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Background: Goal-directed control guides optimal decision-making and it is an important cognitive faculty that protects against developing habits. Previous studies have found some evidence of goal-directed deficits when healthy individuals are stressed, and in psychiatric conditions characterised by compulsive behaviours and anxiety. Here, we tested if goal-directed control is affected by state anxiety, which might explain the former results. Methods: We carried out a causal test of this hypothesis in two experiments (between-subject N = 88; within-subject N = 50) that used the inhalation of hypercapnic gas (7.5% CO2) to induce an acute state of anxiety in healthy volunteers. In a third experiment (N = 1413), we used a correlational design to test if real-life anxiety-provoking events (panic attacks, stressful events) are associated with impaired goal-directed control. Results: In the former two causal experiments, we induced a profoundly anxious state, both physiologically and psychologically, but this did not affect goal-directed performance. In the third, correlational, study, we found no evidence for an association between goal-directed control, panic attacks or stressful life eventsover and above variance accounted for by trait differences in compulsivity. Conclusions: In sum, three complementary experiments found no evidence that anxiety impairs goal-directed control in human subjects. [ABSTRACT FROM AUTHOR]

In recent decades, batch hot-dip galvanized (HDG) steel has proven itself in practical applications due to the good corrosion resistance of its components. Despite the importance of the mechanical-load-bearing capacity of these coatings, the wear behavior has, so far, only been investigated very sporadically and not systematically, so a quantification of the wear behavior and statements on the mechanisms are vague. Therefore, two body wear tests with bonded abrasive grain were carried out. Varying the friction rolls, load, and total number of cycles, the wear behavior was investigated. The mass loss and the layer thickness reduction were measured at different intervals. After the test, the microstructure in the cross-section and the hardness according to Vickers (0.01 HV) were evaluated. The results showed that the wear behavior of HDG coatings against abrasive loads can be characterized with the selected test conditions. Initially, the applied load removed the soft η-phase. As the total number of cycles increases, the η- and ζ-phases deform plastically, resulting in a lower mass reduction compared to that expected from the measured layer thickness. The characteristic structure of a batch HDG coating with hard intermetallic Zn-Fe phases and an outer pure zinc phase has demonstrated effective resistance to abrasion.

Aim: The aim of this qualitative study is to explore patients' perspectives on Acceptance and Commitment Therapy for early stages of psychosis. Therefore, we interviewed participants of the INTERACT study, that quantitatively investigated Acceptance and Commitment Therapy in Daily Life (ACT-DL) in combination with treatment as usual, for early stages of psychosis, comparing it to treatment as usual., Methods: Within 6 months after finishing ACT-DL, we conducted semi-structured, individual interviews with 19 participants. All interviews were audio-recorded and transcribed. Thematic analysis was used for coding and analysis., Results: Two overarching themes were formed: 'the meaning of ACT' and 'what to improve'. Considering the first, participants generally understood and connected with the meaning of ACT, noticing more awareness and acceptance of their thoughts and feelings, and living more in line with their personal values. The second theme included comments on the protocol not being personal or psychosis specific enough and some elements of ACT being too difficult to understand when having active psychotic symptoms., Conclusions: This study suggests that ACT is an acceptable and promising new form of treatment for early stages of psychosis, and it provides relevant information to further develop ACT for this group., (© 2023 The Authors. Early Intervention in Psychiatry published by John Wiley & Sons Australia, Ltd.)

Background/introduction: Penile cancer is a rare disease in demand for new therapeutic options. Frequently used combination chemotherapy with 5 fluorouracil (5-FU) and cisplatin (CDDP) in patients with metastatic penile cancer mostly results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance against standard therapy could help to understand molecular mechanisms underlying chemotherapy resistance and to identify candidate treatments for an efficient second line therapy., Methods: We generated a cell line from a humanpapilloma virus (HPV) negative penile squamous cell carcinoma (UKF-PEC-1). This cell line was subject to chronic exposure to chemotherapy with CDDP and / or 5-FU to induce acquired resistance in the newly established chemo-resistant sublines (PEC-1 r CDDP 2500 , adapted to 2500 ng/ml CDDP; UKF-PEC-1 r 5-FU 500 , adapted to 500 ng/ml 5- FU; UKF-PEC1 r CDDP 2500 / r 5-FU 500 , adapted to 2500 ng/ml CDDP and 500 ng/ml 5 -FU). Afterwards cell line pellets were formalin-fixed, paraffin embedded and subject to sequencing as well as testing for homologous recombination deficiency (HRD). Additionally, exemplary immunohistochemical stainings for p53 and gammaH2AX were applied for verification purposes. Finally, UKF-PEC-1 r CDDP 2500 , UKF-PEC-1 r 5-FU 500 , UKF-PEC1 r CDDP 2500 / r 5-FU 500 , and UKF-PEC-3 (an alternative penis cancer cell line) were tested for sensitivity to paclitaxel, docetaxel, olaparib, and rucaparib., Results and Conclusions: The chemo-resistant sublines differed in their mutational landscapes. UKF-PEC-1 r CDDP 2500 was characterized by an increased HRD score, which is supposed to be associated with increased PARP inhibitor and immune checkpoint inhibitor sensitivity in cancer. However, UKF-PEC-1 r CDDP 2500 did not display sensitivity to PARP inhibitors., (© 2022. The Author(s).)

A majority of the workforce in the humanitarian aid consists of volunteers who partly suffer from health problems related to their voluntary service. To date, only a fraction of the current research focuses on this population. The aim of this qualitative explorative study was to identify burdening and protective organisational factors for health and well-being among humanitarian aid volunteers in a Greek refugee camp. To this end, interviews with 22 volunteers were held on site and afterwards analysed by using qualitative content analysis. We focused on international volunteers working in Greece that worked in the provision of food, material goods, emotional support and recreational opportunities. We identified burdening factors, as well as protective factors, in the areas of work procedures, team interactions, organisational support and living arrangements. Gender-specific disadvantages contribute to burdening factors, while joyful experiences are only addressed as protective factors. Additionally, gender-specific aspects in the experience of team interactions and support systems were identified. According to our findings, several possibilities for organisations to protect health and well-being of their volunteers exist. Organisations could adapt organisational structures to the needs of their volunteers and consider gender-specific factors.

Background: Stroke patients are at risk of acquiring secondary infections due to stroke-induced immune suppression (SIIS). Immunosuppressive cells comprise myeloid-derived suppressor cells (MDSCs) and immunosuppressive interleukin 10 (IL-10)-producing monocytes. MDSCs represent a small but heterogeneous population of monocytic, polymorphonuclear (or granulocytic), and early progenitor cells ("early" MDSC), which can expand extensively in pathophysiological conditions. MDSCs have been shown to exert strong immune-suppressive effects. The role of IL-10-producing immunosuppressive monocytes after stroke has not been investigated, but monocytes are impaired in oxidative burst and downregulate human leukocyte antigen-DR isotype (HLA-DR) on the cell surface. Objectives: The objective of this work was to investigate the regulation and function of MDSCs as well as the immunosuppressive IL-10-producing monocytes in experimental and human stroke. Methods: This longitudinal, monocentric, non-interventional prospective explorative study used multicolor flow cytometry to identify MDSC subpopulations and IL-10 expression in monocytes in the peripheral blood of 19 healthy controls and 27 patients on days 1, 3, and 5 post-stroke. Quantification of intracellular STAT3p and Arginase-1 by geometric mean fluorescence intensity was used to assess the functionality of MDSCs. In experimental stroke induced by electrocoagulation in middle-aged mice, monocytic (CD11b + Ly6G - Ly6C high ) and polymorphonuclear (CD11b + Ly6G + Ly6C low ) MDSCs in the spleen were analyzed by flow cytometry. Results: Compared to the controls, stroke patients showed a relative increase in monocytic MDSCs (percentage of CD11b + cells) in whole blood without evidence for an altered function. The other MDSC subgroups did not differ from the control. Also, in experimental stroke, monocytic, and in addition, polymorphonuclear MDSCs were increased. The numbers of IL-10-positive monocytes did not differ between the patients and controls. However, we provide a new insight into monocytic function post-stroke since we can report that a differential regulation of HLA-DR and PD-L1 was found depending on the IL-10 production of monocytes. IL-10-positive monocytes are more activated post-stroke, as indicated by their increased HLA-DR expression. Conclusions: MDSC and IL-10 + monocytes can induce immunosuppression within days after stroke., (Copyright © 2020 Achmus, Ruhnau, Grothe, von Sarnowski, Bröker, Dressel, Schulze and Vogelgesang.)

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention., Competing Interests: The authors have declared that no competing interests exist.

Background: Granulocytes and monocytes are the first cells to invade the brain post stroke and are also being discussed as important cells in early neuroinflammation after seizures. We aimed at understanding disease specific and common pathways of brain-immune-endocrine-interactions and compared immune alterations induced by stroke and seizures. Therefore, we compared granulocytic and monocytic subtypes between diseases and investigated inflammatory mediators. We additionally investigated if seizure type determines immunologic alterations. Material and Methods: We included 31 patients with acute seizures, 17 with acute stroke and two control cohorts. Immune cells were characterized by flow cytometry from blood samples obtained on admission to the hospital and the following morning. (i) Monocytes subpopulations were defined as classical (CD14 ++ CD16 - ), (ii) intermediate (CD14 ++ CD16 + ), and (iii) non-classical monocytes (CD14 dim CD16 + ), while granulocyte subsets were characterized as (i) "classical granulocytes" (CD16 ++ CD62L + ), (ii) pro-inflammatory (CD16 dim CD62L + ), and (iii) anti-inflammatory granulocytes (CD16 ++ CD62L - ). Stroke patient's blood was additionally drawn on days 3 and 5. Cerebrospinal fluid mitochondrial DNA was quantified by real-time PCR. Plasma High-Mobility-Group-Protein-B1, metanephrine, and normetanephrine were measured by ELISA. Results: HLA-DR expression on monocytes and their subpopulations (classical, intermediate, and non-classical monocytes) was reduced after stroke or seizures. Expression of CD32 was increased on monocytes and subtypes in epilepsy patients, partly similar to stroke. CD32 and CD11b regulation on granulocytes and subpopulations (classical, anti-inflammatory, pro-inflammatory granulocytes) was more pronounced after stroke compared to seizures. On admission, normetanephrine was upregulated in seizures, arguing for the sympathetic nervous system as inducer of immune alterations similar to stroke. Compared to partial seizures, immunologic changes were more pronounced in generalized tonic-clonic seizures. Conclusion: Seizures lead to immune alterations within the immediate postictal period similar but not identical to stroke. The type of seizures determines the extent of immune alterations., (Copyright © 2020 Ruhnau, Tennigkeit, Ceesay, Koppe, Muszelewski, Grothe, Flöel, Süße, Dressel, Podewils and Vogelgesang.)

Acute anxiety impacts cognitive performance. Inhalation of air enriched with carbon dioxide (CO 2 ) in healthy humans provides a novel experimental model of generalised anxiety, but has not previously been used to assess cognition. We used inhalation of 7.5% CO 2 to induce acute anxiety and autonomic arousal in healthy volunteers during neuropsychological tasks of cognitive flexibility, emotional processing and spatial working memory in a single-blind, placebo-controlled, randomized, crossover, within-subjects study. In Experiment 1 (n = 44), participants made significantly more extra-dimensional shift errors on the Cambridge Neuropsychological Test Automated Battery (CANTAB) Intra-Extra Dimensional Set Shift task under CO 2 inhalation compared with 'normal' air. Participants also had slower latencies when responding to positive words and made significantly more omission errors for negative words on the CANTAB Affective Go/No-go task. In Experiment 2 (n = 28), participants made significantly more total errors and had poorer heuristic search strategy on the CANTAB Spatial Working Memory task. In both experiments, CO 2 inhalation significantly increased negative affect; state anxiety and fear; symptoms of panic; and systolic blood pressure/heart rate. Overall, CO 2 inhalation produced robust anxiogenic effects and impaired fronto-executive functions of cognitive flexibility and working memory. Effects on emotional processing suggested a mood-congruent slowing in processing speed in the absence of a negative attentional bias. State-dependent effects of anxiety on cognitive-emotional interactions in the prefrontal cortex warrant further investigation.

Dravet syndrome is a rare and malignant epileptic syndrome that is typically resistant to antiepileptic drugs. There is a growing interest in the antiepileptic properties of neurosteroids. Especially progesterone is known to act as an anticonvulsive agent. However, contraceptives that act as orally given neurosteroids often lead to increased seizures in women with epilepsy.We report on two girls with Dravet syndrome, who gained seizure freedom under therapy with contraceptives. The first patient received an oral contraceptive, a combination of an estrogen, and a gestagen, due to hypermenorrhea. The second received a pure gestagen implant for contraception.The antiepileptic effect of progesterone is mainly attributed to its conversion into allopregnanolone and related neurosteroids, which are positive modulators of gamma-aminobutyric acid A receptors. It is unclear whether the observed effect of these contraceptives containing synthetic progesterone derivatives may be specific for the Dravet syndrome, and it is unclear whether and how this observation can be used therapeutically., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Georg Thieme Verlag KG Stuttgart · New York.)

Despite a wealth of research, the core features of psychopathy remain hotly debated. Using network analysis, an innovative and increasingly popular statistical tool, the authors mapped the network structure of psychopathy, as operationalized by the Psychopathy Checklist-Revised (PCL-R; Hare, 2003) in two large U.S. offender samples (nNIMH = 1559; nWisconsin = 3954), and 1 large Dutch forensic psychiatric sample (nTBS = 1937). Centrality indices were highly stable within each sample, and indicated that callousness/lack of empathy was the most central PCL-R item in the 2 U.S. samples, which aligns with classic clinical descriptions and prototypicality studies of psychopathy. The similarities across the U.S. samples offer some support regarding generalizability, but there were also striking differences between the U.S. samples and the Dutch sample, wherein the latter callousnesss/lack of empathy was also fairly central but irresponsibility and parasitic lifestyle were even more central. The findings raise the important possibility that network-structures do not only reflect the structure of the constructs under study, but also the sample from which the data derive. The results further raise the possibility of cross-cultural differences in the phenotypic structure of psychopathy, PCL-R measurement variance, or both. Network analyses may help elucidate the core characteristics of psychopathological constructs, including psychopathy, as well as provide a new tool for assessing measurement invariance across cultures. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

Background: Dendritic messenger RNA (mRNA) localization and subsequent local translation in dendrites critically contributes to synaptic plasticity and learning and memory. Little is known, however, about the contribution of RNA-binding proteins (RBPs) to these processes in vivo., Results: To delineate the role of the double-stranded RBP Staufen2 (Stau2), we generate a transgenic rat model, in which Stau2 expression is conditionally silenced by Cre-inducible expression of a microRNA (miRNA) targeting Stau2 mRNA in adult forebrain neurons. Known physiological mRNA targets for Stau2, such as RhoA, Complexin 1, and Rgs4 mRNAs, are found to be dysregulated in brains of Stau2-deficient rats. In vivo electrophysiological recordings reveal synaptic strengthening upon stimulation, showing a shift in the frequency-response function of hippocampal synaptic plasticity to favor long-term potentiation and impair long-term depression in Stau2-deficient rats. These observations are accompanied by deficits in hippocampal spatial working memory, spatial novelty detection, and in tasks investigating associative learning and memory., Conclusions: Together, these experiments reveal a critical contribution of Stau2 to various forms of synaptic plasticity including spatial working memory and cognitive management of new environmental information. These findings might contribute to the development of treatments for conditions associated with learning and memory deficits.

Spin fluctuations are a leading candidate for the pairing mechanism in high temperature superconductors, supported by the common appearance of a distinct resonance in the spin susceptibility across the cuprates, iron-based superconductors and many heavy fermion materials. The information we have about the spin resonance comes almost exclusively from neutron scattering. Here we demonstrate that by using low-temperature scanning tunnelling microscopy and spectroscopy we can characterize the spin resonance in real space. We show that inelastic tunnelling leads to the characteristic dip-hump feature seen in tunnelling spectra in high temperature superconductors and that this feature arises from excitations of the spin fluctuations. Spatial mapping of this feature near defects allows us to probe non-local properties of the spin susceptibility and to image its real space structure.

Deregulation of TGF-β superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-β superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-β type II receptor-based, single-chain traps were designed, termed (TβRII)2 and (TβRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TβRII-ED), and a similar or slightly better potency than pan-TGF-β-neutralizing antibody 1D11 or an Fc-fused receptor trap (TβRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TβRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TβRII)2 treatment of mice with established 4T1 tumors (100 mm(3)) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential., (©2012 AACR.)

Dysregulation of cell adhesion molecules is associated with progression of malignant melanoma. Immunohistologic study of benign nevi (BN), dysplastic nevi (DN), and primary superficial spreading melanoma (SSM) was performed for carcinoembryonic antigen (CEA) and CEA cell adhesion molecule-1 (CEACAM1) using monoclonal antibodies. We investigated BN (n = 42), DN (n = 22), thin SSM (n = 21), and thick SSM (n = 21). CEA expression in melanomas and DN was significantly increased compared with BN. CEA expression in thick SSM was significantly higher than in DN. Compared with BN, expression of CEACAM1 in melanomas was significantly increased. CEACAM1 expression in thick SSM was significantly increased compared with DN and thin SSM. Our data support the findings of previous studies indicating that cell adhesion molecules of the CEA family may have a role in the development and progression of cutaneous melanoma and potentially serve as prognostic markers.

Objective: Tumor growth regulation by extracellular matrix components has been one of the main topics on tumor biology in the last years. We aimed to investigate the protein expression pattern of decorin and versican in superficial spreading melanoma (SSM) and its precursors., Patients and Methods: Paraffin-embedded sections of benign nevi (BN), dysplastic nevi (DN), and primary SSM were assessed. Immunohistochemistry was performed for decorin and versican antibodies., Results: We investigated 64 patients with BN (n = 29), DN (n = 15), and SSM (n = 20) with a median Breslow thickness of 0.8 mm (0.2 - 4.6 mm). We did not observe decorin or versican immunoreactivity in melanocytes but in peritumoral stroma. Kruskal-Wallis ANOVA did not reveal significant differences of decorin expression between the groups investigated (P = 0.19). However, compared to BN and DN median expression of versican was significantly increased in SSM (P = 0.016 and P = 0.019, respectively). Decorin as well versican expression of SSM did not significantly correlate with Breslow tumor thickness or Clark level., Conclusion: Our data indicate that decorin is not differentially expressed in peritumoral stroma of SSM, DN, BN, and thus unlikely of pathogenetic significance in melanoma transformation and/or progression. By contrast, we have demonstrated that SSM is associated with a significant overexpression of peritumoral versican suggesting a role for versican in the pathogenesis of melanoma.

The microbiota of ten seeds and ready-to-eat sprouts produced thereof was characterized by bacteriological culture and denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments of the 16S rRNA gene. The predominant bacterial biota of hydroponically grown sprouts mainly consisted of enterobacteria, pseudomonades and lactic acid bacteria (LAB). For adzuki, alfalfa, mung bean, radish, sesame and wheat, the ratio of these bacterial groups changed strongly in the course of germination, whereas for broccoli, red cabbage, rye and green pea the ratio remained unchanged. Within the pseudomonades, Pseudomonas gesardii and Pseudomonas putida have been isolated and strains of the potentially pathogenic species Enterobacter cancerogenes and Pantoea agglomerans were found as part of the main microbiota on hydroponically grown sprouts. In addition to the microbiota of the whole seedlings, the microbiota of root, hypocotyl and seed leafs were examined for alfalfa, radish and mung bean sprouts. The highest and lowest total counts for aerobic bacteria were found on seed leafs and hypocotyls, respectively. On the other hand, the highest numbers for LAB on sprouts were found on the hypocotyl. When sprouting occurred under the agricultural conditions, e.g. in soil, the dominating microbiota changed from enterobacteria to pseudomonades for mung beans and alfalfa sprouts. No pathogenic enterobacteria have been isolated from these sprout types. Within the pseudomonades group, Pseudomonas jessenii and Pseudomonas brassicacearum were found as dominating species on all seedling parts from soil samples. In practical experiments, a strain of P. jessenii was found to exhibit a potential for use as protective culture, as it suppresses the growth of pathogenic enterobacteria on ready-to-eat sprouts.

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of (125)I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized (125)I-225 mAb is recycled to the surface much more efficiently than internalized (125)I-EGF. Also, we found that internalization of (125)I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction., (Copyright 2003 John Wiley & Sons, Ltd.)

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, k(on) and k(off), and equilibrium dissociation constant, K(d), have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca(2+)) binding to the C-terminal TnI regulatory peptides TnI(96-131) and TnI(96-139) of 89nM and 58nM, respectively. The apparent association and dissociation rate constants for each interaction were k(on)=2.3x10(5)M(-1)s(-1), 2.0x10(5)M(-1)s(-1) and k(off)=2.0x10(-2)s(-1), 1.2x10(-2)s(-1) for TnI(96-131) and TnI(96-139) peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: K(d) being equal to 130nM and 56nM for TnC-TnI(96-131) and TnC-TnI(96-139), respectively. Interestingly, although the inhibitory region peptide (TnI(96-115)) was observed to bind with an affinity similar to that of TnI(96-131) by fluorescence analysis (K(d)=380nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate ( approximately 1x10(8) to 1x10(9)M(-1)s(-1)) and a fast off-rate ( approximately 1x10(2)s(-1)). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.