bluuebunny/crossref_metadata_2025 huggingface dataset:
EĞİTİM BİLİMLERİ ÖRNEKLEMİNDE LİSANSÜSTÜ EĞİTİMİN NİTELİKSEL BOYUTUNA İLİŞKİN GÖRÜŞLER (NİTEL BİR ÇALIŞMA)
Authors: Dilci Tuncay
Published: March 2019

Bu çalışmada; lisansüstü eğitiminin sürece ilişkin
işleyişi gözden geçirilerek, eğitim bilimleri alanındaki katılımcıların bakış
açısıyla sorunları ve bu sorunlara ilişkin çözüm önerilerinin geliştirilmesi
amaçlanmıştır. Araştırma tarama modelinde yapılmış olup nitel araştırma deseni
kullanılmıştır. Çalışma grubunu Türkiye’de bulunan 7 Coğrafi bölge içerisinden her bölgeden
lisansüstü eğitim yapan köklü ve geleneksel kültürü oluşmuş üniversitelerden en
az bir üniversite olması koşuluyla belirlenen 15
lisansüstü ve 15 öğretim üyesi olmak üzere 30 katılımcı çalışma grubunu oluşturmaktadır.
Verilerin toplanması amacıyla katılımcılara; yarı yapılandırılmış olarak
hazırlanan; eğitim bilimlerinde lisansüstü eğitim çalışmalarının bilim adamı yetiştirmedeki
rolü ve eğitim hedeflerine katkısı, lisansüstünde görülen genel aksaklıklar, lisansüstü
eğitim geliştirilmesine yönelik çözüm önerilerinin belirlenmesini amaçlayan 4 adet
soru yöneltilmiştir. Soruların içerik ve frekans analizi yapıldıktan sonra
NVİVO paket programı kullanılarak şema halinde görselleştirilmiştir.
Araştırmadan elde edilen bulgulara göre; lisansüstü eğitime girişte yapılan
öğrenci seçiminde, danışman ve öğrencilerin hazırbulunuşluk düzeylerinde,
öğrenme ortamlarının öğretimsel boyutunda, ders içerikleri ve yapılan tezlerin
genel nitelikleri üzerinde bazı aksaklıkların olduğu tespit edilmiştir. Bu
sorunların çözümüne yönelik olarak öğretim üyelerinin ders sayısının
azaltılması, lisansüstü eğitimde öğretim üyelerinin niteliklerini arttırıcı
tedbirlerin alınması, çalışma koşullarının gözden geçirilmesi, lisansüstü
eğitime giriş sınavı olan ALES’ in içerik olarak yeniden yapılandırılarak,
öğrencilerin kişisel özellikleriyle alana ilişkin özelliklerini örtüştürerek
ölçen bir sınav aracı haline getirilmesi gibi öneriler yapılmıştır. 
ÖĞRETİM ETKİNLİKLERİ MODELİ’NE DAYALI ÇEVRİMİÇİ ÖĞRENME ORTAMININ ÖĞRENCİLERİN ÇEVRİMİÇİ ÖĞRENME ORTAMINA YÖNELİK TUTUMLARINA ETKİSİ
Authors: Donmuş Kaya Vildan , Akpunar Bürhan
Published: March 2019

Bu
araştırmanın genel amacı; Gagné’nin Öğretim Etkinlikleri Modeli’ne dayalı
çevrimiçi öğrenme ortamının öğrencilerin çevrimiçi öğrenme ortamına yönelik
tutumlarına etkisini incelemektir. Araştırmanın modeli, öntest-sontest kontrol
gruplu deneysel desen olarak ele alınmıştır. Araştırmanın çalışma grubunu,
2016-2017 öğretim yılı güz dönemi Fırat Üniversitesi Eğitim Fakültesi
İlköğretim Bölümü Matematik Öğretmenliği Programında 2. sınıfta öğrenim gören
ve “Öğretim İlke ve Yöntemleri Dersi”ne devam zorunluluğu olan 61 öğrenci
oluşturmaktadır. Bu öğrenciler Gagné’nin Öğretim Etkinlikleri Modeli’ne dayalı
çevrimiçi öğrenme ortamında ders gören deney grubu ve mevcut öğretimine
çevrimiçi öğrenme ortamında devam eden kontrol gruplarına yansız olarak kura
ile atanmışlardır. Deney grubunda 30, kontrol grubunda 31 öğrenci yer
almaktadır. Deneysel desene ilişkin öğrencilerin çevrimiçi öğrenmeye yönelik
tutumlarının belirlenmesi amacıyla Erdoğan, Bayram ve Deniz (2007) tarafından
geliştirilen “Web Tabanlı Öğretim Tutum Ölçeği” deney ve kontrol grubu
öğrencilerine öntest ve sontest olarak uygulanmıştır. Araştırma verilerinin
analizinde Wilcoxon işaretli sıralar testi, bağımsız gruplar t-testi, bağımlı
gruplar t-testi analizi kullanılmıştır. Araştırmadan elde edilen bulgulara göre;
Gagné’nin Öğretim Etkinlikleri Modeli’ne Dayalı Çevrimiçi Öğrenme Ortamının,
öğrencilerin derse yönelik tutumları üzerinde deney grubu lehine istatistiksel
fark olduğu, öğrencilerin çevrimiçi öğrenme ortamına yönelik tutumlarında ise
istatistiksel olarak anlamlı olmasa da deney grubunun aritmetik ortalamasının
kontrol grubundan yüksek olduğu görülmüştür.  
İHRACAT PAZARLAMASI STRATEJİLERİ İLE FİRMA PERFORMANSI İLİŞKİSİ
Authors: Yücel Atilla
Published: March 2019

Küreselleşme ihracatı birçok firma için önemli bir
faaliyet haline getirmektedir. Bu nedenle, firmaların dış pazarlarda başarılı
olabilmesi için, ihracat performansını etkileyen faktörlerin belirlenmesi önemli
bir unsur olarak ortaya çıkmaktadır. Firmaların ihracat performansını ve
başarısını etkileyen temel belirleyiciler ve tanımlayıcı faktörler birçok
çalışmada ele alınmakla beraber özellikle ihracat performansını etkileyen
değişkenler 5 temel grupta incelenmiştir: Yönetsel Faktörler, örgütsel faktörler,
çevresel faktörler, hedef pazar seçimi,  standardizasyon
ve adaptasyon kararlarını da içeren pazarlama karması değişkenleridir. Firmaların ihracat pazarlaması
mantığıyla dış pazarlarda başarılı olmalarında ve istenilen performansa
ulaşabilmelerinde uygulayacakları pazarlama stratejilerinin rolü büyüktür. Bu çalışmada; ihracat faaliyetinde bulunan firmaların
pazarlama stratejileri ile performansları arasındaki ilişkiler incelenmiştir.
İhracat pazarlaması stratejileri, pazarlama karması elemanları (ürün, fiyat,
dağıtım, tutundurma), pazarlama karmasının adaptasyonu ve standardizasyonu
kararları ve ihracat hedef pazar değişkenleri ( pazar seçimi ve bölümlendirme)
çerçevesinde ele alınarak farklı performans boyutları ile ilişkileri
belirlenmeye çalışılmıştır. 
OSMANLI DEVLETİ’NİN DIŞ TİCARETİNDE KULLANDIĞI 1862 TARİHLİ GÜMRÜK TARİFE DEFTERİ: AMERİKA ÖRNEĞİ
Authors: Fidan Murat
Published: March 2019

Osmanlı
Devleti Amerika Devleti ile imzaladığı 1830 ticaret anlaşmasıyla birlikte
uygulamaya koyduğu gümrük tarifesini dönemin iktisadî şartlarına göre ortaya
çıkan yeni gelişmelere bağlı olarak değiştirmek istemiştir. Bu değişimde iki
önemli etken dikkat çekmektedir. Birinci etkenin ürün fiyatlarındaki artış ile
alınan gümrük vergisinin azalması olacaktır. İkinci etkenin ise dünya iktisadî
şartlarındaki gelişmelere bağlı olarak kadîm iktisadi anlayışını değiştirme
girişimleri olacaktır. Osmanlı devleti Amerika ile gümrük tarifesini 20 Mart
1862 ticaret anlaşması sonrası yenilemiştir. Tarife
Osmanlı Türkçesine göre iki tarafın temsilcilerinin gözetiminde hazırlanmıştır.
Bu çalışmada tarife defterinde yer alan emtia çeşitlerinin Osmanlı Türkçesinden
Latin alfabesine çevirisi yapılırken, orijinal ifadelere sadık kalınmıştır.
Emerging role of alternative splicing of CRF1 receptor in CRF signaling.
Authors: Zmijewski Michał A , Slominski Andrzej T
Published: March 2019

Alternative splicing of mRNA is one of the most important mechanisms responsible for an increase of the genomic capacity. Thus the majority of human proteins including G protein-coupled receptors (GPCRs) possess several isoforms as a result of mRNA splicing. The corticotropin-releasing factor (CRF) and its receptors are the most proximal elements of hypothalamic-pituitary-adrenal axis (HPA) - the central machinery of stress response. Moreover, expression of CRF and regulated activity of CRF receptor type 1 (CRF1) can also play an important role in regulation of local stress response in peripheral tissues including skin, gastrointestinal tract or reproductive system. In humans, expression of at least eight variants of CRF1 mRNA (alpha, beta, c, d, e, f, g and h) was detected and alternative splicing was found to be regulated by diverse physiological and pathological factors including: growth conditions, onset of labor, during pregnancy or exposure to ultraviolet irradiation. The pattern of expression of CRF1 isoforms is cell type specific and recently has been linked to observed differences in responsiveness to CRF stimulation. In the proposed model of regulation of CRF-signaling, isoform CRF1alpha plays a central role. Other isoforms modulate its activity by oligomerization, leading to alteration in receptor trafficking, localization and function. Co-expression of CRF1 isoforms modulates sensitivity of cells to the ligands and influences downstream coupling to G-proteins. The other possible regulatory mechanisms include fast mRNA and/or protein turnover or decoy receptor function of CRF1 isoforms. Taken together, alternative splicing of CRF1 can represent another level of regulation of CRF-mediated stress responses at the central and peripheral levels. Chronic stress or malfunction of the HPA-axis have been linked to numerous human pathologies, suggesting that alternative splicing of CRF1 receptor could represent a promising target for drugs development.
Synthetic derivatives of genistein, their properties and possible applications.
Authors: Rusin Aleksandra , Krawczyk Zdzisław , Grynkiewicz Grzegorz , Gogler Agnieszka , Zawisza-Puchałka Jadwiga , Szeja Wiesław
Published: March 2019

Genistein, the principal isoflavone constituent of soybean, attracts much attention as a natural molecule with significant affinity towards targets of potential medicinal interest, but also as a food supplement or prospective chemopreventive agent. Since its physicochemical properties are considered suboptimal for drug development, much effort has been invested in designing its analogs and conjugates in hope to obtain compounds with improved efficacy and selectivity. The aim of this article is to summarize current knowledge about the properties of synthetic genistein derivatives and to discuss possible clinical application of selected novel compounds. Some basic information concerning chemical reactivity of genistein, relevant to the synthesis of its derivatives, is also presented.
Purification and characterization of an antibacterial protein from dried fruiting bodies of the wild mushroom Clitocybe sinopica.
Authors: Zheng Suyue , Liu Qinghong , Zhang Guoqing , Wang Hexiang , Ng Tzi Bun
Published: March 2019

A novel antibacterial protein with a molecular mass of 44 kDa has been isolated from dried fruiting bodies of the wild mushroom Clitocybe sinopica. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the protein was composed of two subunits each with a molecular mass of 22 kDa. Its N-terminal amino-acid sequence, SVQATVNGDKML, has not been reported for other antimicrobial proteins. The purification protocol included ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antibacterial protein was adsorbed on all three ion exchangers. The antimicrobial activity profile of the protein against tested bacterial and fungal strains disclosed that it possessed potent antibacterial activity against Agrobacterium rhizogenes, A. tumefaciens, A. vitis, Xanthomonas oryzae and X. malvacearum with a minimum inhibitory concentration mostly below 0.6 microM. However, it had no antibacterial activity against Pseudomonas batatae, Erwinia herbicola, Escherichia coli, and Staphylococcus aureus, and no antifungal activity against Setosphaeria turcica, Fusarium oxysporum, Verticillium dahliae, Bipolaris maydis, and B. sativum. The antibacterial antivity against A. tumefaciens was stable after exposure to 20-60 degrees C for 30 min and to pH 4-9 for 1 h.
Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.
Authors: Grodecka Magdalena , Czerwiński Marcin , Duk Maria , Lisowska Elwira , Waśniowska Kazimiera
Published: March 2019

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.
Phosphomannose isomerase gene for selection in lettuce (Lactuca sativa L.) transformation.
Authors: Bríza Jindrich , Růzicková Nina , Niedermeierová Hana , Dusbábková Jana , Vlasák Josef
Published: March 2019

A positive selection system using phosphomannose isomerase was employed for Agrobacterium tumefaciens mediated transformation of lettuce (Lactuca sativa L. var. 'Achát'). It was shown that the mannose-based selection system works very well with the lettuce genotype used, reaching up to 25% transformation efficiency on the medium with 20 g/L mannose and 20 g/L sucrose. The best transformation efficacy with the commonly-used kanamycin at 100 mg/L as a selection agent was 21%. Southern blot analyses of thirteen chosen mannose-resistant regenerants revealed that some of them have clonal origin, about one-half harbour a single T-DNA copy and one plant contains an incomplete T-DNA segment with only the left part of T-DNA with the pmi gene present in the genomic DNA. The following Northern analysis showed transcriptional activity of the introduced pmi gene in all plants analysed with very high differences in the level of pmi specific mRNA. The results demonstrate that both mannose and kanamycin provide comparable transformation efficiencies in our lettuce genotype. An alternative selection method with mannose as a selection agent is now available for lettuce transgenosis.
In vitro reoxygenation following hypoxia increases MMP-2 and TIMP-2 secretion by human umbilical vein endothelial cells.
Authors: Cavdar Zahide , Oktay Gulgun , Egrilmez Mehtap Yuksel , Genc Sermin , Genc Kursad , Altun Zekiye , Islekel Huray , Guner Gul
Published: March 2019

Endothelial cells lining the inner blood vessel walls play a key role in the response to hypoxia, which is frequently encountered in clinical conditions such as myocardial infarction, renal ischemia and cerebral ischemia. In the present study we investigated the effects of hypoxia and hypoxia/reoxygenation on gelatinases (matrix metalloproteinase-2 and -9), their inhibitor (TIMP-2) and activator (MT1-MMP), in human umbilical vein endothelial (HUVE) cells. HUVE cells were subjected to 4 h of hypoxia or hypoxia followed by 4 and 24 h of reoxygenation. The pro- and active forms of MMP-2 and MMP-9 were analyzed by gelatin zymography; TIMP-2 protein level was assayed using ELISA, while MT1-MMP activity was measured using an activity assay. The secretion of MMP-2 proform increased significantly in cells subjected to 4 h of hypoxia followed by 4 or 24 h of reoxygenation, compared with the normoxic group. TIMP-2 protein level also increased significantly in the hypoxia/reoxygenation groups, compared with the normoxic group. There were no statistically significant differences in the levels of active MT1-MMP in all groups. This study indicates that MMP-2 and TIMP-2 could be regarded as important components of a mechanism in the pathophysiology of ischemic injury following reperfusion.
New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures.
Authors: El Tahchy Anna , Boisbrun Michel , Ptak Agata , Dupire François , Chrétien Françoise , Henry Max , Chapleur Yves , Laurain-Mattar Dominique
Published: March 2019

Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d(3)-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling.
Synthesis and biological activity of raltitrexed-carrier conjugates.
Authors: Jagiello Monika , Kanska Urszula , Nevozhay Dmitry , Boratynski Janusz
Published: March 2019

Drugs used in chemotherapy give undesirable side effects, e.g., cardiotoxicity, leucopenia, hair loss and others. Covalent binding of a drug with a carrier may change its biodistribution, elimination and/or rate of transformation in the organism. The aim of this work was to synthesize conjugates of anticancer drug - raltitrexed (RTX) with lysozyme, bovine serum albumin (BSA), and dextran T40 and to investigate their cytotoxicity and influence on the cell cycle in comparison with the free drug. Before conjugation RTX was transformed into anhydride by treatment with dicyclohexylcarbodiimide in dimethylformamide. Activated RTX was added into aqueous solution of carriers at different pH (from 8.5 to 10.5) for 3 to 15 min. The reaction was stopped by reducing the pH to 7.0. Maximum yield of the reaction was obtained at pH 10 for BSA as well as for dextran. The highest level of substitution was obtained after 5 min of the reaction. In in vitro experiments on three cell lines: SW707, LoVo and A549, all conjugates tested had up to a few hundred times higher IC(50) than the free drug. Interestingly, it was noticed that the conjugates based on dextran and albumin were more cytotoxic than the free drug in the highest concentrations tested (1000 and 10000 ng/ml). The influence of RTX and the conjugates on SW707 cell cycle was studied. RTX blocked the cell cycle mostly in the G(0)-G(1) and S phase and increased the percentage of apoptotic cells. Cells in the G(2)-M phase were not observed. The conjugates blocked the cell cycle in the S phase and decreased the percentage of cells in the G(0)-G(1) phase.
One-step purification of vitronectin from human plasma by affinity chromatography on phage-displayed peptides.
Authors: Skurzynski Szymon
Published: March 2019

A novel affinity purification method for rapid isolation of vitronectin (VN) from human plasma is described. Recently we have used phage display technology to obtain clones expressing peptides with high binding activity for VN. The isolated "strong VN binders" were covalently coupled to CNBr-activated Sepharose. Human plasma was applied to the column and bound VN was eluted using 0.5 M acetic acid, giving purity exceeding 90%. The developed method is a convenient alternative to conventional antibody-antigen affinity chromatography techniques for purification of VN, as it offers low ligand cost, is rapid and ensures good protein recovery from human plasma.
Boric acid as a protector against paclitaxel genotoxicity.
Authors: Turkez Hasan , Tatar Abdulgani , Hacimuftuoglu Ahmet , Ozdemir Ebru
Published: March 2019

Paclitaxel (PAC) is an anticancer drug used for treatments of breast, ovarian and lung cancers. However, little data is available in the literature on its potential genotoxicity on healthy human cells. On the other hand, boron deficiency and supplementation exert important biological effects in human and animal tissues. The biological effects of dietary boron are defined, but its interaction with PAC is not known for therapeutic uses. The aim of the present study was to determine whether boric acid (BA) confer a protection against PAC genotoxicity. After the application of PAC (10 or 20 microg/l) and BA (2.5 or 5 mg/l), the genotoxic effects were assessed by sister chromatid exchange (SCE) and micronucleus (MN) tests in human blood cultures. We also analyzed nuclear division index (NDI) in peripheral lymphocytes. Our results showed that PAC significantly (P
Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.
Authors: Milnerowicz Halina , Bizoń Anna
Published: March 2019

Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate).
Identification of hydroxy-plastochromanol in Arabidopsis leaves.
Authors: Szymańska Renata , Kruk Jerzy
Published: March 2019

In the present study we have identified hydroxy-plastochromanol in plants for the first time. This compound was found both in low light and high light-grown Arabidopsis plants, however, under high light stress its level was considerably increased. Hydroxy-plastochromanol accumulated also during ageing of leaves of low light-grown plants, similarly as in the case of other prenyllipids. Our results indicate that hydroxy-plastochromanol found in leaves is probably formed as a result of plastochromanol oxidation by singlet oxygen generated in photosystem II during photosynthesis. These data also support the hypothesis that plastochromanol is an efficient antioxidant in vivo, similarly as tocopherols and plastoquinol.
Myosin VI is associated with secretory granules and is present in the nucleus in adrenal medulla chromaffin cells.
Authors: Majewski Łukasz , Sobczak Magdalena , Redowicz Maria Jolanta
Published: March 2019

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments. Here, MVI, but not myosins IB or IIB, was detected in chromaffin granules isolated from bovine medulla and found to be tightly associated with the granule apical surface. MVI also localized to secretory granules within rat pheochromocytoma PC12 cells as well as to the Golgi apparatus, endoplasmic reticulum and clathrin-coated pits. Notably, it was also found in the nucleus. RT-PCR revealed that MVI splice variants with a large insert (LI), characteristic of polarized cells, were barely detectable in PC12 cells, whereas variants with a small insert (SI) were the major isoforms. The presented data indicate that MVI in adrenal medulla cells is engaged in secretory vesicle trafficking within the cytoplasm and possibly also involved in transport within the nucleus.
TRACER. A new approach to comparative modeling that combines threading with free-space conformational sampling.
Authors: Trojanowski Sebastian , Rutkowska Aleksandra , Kolinski Andrzej
Published: March 2019

A new approach to comparative modeling of proteins, TRACER, is described and benchmarked against classical modeling procedures. The new method unifies true three-dimensional threading with coarse-grained sampling of query protein conformational space. The initial sequence alignment of a query protein with a template is not required, although a template needs to be somehow identified. The template is used as a multi-featured fuzzy three-dimensional scaffold. The conformational search for the query protein is guided by intrinsic force field of the coarse-grained modeling engine CABS and by compatibility with the template scaffold. During Replica Exchange Monte Carlo simulations the model chain representing the query protein finds the best possible structural alignment with the template chain, that also optimizes the intra-protein interactions as approximated by the knowledge based force field of CABS. The benchmark done for a representative set of query/template pairs of various degrees of sequence similarity showed that the new method allows meaningful comparative modeling also for the region of marginal, or non-existing, sequence similarity. Thus, the new approach significantly extends the applicability of comparative modeling.
Lack of correlation between X region spa polymorphism and virulence of methicillin resistant and methicillin sensitive Staphylococcus aureus strains.
Authors: Kurlenda Julianna , Grinholc Mariusz , Szweda Piotr
Published: March 2019

Staphylococcus aureus is an etiological factor of severe infections in both hospital and ambulatory environments. As methicillin resistant Staphylococcus aureus strains spread quickly across healthcare centers resulting in life-threatening infections with increased mortality, they are considered more virulent than MSSA strains. Protein A, encoded by the spa gene, is one of the virulence factors involved in the staphylococcal pathogenesis. It has been suggested that the number of 24-bp tandem repeat units along the X region of the spa gene correlates with the virulence level of the strains. The current work analyzed the relationships between the virulence of MRSA and MSSA strains with region X polymorphism. No obvious correlation was observed.
Determination of antiradical and antioxidant activity: basic principles and new insights.
Authors: Tirzitis Gunars , Bartosz Grzegorz
Published: March 2019

Although the term 'antioxidant' is used very frequently, there are problems with the definition of antioxidants and estimation of antioxidant activity. The distinction between antioxidant and antiradical activities is not always obvious. This minireview discusses critically the principles, advantages and limitations of the most frequently used methods of estimation of antiradical and antioxidant activities.
Effect of carboxymethylated pyridoindoles on free radical-induced haemolysis of rat erythrocytes in vitro.
Authors: Juskova Maria , Snirc Vladimir , Krizanova Ludmila , Stefek Milan
Published: March 2019

Recently novel carboxymethylated pyridoindoles, analogues of the efficient chain-breaking antioxidant stobadine, have been designed, synthesised and characterised as bifunctional compounds with joint antioxidant/aldose reductase inhibitory activities with the potential of preventing diabetic complications. The critical property for the efficacy of the novel aldose reductase inhibitors in vivo is their ability to penetrate into target tissues. In this study, the issue was addressed by measuring the antioxidant activity of compounds 1 [(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid] and 2 [(+/-)-2-benzyl-(4a,9b)-cis-1,2,3,4,4a,9b-hexahydro-1H-pyrido[4,3-b] indole-8-yl acetic acid] in the cellular system of intact erythrocytes exposed to peroxyl radicals generated by thermal degradation of the azoinitiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) in vitro. Isolated washed rat erythrocytes were incubated in the presence of the azoinitiator AAPH and the compounds tested for increasing periods of time up to 4 h at 37 degrees C. The degree of haemolysis was determined by absorbance of the haemoglobin released. The onset of AAPH-induced haemolysis was found to be shifted from the starting zero point by the time interval assigned as a lag period. In the presence of the compounds studied the lag period was prolonged significantly. The free radical-initiated haemolysis was retarded by the compounds studied with decreasing efficiency: stobadine > compound 1 ~ Trolox > compound 2. The results have demonstrated an antioxidant activity of the novel carboxymethylated pyridoindoles developed as potential agents for multitarget pharmacology of diabetic complications.
Growth suppression of human breast carcinoma stem cells by lipid peroxidation product 4-hydroxy-2-nonenal and hydroxyl radical-modified collagen.
Authors: Cipak Ana , Mrakovcic Lidija , Ciz Milan , Lojek Antonin , Mihaylova Boryana , Goshev Ivan , Jaganjac Morana , Cindric Marina , Sitic Sanda , Margaritoni Marko , Waeg Georg , Balic Marija , Zarkovic Neven
Published: March 2019

Breast cancer is a leading cause of mortality and morbidity in women, mostly due to high metastatic capacity of mammary carcinoma cells. It has been revealed recently that metastases of breast cancer comprise a fraction of specific stem-like cells, denoted as cancer stem cells (CSCs). Breast CSCs, expressing specific surface markers CD44(+)CD24(-/low)ESA(+) usually disseminate in the bone marrow, being able to spread further and cause late metastases. The fundamental factor influencing the growth of CSCs is the microenvironment, especially the interaction of CSCs with extracellular matrix (ECM). The structure and function of ECM proteins, such as the dominating ECM protein collagen, is influenced not only by cancer cells but also by various cancer treatments. Since surgery, radio and chemotherapy are associated with oxidative stress we analyzed the growth of breast cancer CD44(+)CD24(-/low)ESA(+) cell line SUM159 cultured on collagen matrix in vitro, using either native collagen or the one modified by hydroxyl radical. While native collagen supported the growth of CSCs, oxidatively modified one was not supportive. The SUM159 cell cultures were further exposed to a supraphysiological (35 microM) dose of the major bioactive lipid peroxidation product 4-hydroxynonenal (HNE), a well known as 'second messenger of free radicals', which has a strong affinity to bind to proteins and acts as a cytotoxic or as growth regulating signaling molecule. Native collagen, but not oxidised, abolished cytotoxicity of HNE, while oxidized collagen did not reduce cytotoxicity of HNE at all. These preliminary findings indicate that beside direct cytotoxic effects of anticancer therapies consequential oxidative stress and lipid peroxidation modify the microenvironment of CSCs influencing oxidative homeostasis that could additionally act against cancer.
Lipid peroxidation product 4-hydroxynonenal as factor of oxidative homeostasis supporting bone regeneration with bioactive glasses.
Authors: Mrakovcic Lidija , Wildburger Renate , Jaganjac Morana , Cindric Marina , Cipak Ana , Borovic-Sunjic Suzana , Waeg Georg , Milankovic Andrea M , Zarkovic Neven
Published: March 2019

Bone regeneration is a process of vital importance since fractures of long bones and large joints have a highly deleterious impact on both, individuals and society. Numerous attempts have been undertaken to alleviate this severe medical and social problem by development of novel bioactive materials, among which bioactive glass is the most attractive because of its osteoconductive and osteostimulative properties. Since lipid peroxidation is an important component of systematic stress response in patients with traumatic brain injuries and bone fractures, studies have been undertaken of the molecular mechanisms of the involvement of 4-hydroxynonenal (HNE), an end product of lipid peroxidation, in cellular growth regulation. We found that HNE generated in bone cells grown in vitro on the surfaces of bioactive glasses 45S5 and 13-93. This raises an interesting possibility of combined action of HNE and ionic bioglass dissolution products in enhanced osteogenesis probably through a mitogen-activated protein kinase (MAPK) pathway. While the proposed mechanism still has to be elucidated, the finding of HNE generation on bioglass offers a new interpretation of the osteoinducting mechanisms of bioglass and suggests the possibility of tissue engineering based on manipulations of oxidative homeostasis.
Induction of CMV-1 promoter by 4-hydroxy-2-nonenal in human embryonic kidney cells.
Authors: Jaganjac Morana , Matijevic Tanja , Cindric Marina , Cipak Ana , Mrakovcic Lidija , Gubisch Wolfgang , Zarkovic Neven
Published: March 2019

Oxidative stress, i.e., excessive production of oxygen free radicals and reactive oxygen species, leads to lipid peroxidation and to formation of reactive aldehydes which act as second messengers of free radicals. It has previously been shown that oxidative stress may be involved in the transcriptional regulation of cytomegalovirus (CMV) immediate early promoter, involved in viral reactivation from latency. In the current study we used a plasmid containing the yellow fluorescent protein (YFP) gene under the control of CMV-1 promoter to monitor the influence of hydrogen peroxide and reactive aldehydes, 4-hydroxy-2-nonenal (HNE) and acrolein, on CMV-1 promoter activation in human embryonic kidney cells (HEK293). While acrolein was ineffective, hydrogen peroxide slightly (50 %) stimulated the CMV promoter. In contrast, HNE had a strong, up to 3-fold, enhancing effect on the CMV-1 promoter within four as well as after 24h of treatment. The most effective was the treatment with 24 microM HNE. This effect of HNE suggests that stressful conditions associated with lipid peroxidation could lead to CMV activation.
Influence of 4-hydroxynonenal and spleen cells on primary hepatocyte culture and a novel liver-derived cell line resembling hepatocyte stem cells.
Authors: Cipak Ana , Borovic Suzana , Jaganjac Morana , Bresgen Nikolaus , Kirac Iva , Grbesa Ivana , Mrakovcic Lidija , Cindric Marina , Scukanec-Spoljar Mira , Gall-Troselj Koraljka , Coric Marijana , Eckl Peter , Zarkovic Neven
Published: March 2019

Liver is a unique mammalian organ with a great capacity of regeneration related to its function. After surgical resection or injury, hepatic cells, especially hepatocytes, can proliferate rapidly to repair the damage and to regenerate the structure without affecting the function of the liver. Loss of catalase activity during regeneration indicates that oxidative stress is present in the liver not only in pathological conditions but also as a 'physiological' factor during regeneration. As we have shown in our previous work, liver stem cell-like cells treated with 4-hydroxynonenal (HNE), a cytotoxic and growth regulating lipid peroxidation product, recover in the presence of spleen cells. In the current study we characterized this novel cell line as liver-derived progenitor/oval-like cells, (LDP/OCs), i.e. functional liver stem-like cells. We showed that LDP/OC were OV6 positive, with abundant glycogen content in the cytoplasm and expressed alpha-fetoprotein, albumin, biliverdin reductase and gamma-glutamyl transferase. Also, we compared their growth in vitro with the growth of cultured primary hepatocytes stressed with HNE and co-cultured with autologous spleen cells. The influence of spleen cells on HNE-treated primary hepatocytes and on LDP/OCs showed that spleen cells support in a similar manner the recovery of both types of liver cells indicating their important role in regeneration. Hence, LDP/OC cells may provide a valuable tool to study cell interactions and the role on HNE in liver regeneration.
Double-edged sword behaviour of gallic acid and its interaction with peroxidases in human microvascular endothelial cell culture (HMEC-1). Antioxidant and pro-oxidant effects.
Authors: Serrano José , Cipak Ana , Boada Jordi , Gonzalo Hugo , Cacabelos Daniel , Cassanye Anna , Pamplona Reinald , Zarkovic Neven , Portero-Otin Manuel
Published: March 2019

A previous report from our group had shown in vitro a direct interaction between peroxidases and dietary antioxidants at physiological concentrations, where in the absence of H(2)O(2), the antioxidants could serve as oxidizing substrates for the peroxidases. However, the physiological relevance of those findings had not been evaluated. The main objective of this study was to determine whether the oxidizing products produced in the interaction between peroxidase and gallic acid at a physiological concentration of 1 microM may promote cell death or survival in a human microvascular endothelial cell line (HMEC-1). Our findings suggested that gallic acid may show a double-edged sword behaviour, since in the absence of H(2)O(2) it may have a pro-oxidant effect which may promote cell injury (evidenced by LDH, Crystal Violet and calcein AM viability/citotoxicity assays), while in the presence of H(2)O(2), gallic acid may act as an antioxidant inhibiting oxidative species produced in the peroxidase cycle of peroxidases. These observations were confirmed with several oxidative stress biomarkers and the evaluation of the activation of cell survival pathways like AKT and MAPK/ERK.
Interval hypoxic training in complex treatment of Helicobacter pylori-associated peptic ulcer disease.
Authors: Semen Khrysyna O , Yelisyeyeva Olha P , Kaminskyy Danylo V , Cherkas Andriy P , Zarkovic Kamelija , Lutsyk O , Cipak Ana , Jaganjac Morana , Zarkovic Neven
Published: March 2019

This study was aimed to demonstrate the efficacy of interval hypoxic training (IHT) in complex treatment of Helicobacter pylori-associated duodenal peptic ulcer disease (DPUD) by parameters of aerobic metabolism and indexes of heart rate variability (HRV). Eighty patients with H. pylori-associated DPUD were included into the study, mean age 32+/-1.8 yrs, duration of the disease up to 10 years (66.3 %). IHT was modulated using Frolov's hypoxicator (TDI-01) for 30 days after standard eradication therapy. Daily hypoxic sessions consisted of three one-minute sessions, one two-minute, and one three-minute sessions separated by one-minute intervals of room-air breathing. Use of IHT resulted in more efficient elimination of clinical symptoms, histological hallmarks of inflammation and signs of oxidative stress in glandulocytes of the gastric mucosa as determined by 4-hydroxynonenal accumulation. Moderate prooxidant activity of IHT was demonstrated by the increased level of TBARS and oxidatively modified products, normalization of hydroperoxides, middle mass molecules and atherogenic beta-lipoproteins with simultaneous increase in catalase activity and mild decline of SOD activity. Therefore, IHT appeared to be accompanied by higher intensity of redox reactions and enhanced regeneratory processes in cells and tissues. Significant increase in HRV was also noted. Such changes were associated with reduction of inflammation signs and modulation of the autonomic homeostasis in DPUD patients. In general, use of IHT in complex treatment of H. pylori in DPUD patients can be recommended to increase resistance to oxidative stress and to modulate autonomic balance and oxidative homeostasis.
Evidence for differential effects of glucose and cycloheximide on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-) machinery members: Superinduction of PPAR-gamma1 and -gamma2 mRNAs.
Authors: Rypka Miroslav , Veselý Jaroslav
Published: March 2019

Quantitative real-time RT-PCR study was conducted to reveal the effects of normal (5 mmol/l) and high (30 mmol/l) glucose without or with oleate (0.3 mmol/l) on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-)alpha, -gamma1, -gamma2, and peroxisome proliferator-activated receptor-gamma coactivator- (PGC-)1alpha and -1beta in commercial human hepatoma-derived HepG2 cells maintained under low-serum condition. Significant decrease in PPAR-gamma1 and PGC-1alpha mRNA levels to about 50 % was observed during the first 4 h incubation period. During the next 4 h period, both PPAR-gamma1 and PGC-1alpha mRNAs were partly but significantly restored in high glucose batches. In this period, the presence of the transcriptional inhibitor actinomycin D revealed a significant protective effect of excess glucose on mature PPAR-gamma1 and PGC-1alpha mRNAs. Furthermore, PPAR-gamma1 and -gamma2 mRNAs were differentially superinduced 1.2-2.5 fold in cells upon the administration of the translational inhibitor cycloheximide. When the cells were co-treated with the combination of cycloheximide and actinomycin D, superinduction was completely suppressed, however. Altogether, the experiments revealed, first, an unexpected protective effect of abundant glucose on PPAR-gamma1 and PGC-1alpha mRNAs in HepG2 cells. Second, we demonstrated cycloheximide-induced, transcription-dependent upregulation of mature PPAR-gamma1 and -gamma2 mRNAs in HepG2 cells associated with preferential expression of the PPAR-gamma2 mRNA variant. The results draw attention to as yet unexplored mechanisms involved in the control of PPAR and PGC genes.
Antioxidant properties of PF9601N, a novel MAO-B inhibitor: assessment of its ability to interact with reactive nitrogen species.
Authors: Bellik Lydia , Dragoni Stefania , Pessina Federica , Sanz Elisenda , Unzeta Mercedes , Valoti Massimo
Published: March 2019

The novel MAO-B inhibitor PF9601N, its cytochrome P450-dependent metabolite FA72 and l-deprenyl were studied as potential peroxynitrite (ONOO(-)) scavengers and nitric oxide synthase (NOS) inhibitors. The scavenging activity of these compounds was evaluated by measuring the oxygen consumption through peroxynitrite-mediated oxidation of both linoleic acid and brain homogenate. FA72, PF9601N and l-deprenyl caused a concentration-dependent inhibition of ONOO(-)-induced linoleic acid oxidation with an IC(50) value of 60.2 microM, 82.8 microM and 235.8 microM, respectively. FA72 was the most potent also in inhibiting ONOO(-)-induced brain homogenate oxidation with an IC(50) value of 99.4 microM, while PF9601N and l-deprenyl resulted weaker inhibitors in the same experimental model, showing an IC(50) value of 164.8 and 112.0 microM, respectively. Furthermore, both the novel MAO-B inhibitor as well as its metabolite were able to strongly inhibit rat brain neuronal NOS (IC(50) of 183 microM and 192 microM, respectively), while l-deprenyl at the highest concentration used (3 mM), caused only a slight decrease of the enzyme activity. Moreover, inducible NOS was strongly inhibited by FA72 only. All these results suggest that PF9601N could be a promising therapeutic agent in neurodegenerative disorders such as Parkinson's disease.
Regulation of sporulation in the yeast Saccharomyces cerevisiae.
Authors: Piekarska Iga , Rytka Joanna , Rempola Bozenna
Published: March 2019

Sporulation of the budding yeast Saccharomyces cerevisiae — equivalent to gametogenesis in higher organisms, is a complex differentiation program induced by starvation of cells for nitrogen and carbon. Such environmental conditions activate coordinated, sequential changes in gene expression leading to production of haploid, stress-resistant spores. Sporulation comprises two rounds of meiosis coupled with spore morphogenesis and is tightly controlled to ensure viable progeny. This review concerns the regulation of differentiation process by nutritional and transcriptional signals.
Evaluation of p-phenylene-bis and phenyl dithiocarbamate sodium salts as inhibitors of mushroom tyrosinase.
Authors: Amin Ehsan , Saboury Ali Akbar , Mansouri-Torshizi Hassan , Zolghadri Samane , Bordbar Abdol-Khalegh
Published: March 2019

Two structurally related compounds, phenyl dithiocarbamate sodium salt (I) and p-phenylene-bis (dithiocarbamate) sodium salt (II) were prepared by reaction of the parent aniline and p-phenylenediamine with CS₂ in the presence of sodium hydroxide. These water soluble compounds were characterized by spectroscopic techniques, IR, ¹H NMR and elemental analysis. The inhibitory effects of both compounds on both activities of mushroom tyrosinase (MT) from Agricus bisporus were studied at two temperatures, 27°C and 37°C. L-3, 4-dihydroxyphenylalanine (L-DOPA), and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic analysis confirmed noncompetitive inhibition mode of I and mixed type of II on both activities of MT; I and II inhibit MT with inhibition constants (K(I)) of 300 µM and 4 µM, respectively. Analysis of thermodynamic parameters indicated predominant involvement of hydrophobic interactions in binding of I and electrostatic ones in binding of II to MT. It seems that II is a more potent MT inhibitor due to its two charged head groups able to chelate copper ions in the enzyme active site. Intrinsic fluorescence studies as a function of concentrations of both compounds showed unexpectedly quenching of emission intensity without any shift of emission maximum. Extrinsic ANS-fluorescence indicated that only binding of I induces limited changes in the tertiary structure of MT, in agreement with the postulated hydrophobic nature of the binding mechanism.
Acaconin, a chitinase-like antifungal protein with cytotoxic and anti-HIV-1 reverse transcriptase activities from Acacia confusa seeds.
Authors: Lam Sze Kwan , Ng Tzi Bun
Published: March 2019

From the seeds of Acacia confusa, a chitinase-like antifungal protein designated as acaconin that demonstrated antifungal activity toward Rhizoctonia solani with an IC₅₀ of 30±4 µM was isolated. Acaconin demonstrated an N-terminal sequence with pronounced similarity to chitinases and a molecular mass of 32 kDa. It was isolated by chromatography on Q-Sepharose, SP-Sepharose and Superdex 75 and was not bound by either ion exchanger. Acaconin was devoid of chitinase activity. The antifungal activity against Rhizoctonia solani was completely preserved from pH 4 to 10 and from 0°C to 70°C. Congo Red staining at the tips of R. solani hyphae indicated inhibition of fungal growth. However, there was no antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, Helminthosporium maydis, and Valsa mali. Acaconin inhibited proliferation of breast cancer MCF-7 cells with an IC₅₀ of 128±9 µM but did not affect hepatoma HepG2 cells. Its IC₅₀ value toward HIV-1 reverse transcriptase was 10±2.3 µM. The unique features of acaconin include relatively high stability when exposed to changes in ambient pH and temperature, specific antifungal and antitumor actions, potent HIV-reverse transcriptase inhibitory activity, and lack of binding by strongly cationic and anionic exchangers.
Human plasma and cerebrospinal fibronectins differ in the accessibility of the epitopes on the N-terminal domains.
Authors: Pupek Małgorzata , Lemańska-Perek Anna , Jasonek Jolanta , Kątnik-Prastowska Iwona
Published: March 2019

Three monoclonal antibodies specific to the central cell-binding and the C- and N-terminal domains of fibronectin (FN) were used to test antigenic epitope accessibility on human plasma and cerebrospinal fibronectins. In the plasma group, the mean N-terminal FN domain immunoreactivity was about one fourth that of the cell-binding and C-terminal domains, whereas in cerebrospinal fluid they were nearly equal. In the presence of 0.5-6 M urea N-terminal domain immunoreactivity in the plasma increased 3-6-fold, but it decreased 0.7-3-fold in the cerebrospinal fluid. Analysis of fibronectin domain immunoreactivities of the cell-binding and N-terminal domains by a panel of specific monoclonal antibodies may reveal N-terminal fibronectin domain accessibility for reaction with biological partner ligand(s) and/or processes in which FN could be implicated. Such determinations may have important clinical implications.