92 research outputs found
dam methylase from E. coli Circular dichroism investigations of the secondary structure and influence of S-adenosylmethionine
No full textAbstractThe enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of α-helix as the amount of S-adenosylmethionine bound to the enzyme was increased
Cellular study on transfused packed red blood cells
No full textThe aim of this PhD thesis was the cellular study of pRBCs intended to transfusion that have been stored in CPDA units and the further correlation of the results with relevant clinical studies. The results of the study were as follow: 1. The oxidation of the RBC is a part of storage lesion. 2. The hemoglobin is increasingly bound to the membrane and the cytoskeleton during storage. 3. Some of the main integral proteins of the RBC membrane and the lipid raft-associated leave the membrane, but other cytoplasmic ones become membrane-associated. 4. There is evidence of FAS oligomerization. 5. At the same time, proteolytic fragments representing active caspase-8 and -3 were evident. 6. RBCs lose their physiological shape during storage. From the membrane spikes of echino- and shero-echinocytes, microvesicles are released. 7. The microvesicles released in the units of pRBCs had diameter of 50-210nm. The microvesicles that were isolated from units stored for short periods (1-2 days) were smaller than those released after 35 days of storage (137±37nm, statistically important difference). The latter were characterized by tails, membrane gaps and formation of protein aggregates. All the microvesicles collected from "fresh" units and the majority of those collected on the last day of storage contained hemoglobin, so they were RBC-derived. 8. The degree of vesiculation was increased during storage. 9. The vesicles do not contain the skeletal proteins but have actin, band-3 and GpA. 10. By Western blot analysis the vesicles do not contain aquaporin, but contain the lipid raft associated proteins and proteins related to the membrane pathway of classical apoptosis. The vesicles contain proteins related to phagocytosis signaling events. 11. The proteins of the microvesicles were heavily carbonylated in relation to the RBC membrane of origin. In conclusion, the protein oxidation is a probably important part of the storage lesion. During storage, various mechanisms of cell signaling are activated and are mainly related to cell aging, cell clearance and, probably, programmed cell death. The stored RBCs-derived vesicles bear the same signaling components with their parent cells and, as a result, they probably are also post-transfusionally removed by the same mechanisms. In addition, the role of vesiculation seems to be at least of dual importance: a) one negative parameter is associated with the resulted permanent membrane loss, but b) another positive parameter is probably associated with the specific protein composition of the collected vesicles: since they contain harmful, oxidized and high components signaling potential their release during storage may prevent the premature removal or death of otherwise functional RBCs. Recent clinical studies, supporting the practice of using more "fresh" pRBCs for transfusion purposes, especially in critically affected patients, corroborated the results of the present study.Σκοπός της παρούσας διατριβής ήταν η κυτταροβιολογική μελέτη των συμπυκνωμένων ερυθροκυττάρων (ΣΕ) που προορίζονται για μετάγγιση προκειμένου να βρεθούν οι άριστες συνθήκες συντήρησης ex vivo που εξασφαλίζουν καλή βιωσιμότητα και λειτουργικότητα. Η μεθοδολογία περιλάμβανε απομόνωση και ανάλυση πρωτεϊνών των ΣΕ σε διάφορα χρονικά σημεία της αποθήκευσης με τεχνικές ηλεκτροφόρησης, ανοσοαποτυπώματος κατά Western και μικροσκοπίας (ηλεκτρονικής, φωτονικής και φθορισμού). Τα αποτελέσματα έδειξαν πως: 1. Η οξείδωση της μεμβράνης, του κυτταροσκελετού και του κυτταροπλάσματος είναι μέρος της αποθηκευτικής βλάβης των ΣΕ. 2. Η αιμοσφαιρίνη μετατοπίζεται από το κυτταρόπλασμα στη μεμβράνη αλλά και στον υπομεμβρανικό κυτταροσκελετό. 3. Οι κύριες πρωτεΐνες του ερυθροκυττάρου καθώς και εκείνες που συσχετίζονται με τις λιπιδικές σχεδίες εγκαταλείπουν τη μεμβράνη και άλλες φαίνεται να μετατοπίζονται από το κυτταρόπλασμα στη μεμβράνη. 4. Υπάρχουν ενδείξεις ενεργοποίησης του μεμβρανικού μονοπατιού της απόπτωσης. 5. Παρατηρήθηκαν πρωτεολυτικά θραύσματα των κασπασών-8 και -3 που αντιπροσωπεύουν τις ενεργοποιημένες μορφές τους. 6. Τα ΣΕ, καθώς αυξάνει ο χρόνος της αποθήκευσης μετασχηματίζονται αρχικά σε ακανθοκύτταρα και στη συνέχεια σε σφαιρο-ακανθοκύτταρα. 7. Τα μικροκυστίδια που απομονώθηκαν από τους ασκούς των ΣΕ είχαν διάμετρο από 50-210nm ενώ τα μικροκυστίδια που απομονώθηκαν από ασκούς αποθηκευμένους για μία ημέρα ήταν μικρότερα σε μέγεθος (137±37 nm) από αυτά που απομονώθηκαν στο τέλος της αποθηκευτικής περιόδου (35η ημέρα, 137±37nm). 8. Η κυστιδιοποίηση αυξάνει με την πάροδο του χρόνου αποθήκευσης. 9. Τα κυστίδια δεν περιέχουν σκελετικά συστατικά ενώ φέρουν ακτίνη, ζώνη-3 και γλυκοφορίνη-Α. 10. Πειράματα ανοσοεντόπισης έδειξαν την απουσία υδατοπορίνης στα κυστίδια, την παρουσία πρωτεϊνών που σχετίζονται με τις λιπιδικές σχεδίες, πρωτεΐνες που συμμετέχουν στο μεμβρανικό μονοπάτι της απόπτωσης και πρωτεΐνες που συμμετέχουν αρνητικά ή θετικά σε πορείες κυτταρικής εκκαθάρισης. 11. Οι κυστιδιακές πρωτεΐνες βρέθηκαν σε μεγάλο βαθμό καρβονυλιωμένες. Συμπερασματικά, τα αποτελέσματα αυτής της μελέτης περιέγραψαν τη σταδιακή αναδιαμόρφωση της ερυθροκυτταρικής μεμβράνης που λαμβάνει χώρα κατά την αποθήκευση. Η πρωτεϊνική οξείδωση φαίνεται να συνιστά σημαντικό καθοριστή της αποθηκευτικής βλάβης των ΣΕ. Κατά την αποθήκευση ενεργοποιούνται διάφοροι μηχανισμοί κυτταρικής σηματοδότησης, που σχετίζονται με τη γήρανση, την εκκαθάριση και ίσως τον προγραμματισμένο κυτταρικό θάνατο των ερυθρών. Τα κυστίδια που παράγονται στον ασκό φέρουν τα ίδια σηματοδοτικά συστατικά με τα κύτταρα προέλευσης τους. Επιπρόσθετα, ο ρόλος της κυστιδιοποίησης κατά την αποθήκευση φαίνεται να είναι διττός: αφενός μεν είναι αρνητικός καθώς προκαλεί μη-αντιστρεπτή μείωση της κυτταρικής επιφάνειας, από την άλλη όμως εξασφαλίζει στο ερυθροκύτταρο την απομάκρυνση οξειδωμένων και επιζήμιων συστατικών τα οποία θα προκαλούσαν τον πρώιμο θάνατο των ερυθροκυττάρων στους ασκούς της μετάγγισης. Τα αποτελέσματα της μελέτης αυτής επιβεβαιώθηκαν πρόσφατα από κλινικές μελέτες που υποστηρίζουν τη χρήση φρέσκων ασκών για μετάγγιση ειδικά σε ασθενείς που είναι ανοσοκατεσταλμένοι ή βρίσκονται σε μονάδες εντατικής θεραπείας
The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4
No full textAbstractThe amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5′-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA inethyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed
In Sickness and in Health: Erythrocyte Responses to Stress and Aging
No full textMature red blood cells (RBC) are the most abundant host cell in our body [...
Exploring Aggressive Behaviors in Greek Secondary Schools: Prevalence, Sociodemographic Factors, and Comparative Analysis with Elementary School Students
No full textThe main objectives of this study are to determine the prevalence of bullying in Greek secondary schools and detect the possible characteristics of bullies’ profiles in Greek school settings. A structured questionnaire was given to one hundred ninety-two (n = 192) educators at Greek junior high schools in urban and rural areas. The educators were asked to report the frequencies and forms of aggressive behavior observed during the 2022–2023 school year, the bullies’ sociodemographic characteristics, and ways of dealing with bullying episodes. The data are presented, after conducting statistical analyses, in comparison with data for elementary school students. The results revealed that higher rates of bullying were reported compared with elementary school children. Moreover, according to teachers’ observations, aggressive behavior is independent of a pupil’s diagnosis, but specific types are correlated significantly with a pupil’s gender, nationality, low academic performance, and popularity. Factor analysis showed two main factors of aggression types, where common points and differences with elementary school students are mentioned. Implementations for the prevention of school bullying are discussed
Synovial Fibroblast Extracellular Vesicles Induce Inflammation via Delivering miR-21-5p in Osteoarthritis
No full textSmall extracellular vesicles (sEVs) derived from different osteoarthritic (OA) tissues regulate OA-related biological processes through transporting their content (proteins, miRNAs, etc.) to recipient cells. This study aimed to characterize the miRNA profile of synovial fibroblasts-derived small EVs (FS_OA_sEVs) and investigate their role in inflammation in chondrocytes. Chondrocytes were isolated from macroscopically preserved and lesioned OA cartilage (C_OAmin and C_OAmax) and synovial fibroblasts from OA synovium. Synovial fibroblasts-derived small EVs (FS_OA_sEVs) were characterized according to ISEV guidelines and used for miRNA profiling and bioinformatics analysis. miR-21-5p was identified as one of the most abundant, and its target genes, such as KLF6, were enriched in OA-related processes including inflammation. Treatment of C_OAmin chondrocytes with FS_OA_sEVs resulted in decreased expression of COL2A1 and ACAN and an increase in catabolic markers MMP-3 and MMP-13. Moreover, C-OAmin receiving FS_OA_sEVs exhibited increased levels of inflammatory markers and miR-21-5p expression, resembling chondrocytes’ phenotype from lesioned OA cartilage, whereas miR-21-5p inhibition reversed their expression of inflammatory markers and miR-21-5p. Compared to C_OA min, C_OAmax chondrocytes exhibited increased miR-21-5p and inflammatory markers expression and decreased KLF6 expression. miR-21-5p inhibition in C_OAmax led to KLF6 upregulation and suppression of inflammatory mediators, whereas co-treatment with siRNA against KLF6 negated this effect, confirming a potential direct regulatory relationship between miR-21-5p and KLF6. Our results provide novel insights into the FS_OA_sEV-mediated inflammation axis, highlighting FS_OA_sEV-derived miR-21-5p as a driver of OA progression via regulating inflammation in chondrocytes
Apolipoprotein J/Clusterin Is a Novel Structural Component of Human Erythrocytes and a Biomarker of Cellular Stress and Senescence
No full textEvaluating Biological Risks in Biomedical Laboratories of Primary Health Care
No full textBiomedical laboratories within primary health care centers are vital for the detection, diagnosis, and management of diseases. However, handling diagnostic samples poses significant biological risks, particularly when biosafety measures are insufficient. This study focuses on analyzing the biological risks in 35 BSL-2 biomedical laboratories situated in health facilities across Athens, Greece, by examining compliance with biosafety regulations, awareness of personnel safety, and practices related to biorisk management. A cross-sectional survey was conducted by combining a customized checklist and a structured health and safety questionnaire, both have been created from the existing literature including the international biosafety guidelines (BMBL 6th ed., WHO Biosafety Program Management, 2020). An expert biorisk management advisor performed on-site evaluations, while 158 laboratory staff members filled out anonymous questionnaires concerning biosafety practices. Findings indicate widespread deficiencies in biosafety culture and risk management. Key gaps were identified in engineering controls (such as restricted access and safety equipment), administrative controls (including risk assessments, standard operating procedures, biosafety manuals, biosafety officers, and ongoing training), personal protective equipment (PPE policies), and emergency preparedness (such as incident reporting, response plans, and occupational health services). Many laboratories failed to meet international biosafety standards set by the WHO, CDC, and ECDC, highlighting the need for urgent improvements. To mitigate these risks, the study recommends the adoption of comprehensive Biorisk Management Systems, enhanced biosafety training, and stricter enforcement of national and European biosafety regulations. Strengthening these measures is essential to protect the laboratory staff, the surrounding community, and the environment from potential biological threats and lab-acquired infections
Evaluating Biological Risks in Biomedical Laboratories of Primary Health Care
No full textBiomedical laboratories in primary health care centers play a critical role in disease detection, diagnosis, and management. However, the handling of diagnostic samples presents significant biological risks, particularly when biosafety measures are insufficient. This study focuses on analyzing the biological risks in 35 BSL-2 biomedical laboratories within health facilities in Athens, Greece, by examining compliance with biosafety regulations, personnel safety awareness, and biorisk management practices. A cross-sectional survey was conducted combining a customized checklist and a structured health and safety questionnaire, both developed based on the existing literature, including the international biosafety guidelines (BMBL 6th ed., WHO Biosafety Program Management, 2020). On-site evaluations were performed by a certified biorisk management advisor, and 158 laboratory professionals anonymously completed questionnaires on biosafety practices. The collected data were analyzed qualitatively, and where possible, quantitatively, by using SPSS software and p-values from the McNemar test. The results revealed widespread deficiencies in biosafety culture and risk management. Key gaps were identified in all layers of engineering controls, administrative controls, personal protective equipment (PPE), and emergency preparedness. Many laboratories failed to meet international biosafety standards set by organizations such as the WHO, CDC, and ECDC, as well as Greek legislation, highlighting the need for urgent improvements. To address these issues and mitigate the observed gaps, the implementation of comprehensive Biorisk Management Systems, enhanced biosafety training, and stricter enforcement of national and European biosafety regulations is strongly recommended. These measures are essential to protect laboratory personnel, the surrounding community, and the environment from lab-acquired infections and other biological threats
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