 0–4 reflecting the size of induration or weal (4 representing the highest reactivity) and using histamine (0.1 mg/ml) reaction scored as a 3 if no interference was present.Patients were tested with 9 common fungal extracts for allergy diagnosis using a skin prick assay. The following extracts were obtained from ALA-Abello ; B. bassiana crude extracts were prepared as described above but were extensively dialyzed against 0.15 N NaCl and filtered through a 0.22 μm filter before use. Subjects were given intradermal injections of 0.1 ml crude extract ranging in concentration from 0.01–1 mg/ml. Control injections included saline and histamine (0.1 mg/ml). Allergenic reactions were allowed to develop for 15–30 min before the height and width of the reactions were recorded.B. bassiana proteins was resolved on SDS-PAGE ; with the most prominent bands located around 64, 45, and 35 kDa. Control experiments omitting either the primary or secondary antibody incubation steps resulted in complete loss of signal. Proteinase K digestion of samples also resulted in loss of all signal  were resolved by SDS-PAGE, blotted to PVDF membranes, and the lanes were cut into separate strips. Each strip was treated with a 1:5 dilution of each respective serum as described in the Materials and Methods . Blots probed with one serum (H) resulted in a large smear ranging from ~30 kDa to 55 kDa. The reason for the observed smear is unknown and efforts to distinguish separate bands by manipulating the concentrations of either sera or extract were unsuccessful. A number of bands (based upon molecular mass) appeared to be common to several sera including proteins of approximately 35, 42–48, and 60 kDa. A number of allergens of high molecular weight (~100–200 kDa) were also visible; however the resolution in this range on the Western blots is poor.In order to determine the variation and distribution of serum IgEs reactive to ies Fig. . The reaies Fig. , lane 2.ies Fig.  and reprB. bassiana crude extracts using an intradermal delivery procedure. Data using 1 mg/ml B. bassiana crude extract and histamine controls are presented in Table B. bassiana extracts  were resolved by SDS-PAGE, blotted to PVDF membranes, and lanes were cut into separate strips. Each strip was treated with a 1:5 dilution pooled sera (serum mix-II) as the primary antibody supplemented with concentrations of fungal crude extracts as described in the Materials and Methods. Fig. B. bassiana extracts were competed with: excess crude extracts from Alternaria alternata , Cladosporium herbarum (lanes 7), Epicoccum purpurascens (Lane 8), Penicillium notatum (lane 9), and Candida albicans (lane 10). There was complete loss of all signals using 2-fold excess B. bassiana extract as the competitor (data not shown). These data indicate that while Beauveria possess many epitopes in common with several other fungi, notably Alternaria and Penicillium, a 35-kDa major reactive band was not inhibited by any extract tested.In order to determine the extent of cross-reactivity of Beauveria could barely be detected in airborne samples, and represented less than 0.1% of the airborne fungal "flora", the incidence of allergic skin test reaction to Beauveria was the highest of all fungal species tested [Beauveria bassiana is imperative.Although it is well known that fungi are important triggers of respiratory allergies, the potential allergenicity of entomopathogenic fungi used in biocontrol has largely been untested. Aerobiological surveys of conidial fungi and skin sensitivity tests to fungal extracts performed in the 1980s in the Netherlands revealed that although s tested ,23,24. IB. bassiana directly by intradermal skin testing of individuals and in vitro by revealing the presence of serum IgEs capable of binding allergens present in fungal crude extracts. Over 20 different IgE binding proteins were observed using Western blots probed with sera from patients displaying mould allergies. Results using individual sera revealed a wide variation in IgE-binding proteins between sera, although several common bands, including a protein with an apparent molecular mass of 35 kDa were visible among the sera of several patients.The present study demonstrated the allergenic potential of in vitro observations were confirmed by intradermal skin testing on individuals using B. bassiana extracts. While the testing sample population was small, these results indicated that our extracts were able to elicit allergic reactions in individuals, including some that have had occupational exposure to the fungus. Concentrations of ~1 mg/ml of B. bassiana extracts were required to elicit indurations equivalent to 0.1 mg/ml histamine in most individuals, indicating the possibility of potent allergens in the Beauveria extract. Interestingly, not all individuals specifically exposed to B. bassiana displayed allergic reactions and individuals J, K, and M  exists between the two fungal species, implying that primary sensitization by one organism can result in an allergic reaction when exposed to the homologous allergen of another organism. Competitive immunoblot inhibition experiments revealed significant epitope homology between B. bassiana and several clinically important fungi responsible for IgE-mediated allergic reactions in atopic individuals. Thus, an allergic reaction to Beauveria exposure may arise in patients sensitized to other fungi. Extracts from A. alternata and E. purpurascens almost completely competed with allergens present in the B. bassiana extract with the notable exception of the ~35 kDa allergen. Competition experiments using A. fumigatus, C. herbarum, C. albicans, and P. notatum extracts also indicated the presence of many shared epitopes, although distinct (non-competed) IgE-binding B. bassiana proteins of 35 kDa, 64 kDa, and >200 kDa molecular mass were detectable. These proteins, particularly the 35 kDa allergens may represent B. bassiana specific allergens. Experiments are underway to characterize the 35 kDa allergen, which may lead to a diagnostic assay for B. bassiana sensitization. Finally, our analysis of potential B. bassiana allergens was limited to cell extracts grown under specific conditions and did not include the culture filtrate. Extracellular proteases, an important class of fungal proteins that can elicit allergenic reactions, have been characterized from a number of fungal species [B. bassiana. A careful examination of culture growth conditions is also warranted in order to provide a standardized reagent for testing purposes.Numerous studies have revealed the presence of cross-reactive proteins among fungal species between genera -22,25-27 species -31, and Beauveria holds promise as an arthropod biological control agent, there have been few reports on the allergenic potential of these organisms. Identification of B. bassiana specific allergens can lead diagnostic methods for determining sensitization to this organism and may provide a rational basis for allergen attenuation in order to yield safer biocontrol products. The observed cross-reactivity among proteins of B. bassiana and the fungi tested, highlight the importance of considering the possibility that multiple fungal sensitivity can occur due to exposure to a single fungus. Further testing should be performed to determine the scope, severity, and range of allergenic reactions to B. bassiana.Although The author(s) declare that they have no competing interests.in vitro experiments. SWH performed the clinical experiments and participated in the design of the study. NOK conceived of the study, and participated in its design and coordination, and drafted the manuscript.GSW carried out the immunoassays and other