Functions of biogenic amines as neurotransmitters in the central nervous system

Biogeni amini kao neurotransmiteri imaju brojne uloge u fiziološkim funkcijama i sudjeluju u širokom spektru ponašanja organizma i raznim kognitivnim fenomenima. Sintetiziraju se iz aminokiselina u živčanom sustavu i mogu se podijeliti na kateholamine, indolamine i imidazolamine. Patogeneza i etiologija raznih neuroloških bolesti i razvoj ovisnosti o drogama i supstancama poput nikotina i kofeina povezane su s promijenjenom signalizacijom biogenih amina. U ovom radu izložen je pregled puteva sinteze biogenih amina iz aminokiselina, specifični receptori na koje se vežu te brojne uloge koje amini vrše kao neurotransmiteri u centralnom živčanom sustavu. Amini su većinom djelovanjem povezani s drugim spojevima i mogu imati inhibitornu ili ekscitacijsku aktivnost. Determinacija specifičnih uloga pojedinog amina otežana je zbog njihovih kompleksnih interakcija i zbog same složenosti živčanog sustava. Mnoge dosad otkrivene funkcije amina zbog toga nisu u potpunosti objašnjene.Biogenic amines as neurotransmitters have numerous roles in physiological functions and take part in a wide spectre of behaviours and many cognitive phenomena. They are synthesized from aminoacids in the nervous system and are divided into catecholamines, indoleamines and imidazoleamines. Patogenesis and etyology of many neurological diseases and development of drug, caffeine and nicotine addictions are connected to changes in biogenic amines signalization pathways. This work represents a review of biogenic amines synthesis pathways, specifical receptors which amines bind to and numerous roles which they carry out as neurotransmitters in the central nervous system. Amine activity is mostly connected to other compounds and they can have inhibitory or excitatory effect. Determination of specific amine roles is made difficult because of their complicated interactions and because of the complexity of the nervous system. This is the reason why many amine functions discovered so far haven't been completely explained

miR-7 AND miR-34a sequence cloning and expression in a1235 glioblastoma cell line

miRNAs are small non-coding RNAs which have an important role in signalling circuits regulating different cell processes. miR-7 and miR-34a are known as tumour suppressors, and both of them can interfere with cell proliferation, differentiation, apoptosis and migration. We constructed plasmids containing pri-miRNA sequences for these two miRNAs and introduced them into the A1235 glioblastoma cell line. Clones containing increased expression of processed miR-7 and miR-34a were obtained. The proliferation and sensitivity to alkylation agent of transfected cells were similar to those of control cells. Our results indicate that an increase in miR-7 and miR34 expression alone in A1235 glioblastoma cells is not sufficient to change their proliferation or sensitivity to the influence of alkylating agents

Cloning of mikroRNA sequence and analysis of their biological activity in glioblastoma cell line

Nekodirajuće RNA (ncRNA) važna su skupina regulatornih molekula koja se sastoji od transkripata dijelova genoma koji ne kodiraju za proteine. Sudjeluju u brojnim procesima u eukariotskim stanicama, poput regulacije ekspresije gena i interferencije s RNA. Jedna skupina malih jednolančanih ncRNA su mikroRNA (miRNA). Geni miRNA uobičajeno se nalaze unutar introna i transkribira ih RNA polimeraza II, nakon čega se transkript cijepa RNazama Drosha i Dicer. Novonastala jednolančana RNA, duga 19 do 25 nukleotida, u citoplazmi formira kompleks s proteinima zvan miRISC (kompleks za utišavanje induciran s miRNA). miRISC regulira ekspresiju gena tako što se veže na ciljanu mRNA koja ima sekvencu komplementarnu miRNA koja se nalazi u kompleksu, čime vodi do degradacije mRNA ili sprječavanja inicijacije translacije. Mnoge miRNA imaju ulogu tumorskih supresora ili onkogena, te se pokazalo da su miR-34a i miR-7 važni tumorski supresori čija je ekspresija utišana u brojnim tumorima čovjeka. Cilj diplomskog rada bio je konstrukcija plazmidnih vektora koji su eksprimirali sekvencu za prekursore miR-34a i miR-7 te proučavanje bioloških promjena u staničnoj liniji glioblastoma A1235 transficiranoj plazmidnim konstruktima. Uzgojene su stanične linije s povećanom ekspresijom miR-34a i miR-7, te je praćena njihova sposobnost proliferacije, otpornost na citostatike i ekspresija proteina koji su potencijalna meta miRNA.Non-coding RNAs (ncRNA) belong to an important group of regulatory molecules which consist of transcripts from non-protein coding genomic regions. They have a role in many biological processes in eukariotic cells, such as gene expression regulation and RNA interference. One group of small single stranded ncRNAs are microRNAs (miRNA). miRNA genes are usually located inside introns and are transcribed by RNA polymerase II, after which the transcipt is processed by Drosha and Dicer RNase. Newly formed single stranded RNA, 19 to 25 nucleotides long, forms a complex with proteins called miRISC (miRNA induced silencing complex) in the cytoplasm. miRISC regulates gene expression by binding with the target mRNA which has a sequence complementary to the miRNA in the complex, leading to mRNA degradation or translation initiation inhibition. Many miRNAs act as tumor supressors or oncogenes, and it was shown that miR-7 and miR-34a are important tumor supressors whose expression is silenced in multiple human cancers. The aim of this thesis was to construct plasmid vectors which expressed precursor sequences of miR-34a and miR-7, and to study biological changes in glioblastoma A1235 cell line transfected with plasmid constructs. Cell lines overexpressing miR-7 and miR-34a were produced, and cell proliferation, resistance to cytostatics and expression of potential protein targets were analyzed

Cloning of mikroRNA sequence and analysis of their biological activity in glioblastoma cell line

Nekodirajuće RNA (ncRNA) važna su skupina regulatornih molekula koja se sastoji od transkripata dijelova genoma koji ne kodiraju za proteine. Sudjeluju u brojnim procesima u eukariotskim stanicama, poput regulacije ekspresije gena i interferencije s RNA. Jedna skupina malih jednolančanih ncRNA su mikroRNA (miRNA). Geni miRNA uobičajeno se nalaze unutar introna i transkribira ih RNA polimeraza II, nakon čega se transkript cijepa RNazama Drosha i Dicer. Novonastala jednolančana RNA, duga 19 do 25 nukleotida, u citoplazmi formira kompleks s proteinima zvan miRISC (kompleks za utišavanje induciran s miRNA). miRISC regulira ekspresiju gena tako što se veže na ciljanu mRNA koja ima sekvencu komplementarnu miRNA koja se nalazi u kompleksu, čime vodi do degradacije mRNA ili sprječavanja inicijacije translacije. Mnoge miRNA imaju ulogu tumorskih supresora ili onkogena, te se pokazalo da su miR-34a i miR-7 važni tumorski supresori čija je ekspresija utišana u brojnim tumorima čovjeka. Cilj diplomskog rada bio je konstrukcija plazmidnih vektora koji su eksprimirali sekvencu za prekursore miR-34a i miR-7 te proučavanje bioloških promjena u staničnoj liniji glioblastoma A1235 transficiranoj plazmidnim konstruktima. Uzgojene su stanične linije s povećanom ekspresijom miR-34a i miR-7, te je praćena njihova sposobnost proliferacije, otpornost na citostatike i ekspresija proteina koji su potencijalna meta miRNA.Non-coding RNAs (ncRNA) belong to an important group of regulatory molecules which consist of transcripts from non-protein coding genomic regions. They have a role in many biological processes in eukariotic cells, such as gene expression regulation and RNA interference. One group of small single stranded ncRNAs are microRNAs (miRNA). miRNA genes are usually located inside introns and are transcribed by RNA polymerase II, after which the transcipt is processed by Drosha and Dicer RNase. Newly formed single stranded RNA, 19 to 25 nucleotides long, forms a complex with proteins called miRISC (miRNA induced silencing complex) in the cytoplasm. miRISC regulates gene expression by binding with the target mRNA which has a sequence complementary to the miRNA in the complex, leading to mRNA degradation or translation initiation inhibition. Many miRNAs act as tumor supressors or oncogenes, and it was shown that miR-7 and miR-34a are important tumor supressors whose expression is silenced in multiple human cancers. The aim of this thesis was to construct plasmid vectors which expressed precursor sequences of miR-34a and miR-7, and to study biological changes in glioblastoma A1235 cell line transfected with plasmid constructs. Cell lines overexpressing miR-7 and miR-34a were produced, and cell proliferation, resistance to cytostatics and expression of potential protein targets were analyzed

Cloning of mikroRNA sequence and analysis of their biological activity in glioblastoma cell line

Nekodirajuće RNA (ncRNA) važna su skupina regulatornih molekula koja se sastoji od transkripata dijelova genoma koji ne kodiraju za proteine. Sudjeluju u brojnim procesima u eukariotskim stanicama, poput regulacije ekspresije gena i interferencije s RNA. Jedna skupina malih jednolančanih ncRNA su mikroRNA (miRNA). Geni miRNA uobičajeno se nalaze unutar introna i transkribira ih RNA polimeraza II, nakon čega se transkript cijepa RNazama Drosha i Dicer. Novonastala jednolančana RNA, duga 19 do 25 nukleotida, u citoplazmi formira kompleks s proteinima zvan miRISC (kompleks za utišavanje induciran s miRNA). miRISC regulira ekspresiju gena tako što se veže na ciljanu mRNA koja ima sekvencu komplementarnu miRNA koja se nalazi u kompleksu, čime vodi do degradacije mRNA ili sprječavanja inicijacije translacije. Mnoge miRNA imaju ulogu tumorskih supresora ili onkogena, te se pokazalo da su miR-34a i miR-7 važni tumorski supresori čija je ekspresija utišana u brojnim tumorima čovjeka. Cilj diplomskog rada bio je konstrukcija plazmidnih vektora koji su eksprimirali sekvencu za prekursore miR-34a i miR-7 te proučavanje bioloških promjena u staničnoj liniji glioblastoma A1235 transficiranoj plazmidnim konstruktima. Uzgojene su stanične linije s povećanom ekspresijom miR-34a i miR-7, te je praćena njihova sposobnost proliferacije, otpornost na citostatike i ekspresija proteina koji su potencijalna meta miRNA.Non-coding RNAs (ncRNA) belong to an important group of regulatory molecules which consist of transcripts from non-protein coding genomic regions. They have a role in many biological processes in eukariotic cells, such as gene expression regulation and RNA interference. One group of small single stranded ncRNAs are microRNAs (miRNA). miRNA genes are usually located inside introns and are transcribed by RNA polymerase II, after which the transcipt is processed by Drosha and Dicer RNase. Newly formed single stranded RNA, 19 to 25 nucleotides long, forms a complex with proteins called miRISC (miRNA induced silencing complex) in the cytoplasm. miRISC regulates gene expression by binding with the target mRNA which has a sequence complementary to the miRNA in the complex, leading to mRNA degradation or translation initiation inhibition. Many miRNAs act as tumor supressors or oncogenes, and it was shown that miR-7 and miR-34a are important tumor supressors whose expression is silenced in multiple human cancers. The aim of this thesis was to construct plasmid vectors which expressed precursor sequences of miR-34a and miR-7, and to study biological changes in glioblastoma A1235 cell line transfected with plasmid constructs. Cell lines overexpressing miR-7 and miR-34a were produced, and cell proliferation, resistance to cytostatics and expression of potential protein targets were analyzed

Evaluation of Anticholinesterase Activity of the Fungicides Mefentrifluconazole and Pyraclostrobin

Triazoles are compounds with various biological activities, including fungicidal action. They became popular through cholinesterase studies after the successful synthesis of the dual binding femtomolar triazole inhibitor of acetylcholinesterase (AChE, EC 3.1.1.7) by Sharpless et al. via in situ click chemistry. Here, we evaluate the anticholinesterase effect of the first isopropanol triazole fungicide mefentrifluconazole (Ravystar®), developed to overcome fungus resistance in plant disease management. Mefentrifluconazole is commercially available individually or in a binary fungicidal mixture, i.e., with pyraclostrobin (Ravycare®). Pyraclostrobin is a carbamate that contains a pyrazole ring. Carbamates are known inhibitors of cholinesterases and the carbamate rivastigmine is already in use for the treatment of Alzheimer’s disease. We tested the type and potency of anticholinesterase activity of mefentrifluconazole and pyraclostrobin. Mefentrifluconazole reversibly inhibited human AChE and BChE with a seven-fold higher potency toward AChE (Ki = 101 ± 19 μM). Pyraclostrobin (50 μM) inhibited AChE and BChE progressively with rate constants of (t1/2 = 2.1 min; ki = 6.6 × 103 M−1 min−1) and (t1/2 = 1.5 min; ki = 9.2 × 103 M−1 min−1), respectively. A molecular docking study indicated key interactions between the tested fungicides and residues of the lipophilic active site of AChE and BChE. Additionally, the physicochemical properties of the tested fungicides were compared to values for CNS-active drugs to estimate the blood–brain barrier permeability. Our results can be applied in the design of new molecules with a lesser impact on humans and the environment

Cholesterol Oxime Olesoxime Assessed as a Potential Ligand of Human Cholinesterases

Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood–brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10–30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes