The validity and internal structure of the bipolar depression rating scale (BDRS): data from a clinical trial of N-acetylcysteine as adjunctive therapy in bipolar disorder

Background: The phenomenology of unipolar and bipolar disorders differ in a number of ways, such as the presence of mixed states and atypical features. Conventional depression rating instruments are designed to capture the characteristics of unipolar depression and have limitations in capturing the breadth of bipolar disorder.

Method: The Bipolar Depression Rating Scale (BDRS) was administered together with the Montgomery Asberg Rating Scale (MADRS) and Young Mania Rating Scale (YMRS) in a double-blind randomised placebo-controlled clinical trial of N-acetyl cysteine for bipolar disorder (N = 75).

Results: A factor analysis showed a two-factor solution: depression and mixed symptom clusters. The BDRS has strong internal consistency (Cronbach's alpha = 0.917), the depression cluster showed robust correlation with the MADRS (r = 0.865) and the mixed subscale correlated with the YMRS (r = 0.750).

Conclusion: The BDRS has good internal validity and inter-rater reliability and is sensitive to change in the context of a clinical trial.

Effects of N-acetylcysteine on substance use in bipolar disorder : a randomized placebo controlled clinical trial

Objective: To evaluate the effect of N-acetylcysteine (NAC) on substance use in a double-blind, placebo-controlled trial of NAC in bipolar disorder. It is hypothesised that NAC will be superior to placebo for reducing scores on the Clinical Global Impressions scale for Substance Use (CGI-SU).

Methods: Participants were randomised to 6-months of treatment with 2 g/day NAC (n = 38) or placebo (n = 37). Substance use was assessed at baseline using the Habits instrument. Change in substance use was assessed at regular study visits using the CGI-SU.

Results: Amongst the 75 participants 78.7% drank alcohol (any frequency), 45.3% smoked tobacco and 92% consumer caffeine. Other substances were used by fewer than six participants. Caffeine use was significantly lower for NAC-treated participants compared with placebo at week 2 of treatment but not at other study visits.

Conclusion: NAC appeared to have little effect on substance use in this population. A larger study on a substance using population will be necessary to determine if NAC may be a useful treatment for substance use.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the p-barrel 1 domain that harborst he GTP-bindings ite). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ2, χ3, χ2′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ2 − 60°, χ390, χ2′ − 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N_Ɛ1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant K_m (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant k_cat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower k_cat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate- stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The α-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (α-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia α-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia α-DsbA1 possesses a second disulfide that is highly conserved in α-proteobacterial DsbAs but not in other DsbAs. The α-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of α-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

'Forbidden' disulfides : their role as redox switches

Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins.

Evolution of distinct EGF domains with specific functions

EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional interdomain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues.

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the ‘entrance’ of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2–9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets

Aims/hypothesis We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure.

Materials and methods Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes.

Results Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-l-cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels.

Conclusions/interpretation Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.

Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis

Aims/hypothesis Islet amyloid in type 2 diabetes contributes to loss of beta cell mass and function. Since islets are susceptible to oxidative stress-induced toxicity, we sought to determine whether islet amyloid formation is associated with induction of oxidative stress.

Methods Human islet amyloid polypeptide transgenic and non-transgenic mouse islets were cultured for 48 or 144 h with or without the antioxidant N-acetyl-l-cysteine (NAC) or the amyloid inhibitor Congo Red. Amyloid deposition, reactive oxygen species (ROS) production, beta cell apoptosis, and insulin secretion, content and mRNA were measured.

Results After 48 h, amyloid deposition was associated with increased ROS levels and increased beta cell apoptosis, but no change in insulin secretion, content or mRNA levels. Antioxidant treatment prevented the rise in ROS, but did not prevent amyloid formation or beta cell apoptosis. In contrast, inhibition of amyloid formation prevented the induction of oxidative stress and beta cell apoptosis. After 144 h, amyloid deposition was further increased and was associated with increased ROS levels, increased beta cell apoptosis and decreased insulin content. At this time-point, antioxidant treatment and inhibition of amyloid formation were effective in reducing ROS levels, amyloid formation and beta cell apoptosis. Inhibition of amyloid formation also increased insulin content.

Conclusions/interpretation Islet amyloid formation induces oxidative stress, which in the short term does not mediate beta cell apoptosis, but in the longer term may feed back to further exacerbate amyloid formation and contribute to beta cell apoptosis.

IgM-mediated autoimmune responses directed against anchorage epitopes are greater in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) than in major depression

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and depression are considered to be neuro-immune disorders (Maes and Twisk, BMC Medicine 8:35, 2010). There is also evidence that depression and ME/CFS are accompanied by oxidative and nitrosative stress (O&NS) and by increased autoantibodies to a number of self-epitopes some of which have become immunogenic due to damage by O&NS. The aim of this study is to examine IgM-mediated autoimmune responses to different self-epitopes in ME/CFS versus depression. We examined serum IgM antibodies to three anchorage molecules (palmitic and myristic acid and S-farnesyl-L-cysteine); acetylcholine; and conjugated NO-modified adducts in 26 patients with major depression; 16 patients with ME/CFS, 15 with chronic fatigue; and 17 normal controls. Severity of fatigue and physio-somatic (F&S) symptoms was measured with the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale. Serum IgM antibodies to the three anchorage molecules and NO-phenylalanine were significantly higher in ME/CFS than in depression. The autoimmune responses to oxidatively, but not nitrosatively, modified self-epitopes were significantly higher in ME/CFS than in depression and were associated with F&S symptoms. The autoimmune activity directed against conjugated acetylcholine did not differ significantly between ME/CFS and depression, but was greater in the patients than controls. Partially overlapping pathways, i.e. increased IgM antibodies to a multitude of neo-epitopes, underpin both ME/CFS and depression, while greater autoimmune responses directed against anchorage molecules and oxidatively modified neo-epitopes discriminate patients with ME/CFS from those with depression. These autoimmune responses directed against neoantigenic determinants may play a role in the dysregulation of key cellular functions in both disorders, e.g. intracellular signal transduction, cellular differentiation and apoptosis, but their impact may be more important in ME/CFS than in depression.

Lower whole blood glutathione peroxidase (GPX) activity in depression, but not in myalgic encephalomyelitis / chronic fatigue syndrome: another pathway that may be associated with coronary artery disease and neuroprogression in depression

BAKGROUND: Major depression and myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) are two disorders accompanied by an upregulation of the inflammatory and oxidative and nitrosative (IO&NS) pathways and a decreased antioxidant status. Moreover, depression is accompanied by disorders in inflammatory and neuroprogressive (IN-PRO) pathways.

METHODS: This study examines whole blood glutathione peroxidase (GPX) in depression and in ME/CFS; GPX is an enzyme that reduces hydroperoxides by oxidizing glutathione and consequently protects the cells from oxidative damage. Blood was sampled in 39 patients with depression, 40 patients with ME/CFS and 24 normal volunteers. Whole blood was analysed for GPX activity using the Ransel assay (Randox). Severity of illness was measured by means of the Hamilton Depression Rating Scale (HDRS) and the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale (FF scale).

RESULTS: We found that whole blood GPX activity was significantly (p=0.001) lower in depressed patients than in normal controls and that there were no significant differences between ME/CFS and controls. In depression and ME/CFS, there were significant and inverse relationships between GPX activity and the FF items, depressed mood and autonomic symptoms. In depression, there were significant and negative correlations between whole blood GPX and the HDRS score and autonomic symptoms.

DISCUSSION: The results show that lowered whole blood GPX activity contributes to the lowered antioxidant status in depression. Since GPX activity is a predictor of neuroprogression and coronary artery disease (CAD), lowered GPX activity in depression contributes to the IN-PRO pathways and the comorbidity between depression and CAD. Our results suggest that patients with depression would benefit from Ebselen or a supplementation with glutathione, N-Acetyl-l-Cysteine and selenium.