Detection and quantitation of iron in ferritin, transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging.

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter 55Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox-active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000 Da) in the neonatal cardiomyocytes.

Integral membrane proteins in proteomics. How to break open the black box?

Integral membrane proteins (IMPs) are coded by 20-30% of human genes and execute important functions - transmembrane transport, signal transduction, cell-cell communication, cell adhesion to the extracellular matrix, and many other processes. Due to their hydrophobicity, low expression and lack of trypsin cleavage sites in their transmembrane segments, IMPs have been generally under-represented in routine proteomic analyses. However, the field of membrane proteomics has changed markedly in the past decade, namely due to the introduction of filter assisted sample preparation (FASP), the establishment of cell surface capture (CSC) protocols, and the development of methods that enable analysis of the hydrophobic transmembrane segments. This review will summarize the recent developments in the field and outline the most successful strategies for the analysis of integral membrane proteins. SIGNIFICANCE: Integral membrane proteins (IMPs) are attractive therapeutic targets mostly due to their many important functions. However, our knowledge of the membrane proteome is severely limited to effectively exploit their potential. This is mostly due to the lack of appropriate techniques or methods compatible with the typical features of IMPs, namely hydrophobicity, low expression and lack of trypsin cleavage sites. This review summarizes the most recent development in membrane proteomics and outlines the most successful strategies for their large-scale analysis.

An activating mutation of GNB1 is associated with resistance to tyrosine kinase inhibitors in ETV6-ABL1-positive leukemia.

Leukemias harboring the ETV6-ABL1 fusion represent a rare subset of hematological malignancies with unfavorable outcomes. The constitutively active chimeric Etv6-Abl1 tyrosine kinase can be specifically inhibited by tyrosine kinase inhibitors (TKIs). Although TKIs represent an important therapeutic tool, so far, the mechanism underlying the potential TKI resistance in ETV6-ABL1-positive malignancies has not been studied in detail. To address this issue, we established a TKI-resistant ETV6-ABL1-positive leukemic cell line through long-term exposure to imatinib. ETV6-ABL1-dependent mechanisms (including fusion gene/protein mutation, amplification, enhanced expression or phosphorylation) and increased TKI efflux were excluded as potential causes of resistance. We showed that TKI effectively inhibited the Etv6-Abl1 kinase activity in resistant cells, and using short hairpin RNA (shRNA)-mediated silencing, we confirmed that the resistant cells became independent from the ETV6-ABL1 oncogene. Through analysis of the genomic and proteomic profiles of resistant cells, we identified an acquired mutation in the GNB1 gene, K89M, as the most likely cause of the resistance. We showed that cells harboring mutated GNB1 were capable of restoring signaling through the phosphoinositide-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, whose activation is inhibited by TKI. This alternative GNB1K89M-mediated pro-survival signaling rendered ETV6-ABL1-positive leukemic cells resistant to TKI therapy. The mechanism of TKI resistance is independent of the targeted chimeric kinase and thus is potentially relevant not only to ETV6-ABL1-positive leukemias but also to a wider spectrum of malignancies treated by kinase inhibitors.

Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib­resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients.

Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.

Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease "shaving" of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original "hppK" method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1-16 transmembrane segments. This method allowed us to identify 13 "missing proteins" - proteins with no previous evidence on protein level. BIOLOGICAL SIGNIFICANCE: Integral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called "missing proteins".

Iron transport in K562 cells: a kinetic study using native gel electrophoresis and 59Fe autoradiography.

The exact mechanisms of iron transport from endosomes to the target iron containing cellular proteins are currently unknown. To investigate this problem, we used the gradient gel electrophoresis and the sensitive detection of 59Fe by autoradiography to detect separate cellular iron compounds and their iron kinetics. Cells of human leukemic line K562 were labeled with [59Fe]transferrin for 30-600 s and cellular iron compounds in cell lysates were analyzed by native electrophoretic separation followed by 59Fe autoradiography. Starting with the first 30 s of iron uptake, iron was detectable in a large membrane bound protein complex (Band I) and in ferritin. Significant amounts of iron were also found in labile iron compound(s) with the molecular weight larger than 5000 as judged by ultrafiltration. Iron kinetics in these compartments was studied. Band I was the only compound with the kinetic properties of an intermediate. Transferrin, transferrin receptor and additional proteins of the approximate molecular weights of 130000, 66000 and 49000 were found to be present in Band I. The labile iron compounds and ferritin behaved kinetically as end products. No evidence for low molecular weight transport intermediates was found. These results suggest that intracellular iron transport is highly compartmentalized, that iron released from endosomal transferrin passes to its cellular targets in a direct contact with the endosomal membrane complex assigned as Band I. The nature of the labile iron pool and its susceptibility to iron chelation is discussed.

[Proteomics and its role in medicine]. / Proteomika a její místo v medicíne.

Proteomics is a new methodological and conceptual approach to the study of live systems, which aims to map the whole proteom - the protein complement of genome. It aspires to describe quantitatively and qualitatively all proteins present at the given moment in the cell, tissue or the organism. The principal tools of proteomics are separation techniques based on electrophoresis and chromatography, which are used to fractionate complex protein mixtures and namely the mass spectrometry used for the identification of individual proteins. Proteomics represents very young and rapid developing specialization, which has already proved its valuable potential for the study of various physiological and pathlogical molecular mechanisms and for the direct use in the diagnostics of serious diseases.

Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique.

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study we describe a new method to reconstruct the interaction of iron with iron-binding proteins. Proteins were separated by native gradient polyacrylamide gel electrophoresis and transfered to polyvinilidene difluoride membrane under native conditions. The immobilized iron-binding proteins are then labeled by 59Fe using a 'titrational blotting' technique and visualized by storage phosphorimaging. At least six proteins, in addition to ferritin and transferrin, are specifically labeled in cellular lysates of human erythroleukemic cells. This technique enables separation and detection of iron-binding proteins or other metal-protein complexes under near-physiological conditions and facilitates identification of weak iron-protein complexes. Using a new native metal blotting method, we have confirmed that specific high molecular weight proteins bind the labile iron pool.

Post-traumatic stress disorder in female survivors of rape attending a genitourinary medicine clinic: a pilot study.

This study describes psychological symptomatology including post-traumatic stress disorder (PTSD) in 19 women attending a specialist sexual assault service within a genitourinary medicine (GUM) clinic. Women were interviewed within one year post-rape (mean = 12 weeks) using standardized questionnaires for PTSD and other psychological symptomatology. Seventeen (89.5%) of 19 women met full criteria for a diagnosis of PTSD. Anxiety predominated amongst other psychological symptomatology. Suicidal ideation was reported by 8 women and one made a suicide attempt following the rape. Although it is acknowledged this is a small, select sample, the high level of psychological trauma found suggests that genitourinary medicine clinics providing for sexual assault require access to mental health professionals.

Factors associated with self-disclosure of HIV serostatus to significant others.

OBJECTIVES: To examine rates and patterns of self-disclosure of HIV serostatus amongst individuals attending an out-patient HIV clinic in East London. DESIGN: A cross-sectional survey design was used. METHODS: A volunteer sample of 95 out-patient HIV clinic attendees completed a self-report questionnaire examining patterns of disclosure to self-identified significant others, reasons for disclosure and non-disclosure, satisfaction with social support (SSQ6), quality of life (MOS-30) and anxiety and depression (HADS). Self-disclosure was examined in relation to cultural background, gender, satisfaction with social support, and medical and psychological variables. RESULTS: Seventy-nine men and 16 women reported a mean disclosure rate of 68% to self-identified significant others. Five individuals had not disclosed their HIV status to anyone; 91% of individuals had informed their partner. Friends were more frequently informed (79%) than family (53%). Ethnicity (p <.001) and length of time since testing HIV seropositive (p <.05) emerged as significant predictors of disclosure. Global satisfaction with social support was negatively correlated with depression but was not associated with the total rate of HIV disclosure. Frequently reported reasons for non-disclosure included wanting to protect others from distress and fear of discrimination. CONCLUSIONS: Self-disclosure of HIV serostatus rates was highest for partners, followed by friends, and lowest for family members. Patterns of disclosure of HIV serostatus varied in relation to ethnicity. Fifteen years into the HIV epidemic, social stigma continues to contribute towards non-disclosure of diagnosis.

Separation of cellular iron containing compounds by electrophoresis.

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by 59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with 59Fe-transferrin were separated on 3-20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.

[Bibliometric indicators for evaluation of research work. 2. Citations and their analysis]. / Bibliometrijski pokazatelji u ocjenjivanju znanstvenog rada. 2. Citati i njihova analiza.

Citations are part of publishing in science, and scientific areas differ in use of citations. Mostly, only previously published papers which had been useful in writing and publishing the new work are cited. Therefore, the number of citations points to effective presence of a paper or an author in a scientific community, and it is used in the evaluation of scientific impact of an author, institution, discipline, country etc. Sources of data for so called citation analyses are citation indexes. Results of citation analyses should be assessed with caution, in the context of other indicators of scientific impact.

[Bibliometric indicators in evaluation of research activity. 1. Publishing and evaluation of research]. / Bibliometrijski pokazatelji u ocjenjivanju znanstvenog rada. 1. Objavljivanje i ocjenjivanje rezultata znanstvenog rada.

Publishing of scientific results is the key phase in scientific work. Reviewing process is a common way of evaluation of published scientific papers, but bibliometric indexes (number of papers, number of citations, impact factor etc.) are used as an adjunct in the assessment of scientific performance in scientific and academic promotion. The article discusses the presence of Croatian medical journals and Croatian medical authors in international indexing, i.e. data bases.

Anterior versus posterior approach in 3D correction of adolescent idiopathic thoracic scoliosis: a meta-analysis.

PURPOSE: Systematic review was conducted to compare effectiveness and safety of anterior and posterior surgical approach in 3D correction of adolescent idiopathic thoracic scoliosis. METHODS: Data sources were MEDLINE and SCOPUS databases. We included studies on the use of either anterior or posterior instrumentation, or their combination, in surgical correction of adolescent idiopathic thoracic scoliosis, with at least 10 enrolled patients, aged less than 20 years at the time of surgery, and a follow-up of at least 24 months. A study was eligible if it reported the number of patients, mean estimate and dispersion of three key outcome measures (frontal and sagittal Cobb angle, apical vertebra rotation according to Perdriolle) at three measurement points (preoperatively, postoperatively, at follow-up). The quality of studies was assessed using the scale by Pilkington. RESULTS: Although 24 articles met the inclusion criteria, no randomized controlled trials (RCT) was identified. None of the articles was of high quality. Both instrumentations provided a similar degree of reduction of frontal Cobb angle. Long-term effects of surgical correction on the sagittal Cobb angle seemed to be more stable in patients treated by posterior approach, while the anterior approach was more effective in the reduction of apical vertebral rotation. The surgery parameters were more favorable for anterior approach, particularly for the number of fused vertebrae. CONCLUSIONS: Although the available evidence favors neither of the two approaches, our study revealed several important issues: the reports are heterogeneous and provide incomplete relevant information. High quality studies, particularly RCT, are called for. LEVEL OF EVIDENCE: Level II.

Fos expression in tyrosine hydroxylase containing hypothalamic neurons in CRH-KO mice: effect of immobilization stress.

OBJECTIVE: Little is known about the response of tyrosine hydroxylase (TH) containing hypothalamic neurons to stress in corticoliberine deficient (CRH-KO) mice. This study was aimed to extend this issue and reveal the data leading to a better understanding of physiological/anatomical plasticity of hypothalamic TH cells in response to acute immobilization stress (IMO) as well as of possible of CRH body deficiency contribution in the regulation of TH cells during stress. We examined the topographic distribution of TH protein immunolabeled perikarya in selected hypothalamic structures including the paraventricular (PVN), supraoptic (SON), periventricular (PeVN), arcuate (ArcN), dorsomedial (DMN), and ventromedial (VMN) nuclei and extrahypothalamic zona incerta (ZI) in CRH-KO and wild type (WT) mice. METHODS: The animals were perfused with fixative 120 min after a single IMO stress. The brains were removed, cryo-sectioned throughout the hypothalamus and Fos-TH co-localizations were processed immunohistochemically. Fos protein was visualized by diaminobenzidine (DAB) intensified with nickel ammonium sulphate, while TH cells were labeled only with DAB chromogen. The evaluation of Fos-TH co-labeled perikarya was performed with the use of computerized Leica light microscope and expressed as the percentage of total amount of TH labeled cells. RESULTS: From the qualitative point of view, the present data indicate similar anatomical distribution of TH immunoreactive perikarya in all brain structures investigated in both WT and CRH-KO mice, while from the quantitative point of view only TH cells in the DMN of CRH-KO mice showed a trend for increased activation by IMO. CONCLUSIONS: In several hypothalamic structures the basic population of TH neurons was not affected by the absence of endogenous CRH. Based on the data of this study it can also be assumed that despite of the presence of direct reciprocal connections between PVN and DMN neurons, PVN CRH neurons possibly are not participating in the regulation of TH neurons in the DMN during IMO stress. KEYWORDS: Hypothalamic nuclei - Fos-immunohistochemistry - Tyrosine hydroxylase - Immobilization stress - CRH knockout mice.

Response of hypothalamic oxytocinergic neurons to immobilization stress is not dependent on the presence of corticotrophin releasing hormone (CRH): a CRH knock-out mouse study.

This study explores the quantitative patterns of immunolabeled Fos protein incidence in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) oxytocinergic (OXY) neurons in response to immobilization (IMO) stress in corticotrophin releasing hormone deficient (CRH-KO) mice. Adult male mice, taken directly from cages or 120 min after a single IMO, were sacrificed by intracardial perfusion with fixative. Coronal brain sections of 30 mum thickness were processed for dual Fos/OXY immunohistochemistry. In control wild type (WT) and CRH-KO mice, scattered Fos immunoreactivity was observed in hypothalamus, including the PVN where scanty Fos signal occurred in both parvocellular and magnocellular PVN subdivisions. Dual Fos/OXY immunostainings revealed higher basal Fos expression in the PVN of control CRH-KO mice. IMO evoked a marked rise in Fos expression in OXY neurons of the PVN and SON in both WT and CRH-KO groups of mice. The present data demonstrate that 1/ CRH deficiency upregulates the basal activity of hypothalamic PVN OXY cells in CRH-KO mice and 2/ IMO stress in both WT and CRH-KO mice affects distinctly the activity of OXY cells in both SON and PVN. Our data indicate that CRH deficiency does not alter the responsiveness of PVN and SON OXY cells to IMO stress.

[Identification of differentially expressed proteins using automatic meta-analysis of proteomics-related articles].

We present here a new method for automatic meta-analysis of proteomic articles using assessment of frequency of individual protein names in the text. The list of all possible human protein names including synonyms was retrieved from UniProt knowledgebase. The retrieved names were searched in full-texts of peer-reviewed publications from electronic version of "Proteomics" journal and from PubMedCentral. In the automatic mode we have confirmed the earlier list of proteins [Petrak et al., Proteomics (2008) 8, 1744] most frequently reported as differentially expressed (DEPs) in human tissues. We have also verified, that the most recurrent proteins were reported in proteomic papers regardless of tissue, experimental goals or, to some extent, experimental methods employed. Frequently reported DEPs were: annexins, peroxiredoxins, alpha-enolase, triosephosphate isomerase, and HSP60. Besides, serum albumin, cathepsin D and vimentin were observed with relatively high frequency. The DEPs were reported in papers related to oncological, cardiovascular and neuronal diseases, and were involved in such biological processes as inflammation, cell regulation, immune responce and signal transduction. We conclude that automatic meta-analysis of proteomic papers enabled extraction of frequently reported proteins that are most likely the differentially expressed ones.

Sex and relationships for HIV positive women since HAART: a quantitative study.

OBJECTIVE: To investigate current levels of sexual activity, enjoyment, condom use, and other factors affecting sexual behaviour in a sample of women living with HIV. METHOD: Participants were self selected. A cross sectional design using semi-structured questionnaires was employed. 82 HIV positive women completed questionnaires asking about demographics, relationships, sexual behaviour, and safer sex practices. The Hospital Anxiety and Depression Scale (HADS) and Golombok-Rust Inventory of Sexual Satisfaction (GRISS) were administered. RESULTS: 28% of women had had no sexual partners since diagnosis. Mean time diagnosed was 69 months, range 4-191 months. Time since diagnosis was not associated with having had a sexual partner. 59% of women had a current sexual partner, half reporting intercourse in the past month. Infrequent sex (84%), avoidance (84%), non-communication (69%), and dysfunction (60%) were among the most prevalent sexual difficulties. Endorsement of HIV impaired sexual enjoyment was associated with reduced sexual frequency (p = 0.006) and sexual dysfunction (p = 0.042). Sexual dissatisfaction was associated with infrequency of sex (p = 0.037), avoidance (p = 0.02), and non-communication (p = 0.032). Clinically significant levels of anxiety and depression were reported in 60% and 38% of cases, respectively. Depression was associated with avoidance of sex and higher total GRISS scores (p = 0.006 and p = 0.042). 60% of respondents stated that they "always" used condoms; a trend was observed between reduced condom use and higher levels of depression and anxiety (p = 0.09 and p = 0.06, respectively). CONCLUSION: Sexual difficulties, including abstinence, were prevalent in this sample indicating the potential for interventions addressing the psychosexual needs of HIV positive women and their partners.