Cloning characterization and expression of a novel metallothionein gene (mt-d) from triticum durum

Two different metallothionein genes, labelled as mt-d and mt-a were identified in wheat (Triticum durum and Triticum aestivum) genomic DNA sequences were characterized. mt-d and mta, were found to contain 416 and 399 nucleotides, respectively. Nucleic acid sequence alignment showed 95 % similarity between the two. Sequencing results showed that the difference resulted from two extra TTTTTA repeats in the intron regions. cDNAs encoding mt-genes were identifed by RT-PCR. Gene alignment algorithms strongly suggested that both of these cDNAs (mt-a and mt-d) encoded an open reading frame of 75 amino acids with two cysteine-rich domains featuring Cys-XCys motifs at the amino- and carboxy termininus. The deduced amino acid sequences of mt-a and mt-d genes show striking similarity to the MT-like proteins described within the Class II as Type 1 MTs and showed 100 % similarity with each other as deduced from cDNA sequencing results. These results indicate that mt-d from T.durum forms a “novel type 1” MT. For further studies of mt-d expression, localization of the durum metallothionein protein (dMT) and its interactions with other proteins mt-d gene was inserted into the 5’ MCS of pGFPuv vector. Verification was based on sequence data and restriction enzyme analysis. However, expression could not be validated by neither by visual detection of GFP expression nor by SDS-PAGE analysis. A more detailed sequence analysis indicated that the problem was due to a point mutation within the coding sequence of the GFPuv, resulting in a stop codon and premature termination of the fusion protein. Results presented here show the presence of metallothionein gene in the wheat Triticum durum. Although our attempts to express the gene as a fusion protein together with GFP to facilitate its localization in different systems was not successful it will be important in future studies to pursue this goal and achieve expression of labelled protein in plant systems to gain insights into its exact function in plants

Akasya ağacı toprağından izole edilen bacillus sp. tarafından üretilen endüstriyel enzimlerin araştırılması

ÖZETAkasya Ağacı Toprağından İzole Edilen Bacillus Sp. Tarafından Üretilen Endüstriyel Enzimlerin AraştırılmasıMaltooligosakkarit üreticisi olan yeni bir α-amilaz üreticisi Akasya ağacı (Acacia cyanophylla Lindley ) toprağıdan izole edilmiştir. Fiziksel karakteristiğine ve 16S rRNA gen sekansı bakıldığında izolatın Bacillus genusuna ait olduğu belirlenmiş ve Bacillus subtilis SDP1 olarak adlandırılmıştır. En yüksek enzim üretimine çözülebilir nişatanın karbon kaynağı, maya özütünün de azot kaynağı olduğu besiyerinde ulaşılmıştır. SDP1 α-amilaz enzimi yaklaşık 61kDa’luk bir moleküler ağırlığa sahiptir. Enzimin optimum pH’sı 7.0 olmakla birlikte pH 5.0 ile pH 9.0 arasında oldukça aktiftir. Bununla birlikte pH 6.0 ile pH 8.0 arasında da çok kararlı bir enzimdir. Ham enzimin optimum sıcaklığı 60°C olup 50°C’de 1 ve 2 saatlik inkübasyonlar sonucunda başlangıç aktivitesinin sırasıyla %83 ve %74’ünü korumaktadır. SDP1 α-amilaz enzimi DEAE-selüloz iyon değiştirme kromatografisi kullanılarak saflaştırılmıştır. Saflaştırma sonucunda ham fermentasyon sıvısı yaklaşık % 26 verim ile 9,9 kat saflaştırılmıştır. Saflaştırılmış enzimin spesifik aktivitesi 191.8 U mg-1 olarak hesaplanmıştır. Manyetik alginat mikrokürelerine immobilize edilen SDP1 α-amilaz enziminin 70°C’deki termal kararlılığı yaklaşı 7 kat artmaktadır. Hem çözülebilir hem de buğday nişastanın SDP1 α-amilazı ile hidrolizi sonucunda ana ürün olarak maltoz, maltotrioz ve maltotetraoz oluştuğu ince tabaka kromatografisi ile belirlenmiştir. Bu da SDP1 α-amilaz enziminin maltooligosakkarit üreten bir enzim olduğunu göstermektedir. Aktivite ve stabilitesi için kalsiyuma ihtiyaç duymaması ve yeterli miktarda küçük maltotrioz ve maltotetraoz gibi küçük maltooligosakkaritler üretebilmesi SDP1 α-amilazını özellikle gıda ve fırıncılık sektörü için potensiyel bir aday yapmaktadır. Bu tez çalışması Marmara Üniversitesi, Bilimsel Araştırma Projeleri Birimi tarafından FEN-C-DRP-171108-0267 numaralı proje ile desteklenmiştir.ABSTRACTInvestigating Of Industrial Enzymes From Bacillus Sp Isolated From Acacia Cyanophylla Lindley A maltooligosacharide-forming α-amylase producer was isolated from the rhizosphere of Acacia cyanophylla Lindley from Çukurova region of Turkey. According to physiological characteristics and 16S rRNA gene sequence, this strain belonged to the genus Bacillus hence it was designated as Bacillus subtilis SDP1. Highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37 °C. The α-amylase had an apperent molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and it was highly active at pH ranging from 5.0 to 9.0. It was also stable at pH ranging from 6.0 to 8.0. The optimum temperature of the crude enzyme was 60°C and it retained 83% and 74% of its initial activity after 1h and 2h incubation periods at 50°C. SDP1 α-amylase purified to homogenity with single step ion-exchange chromatography. The purity of the enzyme was about 9.9-fold greater than that of the crude supernatant liquid. The yield of the purified enzyme preparation was about 26% with respect to the culture supernatant. The specific activity of the purified enzyme was about 191.8 U mg−1. Immobilization of SDP1 α-amylase stability with magnetic alginate beads enhanced the enzyme stability ~7 fold at 70°C. The unique features of calcium independence and efficient production of small maltooligosaccharides such as maltotetraose and maltotriose make SDP1 amylase potentially useful in food industries.This thesis has been supported by Marmara University, Scientific Research Project Unit with a project number FEN-C-DRP-171108-0267

Akasya ağacı toprağından izole edilen bacillus sp. tarafından üretilen endüstriyel enzimlerin araştırılması

Akasya Ağacı Toprağından İzole Edilen Bacillus Sp. Tarafından Üretilen Endüstriyel Enzimlerin Araştırılması Maltooligosakkarit üreticisi olan yeni bir α-amilaz üreticisi Akasya ağacı (Acacia cyanophylla Lindley ) toprağıdan izole edilmiştir. Fiziksel karakteristiğine ve 16S rRNA gen sekansı bakıldığında izolatın Bacillus genusuna ait olduğu belirlenmiş ve Bacillus subtilis SDP1 olarak adlandırılmıştır. En yüksek enzim üretimine çözülebilir nişatanın karbon kaynağı, maya özütünün de azot kaynağı olduğu besiyerinde ulaşılmıştır. SDP1 α-amilaz enzimi yaklaşık 61kDa’luk bir moleküler ağırlığa sahiptir. Enzimin optimum pH’sı 7.0 olmakla birlikte pH 5.0 ile pH 9.0 arasında oldukça aktiftir. Bununla birlikte pH 6.0 ile pH 8.0 arasında da çok kararlı bir enzimdir. Ham enzimin optimum sıcaklığı 60°C olup 50°C’de 1 ve 2 saatlik inkübasyonlar sonucunda başlangıç aktivitesinin sırasıyla %83 ve %74’ünü korumaktadır. SDP1 α-amilaz enzimi DEAE-selüloz iyon değiştirme kromatografisi kullanılarak saflaştırılmıştır. Saflaştırma sonucunda ham fermentasyon sıvısı yaklaşık % 26 verim ile 9,9 kat saflaştırılmıştır. Saflaştırılmış enzimin spesifik aktivitesi 191.8 U mg-1 olarak hesaplanmıştır. Manyetik alginat mikrokürelerine immobilize edilen SDP1 α-amilaz enziminin 70°C’deki termal kararlılığı yaklaşı 7 kat artmaktadır. Hem çözülebilir hem de buğday nişastanın SDP1 α-amilazı ile hidrolizi sonucunda ana ürün olarak maltoz, maltotrioz ve maltotetraoz oluştuğu ince tabaka kromatografisi ile belirlenmiştir. Bu da SDP1 α-amilaz enziminin maltooligosakkarit üreten bir enzim olduğunu göstermektedir. Aktivite ve stabilitesi için kalsiyuma ihtiyaç duymaması ve yeterli miktarda küçük maltotrioz ve maltotetraoz gibi küçük maltooligosakkaritler üretebilmesi SDP1 α-amilazını özellikle gıda ve fırıncılık sektörü için potensiyel bir aday yapmaktadır. Bu tez çalışması Marmara Üniversitesi, Bilimsel Araştırma Projeleri Birimi tarafından FEN-C-DRP-171108-0267 numaralı proje ile desteklenmiştir. ABSTRACT Investigating Of Industrial Enzymes From Bacillus Sp Isolated From Acacia Cyanophylla Lindley A maltooligosacharide-forming α-amylase producer was isolated from the rhizosphere of Acacia cyanophylla Lindley from Çukurova region of Turkey. According to physiological characteristics and 16S rRNA gene sequence, this strain belonged to the genus Bacillus hence it was designated as Bacillus subtilis SDP1. Highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37 °C. The α-amylase had an apperent molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and it was highly active at pH ranging from 5.0 to 9.0. It was also stable at pH ranging from 6.0 to 8.0. The optimum temperature of the crude enzyme was 60°C and it retained 83% and 74% of its initial activity after 1h and 2h incubation periods at 50°C. SDP1 α-amylase purified to homogenity with single step ion-exchange chromatography. The purity of the enzyme was about 9.9-fold greater than that of the crude supernatant liquid. The yield of the purified enzyme preparation was about 26% with respect to the culture supernatant. The specific activity of the purified enzyme was about 191.8 U mg−1. Immobilization of SDP1 α-amylase stability with magnetic alginate beads enhanced the enzyme stability ~7 fold at 70°C. The unique features of calcium independence and efficient production of small maltooligosaccharides such as maltotetraose and maltotriose make SDP1 amylase potentially useful in food industries. This thesis has been supported by Marmara University, Scientific Research Project Unit with a project number FEN-C-DRP-171108-0267

The relationship with calcium metabolism and bone turnover of ultrasonographic dimensions of parathyroid glands in end-stage chronic renal failure and the role of erythropoetin treatment

AMAÇ: Çalışmanın amacı, son dönem kronik böbrek yetmezliği olan hastalarda paratiroid bez boyutlarının hiper-paratiroidi ve kemik hastalığı ile ilişkisini belirlemek ve eritropoetin tedavisi ile ilişkisini ortaya koymaktır. GEREÇ VE YÖNTEM: Çalışmaya sürekli ayaktan periton diyalizi (SAPD) ünitesinde renal replasman tedavisi almakta olan 20 hasta, hemodiyaliz ünitesinden 40 hasta ve evre 4 prediyaliz (GFR 15-29 ml/dk) olan 20 hasta alındı. Hastalara paratiroid ultrasonografi, biyokimya - hematoloji tetkikleri ve kemik mineral dansitometri (KMD) ölçümleri yapıldı. BULGULAR: Prediyaliz hastaların parathormon (PTH) değerleri SAPD ve hemodiyaliz hastalarına göre daha düşük saptandı (sırasıyla p=0.002, p=0.001). Prediyaliz hastalarda adenom sayısı, periton diyalizi hastalarına göre düşük olduğu bulundu (p=0.015). Prediyaliz hastalarının paratiroid adenom volümü, hemodiyaliz hastalarına göre daha düşük saptandı (p=0.032). Son bir yılda kullanılan total eritropoetin (EPO) dozu; PTH düzeyi, adenom sayısı ve adenom volümünün yanı sıra, PTH’nın arttırdığı kemik yapım markerları olan alkalen fosfataz ve osteokalsin düzeyi ile de pozitif korelasyon göstermiştir (sırasıyla r=0.257 p=0.021, r=0.312 p=0.005). Ortalama PTH değerleri 496.5±439.7 pg/ml olarak bulundu ve adenom volümü ile PTH salgısı arasında pozitif korelasyon mevcuttu (p=0.001). SONUÇ: PTH sekresyonunun en yüksek otonom değerlere sahip olan hastalar, en fazla eritropoetin kullanmış hastalardır. Bu bulgular, eritropoetinin paratiroid gland büyüklüğünü arttırmanın yanı sıra otonomitesini de arttırdığını göstermektedir.OBJECTIVE: The aim of the study is to determine the association of parathyroid gland dimensions with hyperparathyroidism and bone disease in patients with end stage chronic renal failure and to demonstrate its association with erythropoietin treatment. MATERIAL AND METHODS: Twenty patients undergoing renal replacement therapy at the continuous ambulatory peritoneal dialysis (CAPD) unit, 40 patients from hemodialysis unit and 40 patients with stage 4 predialysis (GRF 15-29 ml / min) were included in the study. Patient parathyroid ultrasonography, biochemistry-hematology tests and bone mineral densitometry (BMD) measurements were performed. RESULTS: The PTH values of predialysis patients were lower than those of SAPD and hemodialysis patients (respectively p=0.002, p=0.001). Patients with predialysis had a lower number of adenomas than patients with peritoneal dialysis (p=0.015). The parathyroid adenoma volume of patients with predialysis was lower than that of hemodialysis patients (p=0.032). The total erythropoetin ( EPO) dose used in the last year; showed positive correlation with PTH level, adenoma number, adenome volume, and bone building markers (osteocalcin and alkaline phosphatase) (respectively r=0.257 p=0.021, r=0.312 p=0.005). Mean PTH values were 496.5 ± 439.7 pg / ml and there was a positive correlation between adenoma volume and PTH secretion (p=0.001). CONCLUSIONS: Patients with the highest autonomic levels of PTH secretion were the patients with the most erythropoetin use. These findings suggest that erythropoetin not only increases parathyroid gland size but also increases autonomy

The diagnostic significance of NT-proBNP and troponin I in emergency department patients presenting with palpitations

OBJECTIVE: This prospective study investigated the diagnostic significance of the N-terminal pro-brain natriuretic (NT-proBNP) and troponin I peptides in emergency department patients presenting with palpitations. METHODS: Two groups of patients with palpitations but without documented supraventricular tachycardia were compared: a group with supraventricular tachycardia (n&#8202;=&#8202;49) and a control group (n&#8202;=&#8202;47). Both groups were diagnosed using electrophysiological studies during the study period. Blood samples were obtained from all of the patients to determine the NT-proBNP and troponin I levels within the first hour following arrival in the emergency department. RESULT: The mean NT-proBNP levels were 207.74&#177;197.11 in supraventricular tachyarrhythmia group and 39.99&#177;32.83 pg/mL in control group (p<0.001). To predict supraventricular tachycardia, the optimum NT-proBNP threshold was 61.15 pg/mL, as defined by the receiver operating characteristic (ROC) curve, with a non-significant area under the ROC curve of 0.920 (95% CI, 0.86-0.97, p<0.001). The NT-proBNP cut-off for diagnosing supraventricular tachycardia had 81.6% sensitivity and 91.5% specificity. Supraventricular tachycardia was significantly more frequent in the patients with NT-proBNP levels &#8805;61.15 pg/mL (n&#8202;=&#8202;44, 90.9%, p>0.001). The mean troponin I levels were 0.17&#177;0.56 and 0.01&#177;0.06 pg/mL for the patients with and without supraventricular tachycardia, respectively (p<0.05). Of the 96 patients, 21 (21.87%) had troponin I levels &#8805;0.01: 2 (4.25%) in the control group and 19 (38.77%) in the supraventricular tachycardia group (p<0.001). CONCLUSION: Troponin I and, in particular, NT-proBNP peptide were helpful for differentiating supraventricular tachycardia from non- supraventricular tachycardia palpitations. Further randomized, large, multicenter trials are needed to define the benefit and diagnostic role of NT-proBNP and troponin I in the management algorithm of patients presenting with palpitations in emergency departments

The neutralization effect of montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).Scientific Research Projects Commission of Bahcesehir Universit