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 (note: patients with PD are not 5 + 7 + 9 + 3 = 24, but only 17 as mentioned above, because there is ovelapping between depressive syndromes). 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 (B, C and D). Groups B and C may represent two distinct types of vulnerability to stress (hypercortisolemia, DST non suppression and PD), 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 (glutaredoxins 1, 2, and 3 and glutathione reductase) 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 (expected mass, 84,542, −0.01% deviation), 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 (expected mass, 306 Da). 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 (643, 645, and 726) 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 [For  –280 mV ), more tHowever, these calculations assume that the oxidation state of MetE is in equilibrium with GSH and GSSG. Deviations from theoretical values could occur if MetE were oxidized or reduced in vivo by a mechanism other than simple thiol-disulfide exchange. Comparison of the actual amount of oxidized MetE to measurements of the physiological concentrations of GSH and GSSG could therefore provide an indication of the in vivo mechanism of oxidation. Nevertheless, while MetE inactivation by GSSG may be energetically favorable under oxidizing conditions, the apparently slow kinetics make it difficult to conclude whether or not oxidation actually occurs in vivo.Thus, to determine whether our observations are physiologically relevant, we needed to develop a method whereby the oxidation of MetE could be directly observed in vivo. To this end, thiol-trapping experiments were performed as outlined in trxA gor strains of E. coli. Strains mutant in genes for one of the thioredoxins (trxA) and glutathione reductase (gor) are impaired in their ability to reduce disulfide bonds, resulting in a highly oxidizing cytoplasmic environment (trxA gor mutant strain (WP843) with that in the isogenic wild-type strain (DHB4) revealed that approximately 50%–60% of the MetE in the trxA gor strain was oxidized in vivo  and gor experience more severe disulfide stress than trxA gor strains (trxB gor strain containing a plasmid specifying MetE (which expresses MetE independently of the cellular methionine concentration) was grown in Luria-Bertani (LB) medium containing DTT. Oxidized MetE was observed within 90 min of DTT removal, while MetE was fully reduced in the presence of DTT  within 2 min of diamide treatment.In order to establish a direct link between oxidative inactivation of MetE and the methionine auxotrophy observed during transient oxidative stresses, we performed thiol-trapping experiments on cells challenged with diamide. When wild-type n growth C and 11DE. coli, only the activities of OxyR and PAPS reductase have been reported to be modulated by formation of mixed disulfides with glutathione  growing in LB medium were previously reported to be 223 ± 35 and 18 ± 7, respectively  should be reduced. Similarly, based on the equilibrium constant for thiol-disulfide exchange with MetE, only approximately 7% of the MetE is expected to be oxidized in the trxA gor strain (WP843). Yet 50%–60% of the MetE was found to be oxidized in this strain by thiol-trapping experiments. A [GSH]/[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 (2O2 to purified MetE appeared to cause irreversible oxidation, presumably because of the formation of sulfinic (–SO2) 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 (Schircks Laboratories, Jona, Switzerland), as described previously (L-Homocysteine was prepared by hydrolysis of L-homocysteine thiolactone (Electrophoresis-grade urea, Chaps, ampholytes (pH 4–8), ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions (Ann Arbor, Michigan, United States). Iodoacetate and iodoacetamide were purchased from Fluka (Sigma-Aldrich, St. Louis, Missouri, United States). (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) (E. coli strain GW2531. Mutations were confirmed by DNA sequencing of the entire metE gene.Wild-type K-12 strain W3110 was obtained from F. C. Neidhardt (University of Michigan, Ann Arbor, Michigan, United States). 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 (L-homocysteine, 66 μM (6S)-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 (Millipore, Billerica, Massachusetts, United States), 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 (Lexington, Kentucky, United States) 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 (Easton, Maryland, United States) 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 (2 mM GSSG, no GSH), and A350(red) is for completely reduced MetE (2 mM GSH, 4 μM [trace] GSSG). 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 (0.1 M Tris chloride [pH 8.7] containing 0.2 M potassium chloride, 1 mM EDTA, 8 M urea, and 10 mM DTT), and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate (104 mg dissolved in 0.5 ml of 1 M potassium hydroxide, followed by addition of 0.5 ml of 1 M Tris chloride [pH 7.3]) 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 (14,000g) 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 (upper, 0.1 M sodium hydroxide) and anode (lower, 10 mM phosphoric acid) 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 (Amersham Pharmacia, Piscataway, New Jersey, United States) according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE (1:3,000) and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody (ECF western blotting kit, Amersham Pharmacia) 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 (Osaka, Japan) and HindIII from New England BioLabs (Beverly, USA).Glucose, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), and magnesium chloride were obtained from Kanto Kagaku (Tokyo, Japan), 3-(N-morpholino) propane sulfonate (MOPS) from Dojin (Kumamoto, Japan), and pyrroloquinoline quinone from Mitsubishi Gas Chemical Company (Tokyo, Japan). 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., (Lenoir, NC). Extracts were resuspended in TE (40 mM Tris-HCl, pH 8.0, 1 mM EDTA) 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 (10,000 × g, 10 min) 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 (30 min, 4000 × g), 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 (15 min, 10,000 × g) 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 (30 min, 100,000 × g). 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 (SDS-PAGE, 10% Bis-tris gel, Invitrogen, Carlsbad, CA) using standard protocols. Gels were stained with Gelcode blue stain reagent (Pierce, Rockford, IL) 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 (Biorad, Hercules, CA).B. bassiana crude extracts was treated with Proteinase K (ICN-Biomed, Aurora, Oh) following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution (10 mg/ml in 50 mM Tris-HCl, pH 7.5) for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H (EndoH, New England Biolabs, Beverly, MA) and peptide: N-Glycosidase F (PNGaseF, New England Biolabs) according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) at 100°C for 10 min prior to the addition of the EndoH (5 μl of 10 × G5 Reaction Buffer, 50 mM sodium citrate, pH 5.5) 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 (mixed, P. chrysogenum and P. notatum). 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 (Round Rock, TX); 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


Early identification of permanent hearing impairment in children enables appropriate intervention which reduces adverse developmental outcomes. The UK Government has introduced a universal hearing screening programme for neonates. All involved health professionals, including those in Primary Care, need to be aware of the service to enable them to offer appropriate support to their patients. A programme of information dissemination within Primary Care was therefore undertaken. The aim of the current study was to determine the extent to which the information had reached General Practitioners (GPs), the GPs' preferred mode of dissemination and the sources from which GPs accessed informationPostal questionnaire survey of a randomised sample of 1000 GPs in the Phase I pilot sites of the Neonatal Hearing Screening Programme (NHSP).Responses were received from 54.2% of the sample. Just under 50% of those responding had received information, 62.2% of respondents said they would like to receive more information and the preferred methods of dissemination were the written word and web-sites to allow access when needed. Few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject. Many are keen to delegate detail to a third party, usually the health visitor, who has traditionally had responsibility for hearing screening.Dissemination efforts for service developments of relevance to GPs should concentrate on advertising a website address via brief but memorable posted literature and/or articles in relevant journals and magazines. The website should be GP-friendly, and have a dedicated area for GPs including information of specific relevance and downloadable information sheets. Approximately 1.65 per 1000 babies born in the UK have a significant permanent hearing impairment >40 dB HL) detectable at birth . Thus, b0 dB HL dThis study uses the introduction of the Newborn Hearing Screening Programme (NHSP) to illustrate the issues around dissemination to GPs ,7. IntroThe phased implementation of NHSP in the UK began in 2001 in 23 pilot sites and was accompanied by a campaign of information dissemination including distribution of leaflets and posters to all general practices, the development of a dedicated website and appointment of local co-ordinators. The aim of the current study was to determine the extent to which the dissemination information had reached the GPs, the GPs' preferred mode of dissemination and the sources from which GPs accessed information.We designed and piloted a one-page questionnaire see  and usedOf the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website.When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences.To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%).In open comments (N = 84 respondents, 16.6%) it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening.The response rate of more than 50% is acceptable for a survey of GPs. A second mailing might have improved this but its advantage was weighed against the attraction of anonymity. To minimise the number of questions and encourage response, no demographic details were requested. Analysis of any response bias by age of practitioner, size of practice etc was therefore not possible. A response might be more likely from people who knew something of the subject and our estimates for the level of awareness might therefore be high but it is likely that the views expressed concerning resources accessed and preferred channels of dissemination are representative.Although considerable effort had been expended in distributing letters, leaflets and posters to every general practice, less than half the respondents said they had seen it. It is possible that mail had been screened and intercepted by administrative staff in the practice or rejected as "junk mail" by the GP. It is important therefore to ensure such mailed information is identified as relevant. Some health service developments will not be high on the average GP's agenda. In terms of neonatal hearing screening s/he will see only 20 newborns per year on average and approximately one permanently hearing-impaired child in a working lifetime.Nearly two-thirds of respondents said they would like to receive more information even though many complained about "information overload". This is a weakness inherent in surveys of this nature where stated attitudes may reflect socially desirable responses rather than respondents' true attitudes. Further exploration of this issue using qualitative techniques of personal interview would contribute to the general issue of information dissemination to GPs.GPs cannot know everything about everything and do not necessarily need to but they do need to know where they can find the relevant information and to be able to access it easily . Our surWe suggest that any dissemination effort for service developments of relevance to GPs should concentrate on advertising the website address via brief but memorable posted literature and/or articles in relevant journals and magazines. It is also crucial that any website be GP-friendly by having a dedicated area for GPs including information of specific relevance and downloadable information sheets.The author(s) declare that they have no competing interests.PM designed and carried out the survey, performed the analyses and wrote the original report. HF supervised the design and implementation of the study and drafted the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:A survey of awareness of the NHS Newborn Hearing Screening Programme (NHSP) amongst General Practitioners (GPs) QuestionnaireClick here for file


Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.We have elucidated the kinetics of histone methylation during X inactivation using an inducible  In female mammals, one X chromosome is inactivated. Anton Wutz and colleagues find evidence for a very early event that 'marks' the chromosome for future stable inactivation Xist RNA (Xist RNA attaches to chromatin and spreads from its site of transcription in cis over the entire inactive X chromosome (Xi), mediating transcriptional repression. Xist is essential for initiation of silencing, but not for the maintenance of transcriptional repression on the Xi at later stages of cellular differentiation  and lysine 27 (H3-K27) methyltransferase activity , revealing a novel chromosomal memory that is established by Xist expression at an early time point in ES cell differentiation independent of transcriptional repression. Our analysis suggests that this chromosomal memory might have an important role in the transition from the initiation phase to the maintenance phase of X inactivation.We have previously shown that chromosomal silencing can be recapitulated in embryonic stem (ES) cells by expressing romosome , and thiXist RNA . Initiatpression . TherefoXist RNA in the initiation of chromosomal silencing and for analysing the kinetics and relevance of chromosomal modifications. We aimed to delineate a pattern of histone methylation states that define the initial decision for facultative heterochromatin. To achieve this we performed immunofluorescence staining against the various modification states on histone H3 and H4 lysine residues in clone 36 ES cells, in which Xist expression can be induced from a transgene integrated on Chromosome 11 by addition of doxycycline to the culture medium  experiments using antibodies specific for H3K27m3, H4K20m1, H3K4m3, H3K4m2, and H3K9m2 in both undifferentiated and differentiated clone 36 ES cells in the presence or absence of doxycycline . We obseosome 11 . A controsome 11 F and 2L.d sample B. This induction E and 2K.Xist RNA expression in undifferentiated clone 36 ES cells  showing H3K27m3 staining compared to the wild-type Xist RNA , which is cointegrated with the Xist cDNA transgene on Chromosome 11 and can be silenced by transgenic Xist expression. Xist expression was induced either from the beginning or at 24, 48, 72, 96, or 120 h after the onset of differentiation. The ability of Xist to initiate silencing at various time points was monitored by measuring puro expression, and H3K27m3 was analysed in parallel in all cultures at 12 d after differentiation  and a homozygous insertion of the tetracycline-responsive transactivator in the ROSA26 locus . In uninduced cultures and control male fibroblasts no H3K27m3 foci were detected by immunofluorescence in interphase nuclei. However, upon Xist induction 5% (after 48 h of Xist induction) or 15% (after 72 h) of the cells showed focal H3K27m3 staining  and 52% (day 16.5 embryos) of the cells expressed Xist, as determined by RNA fluorescent in situ hybridization (FISH) analysis . We then analysed the kinetics of loss of methylation in differentiated ES cells. Clone 36 ES cells differentiated for 4 d in the presence of doxycycline were differentiated for 24, 48, and 72 more hours in the absence of doxycycline, and H3K27m3 was measured . In cells that had been continuously differentiated in the presence of doxycycline, methylation was detected in 73%–78% of the nuclei. Our data show that efficient methylation at late time points in differentiation did not require continuous Xist expression. Efficient remethylation occurred on a chromosome that had been exposed to Xist in early ES cell differentiation, consistent with the idea that Xist triggers a chromosomal change in early differentiation that is remembered until later time points to enhance H3K27m3 reestablishment. Importantly, the silencing-deficient Xist mutant RNA in J1:XistΔSX-tetOP ES cells gave identical results, showing that this memory is established independent of silencing.To test whether continuous ectively . ParalleXist was turned off until day 8 of differentiation, when doxycycline was added back, and remethylation was assayed by immunofluorescence at day 13 in differentiation . We attribute the different observations in other studies to the various antisera used. We supply peptide blot analysis for our antisera that suggest that the antibodies are highly specific . We suggest that Xist expression and H3K27m3 might be the signal to recruit a chromosomal memory mediating the lock-in of X inactivation . For sections, embryos were sexed (Clone 36 ES cells  and J1:Xeletions  were culeviously . Primaryeviously . Xist exre sexed  and fixe2), centrifuged for 10 min at 1,200 rpm onto Menzel SuperFrost slides (Roth, Karlsruhe, Germany) using a Cytospin 3 centrifuge (Thermo Shandon, Pittsburgh, Pennsylvania, United States). Staining was performed as described previously , blocked in blocking solution (PBS, 3% [wt/vol] BSA), and incubated with primary antibodies for 3 h. After washing three times for 10 min in TBST (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.1% Tween 20) and incubation with secondary antibodies , detection was performed using ECL reagent . Rabbit polyclonal α-Eed (1:3,500), rabbit polyclonal α-Ezh2 (1:1,000), goat polyclonal α-histone H3 , and rabbit polyclonal α-histone H4  were used.For DNA FISH analysis, biotin-labelled STAR*FISH mouse whole chromosome-specific probes  were detected with streptavidin, Alexa Fluor 633 conjugateS-21375 (Molecular Probes). RNA FISH probes were generated by random priming  using Cy3-dCTP (Amersham). Hybridisation and washing were carried out as described previously . SpecimeCells were cross-linked with 1% formaldehyde for 10 min at RT and quenched with 125 mM glycine, and whole-cell extracts were prepared. ChIPs were performed in duplicates as described previously . BrieflyFigure S1Immunodotblot analysis  of the a(611 KB PDF).Click here for additional data file.Figure S2Xist for 3 d using H3K27m1 (A), H3K27m2 (B), H3K27m3 (C), H3K9m1 (D), H3K9m2 (E), H3K9m3 (F), H4K20m1 (G), H4K20m2 (H), H4K20m3 (I), H3K4m3 (J), and H4Ac (K) antisera. Chromosome 11 was identified by a DNA FISH probe  in (J) and (K). Clone 36 ES cells grown in the absence of doxycycline are used as a control for the H4Ac staining without Xist expression (L).Immunofluorescence staining of metaphase spreads of clone 36 ES cells induced to express (4.8 MB TIF).Click here for additional data file.Figure S3Xist expression is shown.(A) The kinetics of H3K27m3 was measured in undifferentiated clone 36 ES cells. The number of cells showing H3K27m3 staining 6, 12, 24, and 48 h after induction of n > 150) showing H3K27m3 staining was analysed in undifferentiated clone 36 ES cells, which expressed Xist for 3 d (lane 1) or were further grown without inducer for 24 h (lane 2) or 48 h (lane 3). This experiment complements data presented in (B) The stability of H3K27m3 was determined in undifferentiated ES cells. The percentage of metaphase chromosome spreads n > 10 showingXist expression. No progressive accumulation over time was observed, indicating that the steady state of H3K27m3 has been reached at 3 d Xist expression. However, a marked increase in methylation is observed in J1:XistΔSX-tetOP ES cells upon differentiation for 2 d (hatched bar).(C) Levels of H3K27m3 were measured in undifferentiated ES cells after 3 and 10 d of Xist for 3 and 10 d (percentage of nuclei showing a staining is given). Analysis of H3K27m3 and H4 acetylation using an antiserum specific for multiply acetylated forms of H4 in clone 36 and J1:XistΔSX-tetOP ES cells that were grown for 4 d in the presence of doxycycline and then shifted to differentiation conditions for 2 d more in the presence of doxycylcine.(D) Combined Xist RNA FISH (red) immunofluorescence analysis of Ezh2 and H4K20m1 in undifferentiated J1:XistΔSX-tetOP cells expressing (E) Male primary mouse fibroblasts (PMEFs) hemizygous for the inducible Xist-tetOP allele and homozygous for the tetracycline-inducible transactivator were induced with doxycycline for 2 d (lane 1) or 3 d (lane 2), and the number of cells showing H3K27m3 staining in interphase was analysed. Control female PMEFs showed a methylation signal in the large majority of cells (lane 3); uninduced male PMEFs were always negative.Xist RNA triggers less pronounced and less dense foci of H3-K27 trimethylation (green) compared to the female wild-type control.(F) Representative indirect immunofluorescence of uninduced (top) and induced (bottom) male Xist-tetOP PMEFs. The inducible Xist expression, H4-K20 monomethylation (green) is observed in interphase Xist-tetOP PMEFs (left). Focal enrichment colocalises with the site or Xist RNA clusters (red) on the X chromosome. Female wild-type PMEFs (right).(G) Upon (3.0 MF TIF).Click here for additional data file.Figure S4Xist cDNA in given (top): repeats A to E are indicated by arrays of triangles, sequences mediating localisation to chromatin are indicated by boxes underneath (degree of hatching represents importance), and the location of the deletion is indicated by a coloured box. RNA localisation was analysed by FISH (lower left), showing that the RNA localises in small clusters in some cells. The ability of the RNA to induce silencing was measured by cell survival of differentiating cultures under induced versus uninduced conditions (lower right). Controls are cells either having a fully functional Xist cDNA transgene (Xist) or a cDNA lacking repeat A that is incompetent to induce silencing (ΔSX). The ΔXSa RNA shows poor silencing activity, presumably as a consequence of its failure to localise well to the chromosome.The XistΔXSa transgene was integrated by Cre-mediated recombination into the Hprt locus on the single X chromosome in T20 ES cells . A schem(1.5 MB TIF).Click here for additional data file.Figure S5PR/SET domain-containing protein 07 (Pr-Set7), indicating SET domain in black (gi:38080595). Below, region tested for histone methyltransferase (HMTase) activity.(A) Schematic presentation of full-length mouse Pr-Set7 (arrow), H4 peptides (arrowhead), and histones used for in vitro reactions with S-adenosyl-[methyl-14C]-L-methionine as methyl donor. Fluorography (right) indicates HMTase activity on the unmodified H4 peptide comprising residues 12–31 of the histone H4 N-terminus. Notably, no further methyl groups could be transferred to the same peptide if it had been synthetically monomethylated at residue H4 lysine 20 (K20m1) before usage in the in vitro reaction. Free histones are not accepted as substrate.(B) Coomassie stain (left) shows purified recombinant GST-tagged GST–Pr-Set7 selective for the unmodified histone H4 peptide (12–31). H4-K20 monomethylation obviously is the terminal state for Pr-Set7, because synthetically mono- (K20m1), di- (K20m2), and trimethylated H4 peptides (K20m3) could not be significantly methylated.(C) Fluorography indicates histone H4 HMTase activity of (948 KB TIF).Click here for additional data file.Protocol S1(22 KB DOC).Click here for additional data file.


To evaluate the association between mood and anxiety disorders and thyroid autoimmunity in a community sample. Methods: A community based sample of 222 subjects was examined. Psychiatric diagnoses were formulated using the International Composite Diagnostic Interview Simplified (CIDIS), according to DSM-IV criteria. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO).16.6% of the overall sample had an anti-TPO value above the normal cut-off. Subjects with at least one diagnosis of anxiety disorders (OR = 4.2, C.L. 95% 1.9–38.8) or mood disorders (OR = 2.9, Cl 95% 1.4–6.6, P < 0.011) were positive for serum anti-TPO more frequently than subjects without mood or anxiety disorders. A statistically significant association with anti-TPO+ was found in Anxiety Disorder Not Otherwise Specified (OR = 4.0, CL 95% 1.1–15.5), in Major Depressive Episode (OR = 2.7, CL 95% 1.1–6.7) and Depressive Disorder Not Otherwise Specified (OR = 4.4, S CL 95% 1–19.3).The study seems to suggest that individuals in the community with thyroid autoimmunity may be at high risk for mood and anxiety disorders. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. Should our results be confirmed, the findings may be of great interest for future preventive and case finding projects. Autoimmune thyroid disease may be linked to depression  and anxiThe purpose of this investigation was to evaluate the relationship between mood and anxiety disorders and thyroid autoimmunity in a community survey. This research was carried out on the data base of two epidemiological studies aimed at defining the prevalence of psychiatric  and thyrThis paper present the results of the cross psychiatric and endocrinological evaluation from the common areas of the two surveys.The sample was extracted by randomization (1/10) subsequent to stratification according to age and sex, from the records of 2 Sardinian villages. Probands were interviewed face to face in their homes by specifically trained physicians. Two standardized forms were used to acquire information concerning: demographic data, state of health and use of social and health services. Psychiatric diagnosis was made using the Italian Simplified version of the Composite International Diagnostic Interview (CIDIS) . The comAnti-thyroid peroxidase autoantibodies (anti-TPO), considered as the most sensitive and specific marker of thyroid autoimmunity  was deteAll subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). FT4 and FT3 were measured by means of a chromatographic method based on separation of free T4 on Lisophase columns . TSH was measured by a chemiluminescent method  with normal values ranging from 0.3–3.0 μU/ml. Thyroid echography was performed using a "real time" echograph .2 test in 2 × 2 tables. Odds Ratio confidence intervals were calculated through application of the method of Miettinen [The association of anti- TPO+ with the main diagnoses deriving from CIDIS interview was calculated using Odds Ratio. Statistical significance was calculated using the Xiettinen .Multivariate Logistic Regression was performed in order to evaluate the possible influences of gender and age on the association between anti-TPO+ and mood or anxiety disorders. The analysis was carried out considering mood (or anxiety) disorders as dependent variable, and anti-TPO+ (presence vs absence), gender  and age (≤ 44 vs > 44) and their second order interactions as independent variables, by means of backward stepwise procedure; interactions lacking evidence of association (p > 0.20) were eliminated from the models.From a total of 261 subjects identified (age >18 years), 222 (85.1%), 127 females (57.2%), and 95 males (42.7%); over 44 years 127 (57.2%), 79 females (62.2%,) 48 males (37.7%), agreed to take part in the study whilst 20 (8,7%) refused to participate and 19 (7.3%) could not be traced. The final sample did not differ respect to the population of origin with reference to the variables applied in stratification.The lifetime prevalence of anxiety disorders in the sample was: Generalized Anxiety Disorder (GAD) 11.3%, Panic Disorder (PD) 2.7%, Anxiety Disorder Not Otherwise Specified (ADNOS) 5.4%, Social Phobia (SP) 5.4%; 18.5% had been diagnosed with at least one of these anxiety disorders. With regard to mood disorders, Major Depressive Episode (MDE) was present in 14.4%, Dysthymic Disorder (DD) in 2.7%, Depressive Disorder Not Otherwise Specified (DDNOS) in 4.0%; 18.9% had at least one of the above mentioned depressive disorders. 1.1% of the overall sample was affected by hypothyroidism, 16.6% had an anti-TPO value above the normal cut-off (anti-TPO+).Table The Multivariate Logistic Regression showed that gender and age do not interact with anti-TPO either in mood , or in anxiety disorders . Interactions were therefore eliminated from the models.The final Logistic Regression models clearly indicated that gender and age do not influence the risk of one mood or anxiety diagnosis either as independent variables or as confounders Table .The present study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+ in a general population sample which had not been selected from medical or psychiatric health facilities. This association is independent by gender and age. Regarding specific diagnosis, MDE, DDNOS and ADNOS were associated with anti-TPO+.This finding is consistent with several previous clinical studies providing evidence for a significant association of mood disorders or post-partum depression and symptomless autoimmune thyroiditis with or without sub-clinical hypothyroidism . AssociaA sub-clinical dysfunction of axis Thyrotropin Releasing Hormone (TRH) – Thyroid Stimulating Hormone (TSH) with consequent alteration of circadian rhythms of TSH has been hypothesized in some depressive disorders. Indeed, this hypothesis may explain why some forms of mood disorders were associated with anti-TPO+ or thyroid autoimmunity without hypothyroidism, as defined by routine blood tests. A slight reduction in thyroid hormone secretion such as that found in sub-clinical hypothyroidism may affect cognition and mood . At variMoreover, a study carried out in a large community sample found no association between thyroid dysfunction, including hypothyroidism defined by thyroid blood test, and the presence of depression or anxiety symptoms . This suWith regard to the public heath aspects of this research, should these findings be confirmed, they would constitute a most important public health issue, due to the high attributable risk found. The attributable risk is a useful measure to document the burden of risk to a community. Attributable risk depends both on the magnitude of relative risk and on the prevalence of the risk factor in the population . The higThe potential of the study is reduced by the small sample size, particularly regard to psychiatric diagnoses less frequently observed in the general population, such as Panic Disorder; the extension of the findings is therefore rather limited.This study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. If the findings are confirmed, they may prove to be of interest for future projects of case finding: a systematic screening for mood disorders in anti-TPO+ subjects and a systematic evaluation for thyroid diseases and thyroid autoimmunity in subjects with mood disorders may be advisable.None declared.MGC conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. AL, SM and CS participated in the statistical analysis and drafted the manuscript. MCH, SM, MC, BC, LDO participated in its design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here: