Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

A First Insight on the Population Structure of Mycobacterium tuberculosis Complex as Studied by Spoligotyping and MIRU-VNTRs in Santiago,Chile

Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or demographics of Chile related to probable human migrations and evolutions.     

Assessment of Patient Empowerment - A Systematic Review of Measures

BackgroundPatient empowerment has gained considerable importance but uncertainty remains about the best way to define and measure it. The validity of empirical findings depends on the quality of measures used. This systematic review aims to provide an overview of studies assessing psychometric properties of questionnaires purporting to capture patient empowerment, evaluate the methodological quality of these studies and assess the psychometric properties of measures identified.MethodsElectronic searches in five databases were combined with reference tracking of included articles. Peer-reviewed articles reporting psychometric testing of empowerment measures for adult patients in French, German, English, Portuguese and Spanish were included. Study characteristics, constructs operationalised and psychometric properties were extracted. The quality of study design, methods and reporting was assessed using the COSMIN checklist. The quality of psychometric properties was assessed using Terwee’s 2007 criteria.Findings30 studies on 19 measures were included. Six measures are generic, while 13 were developed for a specific condition (N=4) or specialty (N=9). Most studies tested measures in English (N=17) or Swedish (N=6). Sample sizes of included studies varied from N=35 to N=8261. A range of patient empowerment constructs was operationalised in included measures. These were classified into four domains: patient states, experiences and capacities; patient actions and behaviours; patient self-determination within the healthcare relationship and patient skills development. Quality assessment revealed several flaws in methodological study quality with COSMIN scores mainly fair or poor. The overall quality of psychometric properties of included measures was intermediate to positive. Certain psychometric properties were not tested for most measures.DiscussionFindings provide a basis from which to develop consensus on a core set of patient empowerment constructs and for further work to develop a (set of) appropriately validated measure(s) to capture this. The methodological quality of psychometric studies could be improved by adhering to published quality criteria.     

Neural Circuits for Cognitive Appetite Control in Healthy and Obese Individuals: An fMRI Study

The mere sight of foods may activate the brain’s reward circuitry, and humans often experience difficulties in inhibiting urges to eat upon encountering visual food signals. Imbalance between the reward circuit and those supporting inhibitory control may underlie obesity, yet brain circuits supporting volitional control of appetite and their possible dysfunction that can lead to obesity remain poorly specified. Here we delineated the brain basis of volitional appetite control in healthy and obese individuals with functional magnetic resonance imaging (fMRI). Twenty-seven morbidly obese women (mean BMI = 41.4) and fourteen age-matched normal-weight women (mean BMI = 22.6) were scanned with 1.5 Tesla fMRI while viewing food pictures. They were instructed to inhibit their urge to eat the foods, view the stimuli passively or imagine eating the foods. Across all subjects, a frontal cortical control circuit was activated during appetite inhibition versus passive viewing of the foods. Inhibition minus imagined eating (appetite control) activated bilateral precunei and parietal cortices and frontal regions spanning anterior cingulate and superior medial frontal cortices. During appetite control, obese subjects had lower responses in the medial frontal, middle cingulate and dorsal caudate nuclei. Functional connectivity of the control circuit was increased in morbidly obese versus control subjects during appetite control, which might reflect impaired integrative and executive function in obesity.     

The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach

The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5''-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb²⁺-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.     

An Extended,Boolean Model of the Septation Initiation Network in S.Pombe Provides Insights into Its Regulation

Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.     

S-nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface

S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.     

Anti-neoplastic effect of protein kinase CK2 inhibitor, 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), on growth and hormonal activity of human adrenocortical carcinoma cell line (H295R) in vitro

Several studies indicate the involvement of protein kinases in the progression of various malignancies. Kinase inhibitors are therefore becoming important anticancer drugs. CK2 kinase (casein kinase-2) has been suggested to be a constituent of a neoplastic milleu, and its inhibition might represent a new approach to cancer therapy. Adrenocortical carcinomas (ACCs) are highly malignant neoplasms with poor overall prognosis. We have examined the effects of 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), a potent CK2 inhibitor, on the H295R human adrenocortical cancer cell line. Treatment with DMAT decreases the secretion of aldosterone, dehydroepiandrosterone sulfate, and androstendione and results in an accumulation of 17-OH-progesterone. Cell growth as measured by the MTT and 5-bromo-2′-deoxyuridine incorporation assays is inhibited, and cell cycle analysis has revealed a slight induction of apoptosis. Thus, CK2 kinase activity is probably involved in human ACC endocrine activity and growth.     

The ��TuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human ��TuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.