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Connection of redox parameters in selected strains Drosophila melanogaster with high and low preference for metamphetamine
No full textMetamfetamin (METH) je psihostimulans koji se široko zlouporabljuje
diljem svijeta. Sve je veća prevalencija ovisnosti koja može potencijalno biti
povezana kod potomaka čiji su roditelji bili ovisni što potomke čini rizičnom
skupinom. METH u organizmu djeluje na morfološke i funkcionalne promjene
neuralnih mreža – neuralnu plastičnost. Naš model za istraživanje neuralne
plastičnosti je samoadministracija METH-a koju koristimo za provedbu
umjetne selekcije kako bi istražili neurobiološku osnovu razlike u visokoj (HP)
i niskoj (LP) preferenciji.
Cilj ovog eksperimentralnog rada bio je utvrditi hoće li preferencijalna
samoadministracija METH-a pokazati promjene u redoks parametrima kod F1
generacije potomaka 22. i 28. generacije HP i LP soja, te hoće li ti redoks
parametri biti stabilni kod F2 i F3 potomaka 22. i 28. generacije HP i LP sojeva
nakon prestanka selekcije.
Dobiveni rezultati prikazuju da mjerenjem koncentracije vodikovog
peroksida, naprednog produkta glikacije i specifične enzimske aktivnosti
katalaze razine su bile niže u homogenatima cijelog tijela kod F1 potomaka
selektiranih sojeva 22. i 28. generacije HP i LP soja u odnosu na kontrolnu
skupinu. Mjerenjem specifične enzimske aktivnosti superoksid dismutaze
(SOD) nisu opažene razlike između F1 potomaka eksperimentalnih skupina,
razlike nije bilo niti u odnosu na kontrolnu skupinu. Kod F2 i F3 potomaka 22.
generacije HP i LP soja nema razlike u mjerenim redoks parametrima, osim u
SOD specifičnoj enzimskoj aktivnosti, dok kod F2 i F3 potomaka 28. generacije
HP i LP soja se svi parametri mijenjaju osim SOD specifične enzimske
aktivnosti.
U ovom eksperimentalnom radu je prikazano da konzumacija METH-a
roditeljske generacije djeluje na promjenu mjerenih redoks parametara kod
potomaka. Nestabilnost u 28. generaciji HP i LP soja ukazuje na potencijalne
epigenetske promjene. Kako one nisu uočene u 22. generaciji pretpostavka je
da je preferencija 22. generacije posljedica obogaćenja genskih varijanti koje
su rezultirale HP i LP fenotipom. Kako bi se ova pretpostavka istražila potrebno
je provesti buduća istraživanja.Metamphetamine (METH) is a psychostimulant that is widely abused
around the world. There is an increasing prevalence of addiction that can
potentially be associated with offspring whose parents were addicted, making
the offspring a risk group. METH in the body affects the morphological and
functional changes of neural networks – neural plasticity. Our model for
studying neural plasticity is METH self-administration, which we use to
implement artificial selection to investigate the neurobiological basis of the of
the difference in high (HP) and low (LP) preference.
The aim of this experimental work was to determine whether
preferential METH self-administration will show changes in the redox
parameters of F1 filial generation of the 22nd and 28th generations of HP and
LP strains, and whether these redox parameters will be stable in the F2 and
F3 filial generation of the 22nd and 28th generations of HP and LP strains after
the end of selection.
The obtained results show that by measuring the concentration of
hydrogen peroxide, advanced glycation product and catalase specific enzyme
activity, the levels were lower in the whole body homogenates of the F1 filial
generation of the selected strains of the 22nd and the 28th generations of the
HP and LP strains compared to the control group. By measuring the superoxide
dismutase (SOD) specific enzyme activity, no differences were observed
between the F1 filial generation of the experimental groups, nor was there a
difference compared to the control group. In the F2 and F3 filial generations
of the 22nd generation HP and LP strains, there is no difference in the
measured redox parameters, except for SOD specific enzyme activity, while
in the F2 and F3 filial generations of the 28th generation HP and LP strains, all
parameters change except SOD specific enzyme activity. In this experimental work, it was shown that METH consumption of the
parental generation affects the change of measured redox parameters in the
filial generations. Instability in the 28th generation HP and LP strains indicates
potential epigenetic changes. Since they were not observed in the 22nd
generation, the assumption is that the preference of the 22nd generation is
due to the enrichment of gene variants that resulted in the HP and LP
phenotype. In order to investigate this assumption, it is necessary to conduct
future research
Bolesti povezane s bakterijom <i>Porphyromonas gingivalis</i>
No full textPorphyromonas gingivalis is a Gram-negative bacterium that is classified as an oral pathogen and, together with the bacteria Treponema denticola and Tannerella forsythia, forms a complex that promotes periodontal diseases such as periodontitis and gingivitis. The main virulence factors of P. gingivalis are cysteine proteinases, gingipains, fimbriae, capsule, lipopolysaccharide layer, and outer membrane vesicles. Some of these virulent factors promote the recruitment of the innate and acquired immune system cells, but still, P. gingivalis can penetrate the bloodstream and potentially participate in the promotion of other diseases such as cancers, cardiovascular diseases, metabolic disorders, and neurological diseases. Molecular mechanisms that can affect the occurrence of these diseases mostly include the activation of the pro-inflammatory response via the NF-κB pathway. Although there are indications of a connection between the bacterium Porphyromonas gingivalis and the previously mentioned diseases, their cause-and-effect relationship has not yet been confirmed. However, the possibilities of using drugs that target the removal of the P. gingivalis bacterium as a potential method of suppression or prevention of the mentioned diseases should be researched.Porphyromonas gingivalis je Gram-negativna bakterija koja se klasificira kao oralni patogen te zajedno s bakterijama Treponema denticola i Tannerella forsythia čini kompleks koji potiče parodontne bolesti kao što su parodontitis i gingivitis. Glavni faktori virulencije P. gingivalis su cisteinske proteinaze, gingipaini, fimbrije, kapsula, lipopolisaharidni sloj i vezikule vanjske membrane. Neki od tih virulentnih faktora potiču regrutaciju stanica urođenog i stečenog imunosnog sustava, no ipak, P. gingivalis može prodrijeti u krvotok te potencijalno sudjelovati u promicanju drugih bolesti kao što su tumori, kardiovaskularne bolesti, poremećaji u metabolizmu i neurološke bolesti. Molekularni mehanizmi koji mogu utjecati na pojavu tih bolesti većinom uključuju aktivaciju proupalnog odgovora imunosnog sustava preko NF-κB puta. Iako postoje indikacije o povezanosti bakterije Porphyromonas gingivalis i navedenih bolesti, još nije potvrđena njihova uzročno-posljedična veza. Ipak, treba istražiti mogućnosti uporabe lijekova koji ciljaju na uklanjanje bakterije P. gingivalis kao potencijalnu metodu suzbijanja ili prevencije navedenih bolesti
Imunomodulatory drugs in multiple sclerosis therapy
No full textMultipla skleroza (MS) je progresivna autoimunosna demijelinizacijska bolest središnjeg živčanog sustava. Imunološki posredovanim reakcijama dolazi do oštećenja mijelinske ovojnice, a posljedično i aksona što u konačnici dovodi do znatne onesposobljenosti bolesnika. Točan uzrok bolesti nije poznat no smatra se da je pojava multiple skleroze rezultat interakcije genetskih i okolišnih čimbenika. Simptomi prema kojima se može prepoznati bolest su optički neuritis, slabost ekstremiteta, poremećaj hoda i vrtoglavice.
Prvi izbor za liječenje multiple skleroze je terapija koja modificira tijek bolesti odnosno imunomodulacijska terapija. Imunomodulacijskim pristupom liječenja smanjuje se učestalost recidiva, usporava se progresija bolesti te novi broj lezija. Trenutno je preko 10 lijekova odobreno za liječenje multiple skleroze koji se mogu uzimati intravenozno, injektiranjem u masno područje tijela (subkutano), oralno ili topikalno. Fingolimod, teriflunomid i dimetil-fumarat primjenjuju se oralno, natalizumab, alemtuzumab i okrelizumab pimjenjuju se putem intravenske infuzije dok se interferoni primjenjuju injektiranjem.
Terapijski pristup je složen te zahtjeva pristup svakom bolesniku kao pojedincu. Koju terapiju će bolesnik primiti ovisi o tome koje kriterije za indiciranje terapije ispunjava, a kada kriteriji budu jasno definirani započinje se liječenje prvom, a ako je potrebno i drugom linijom terapije.Multiple sclerosis (MS) is a progressive autoimmune demyelinating disease of the central nervous system. Immune-mediated reactions lead to damage to the myelin sheath, and consequently to the axon, which ultimately leads to considerable disability of the patient. The exact cause of the disease is unknown, but it is believed that the appearance of multiple sclerosis is the result of the interaction of genetic and environmental factors. Symptoms by which the disease can be recognized are optic neuritis, limb weakness, gait disturbance and dizziness.
The first choice for the treatment of multiple sclerosis is therapy that modifies the course of the disease, or immunomodulation therapy. The immunomodulation approach to treatment reduces the frequency of relapse, slows down the progression of the disease and the number of new lesions. There are currently over 10 drugs approved for the treatment of multiple sclerosis that can be taken intravenously, by injection into a fatty area of the body (subcutaneously), orally or topically. Fingolimod, teriflunomide, and dimethyl fumarate are administered orally, natalizumab, alemtuzumab, and ocrelizumab are administered via intravenous infusion, while interferons are administered by injection.
The therapeutic approach is complex and requires an approach to each patient as an individual. Which therapy the patient will receive depends on which criteria for indicating the therapy he meets, and when the criteria are clearly defined, the treatment begins with the first and, if necessary, with the second line of therapy
Synthesis of 5-(4-(cis-9,10-epoxyoctadecanamido)phenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride
No full textFotodinamička terapija (PDT) je klinički odobren, minimalno invazivan terapeutski postupak kojeg karakterizira citotoksična aktivnost protiv tumorskih stanica i raznih patogena. Temelji se na dinamičkim interakcijama između fotosenzibilizatora (PS), svjetlosti određene valne duljine te molekularnog kisika koje dovode do razaranja ciljanog tkiva.
Jedna od ključnih komponenti PDT-a je odabir i dizajn PS-a. Većina PS-a koji se koriste u PDT-u temelji se na porfirinskoj strukturi zbog njihovih povoljnih fotofizikalnih svojstava. Za kliničku primjenu važni su amfifilni porfirini budući da se njima omogućava pasivno ciljanje tumora koje je prethodno dokazano učinkovitijim nego aktivno. Njihovi hidrofobni dijelovi omogućavaju im ulazak i akumulaciju u stanice, dok ih hidrofilni dijelovi čine topljivima u vodi što je važno kako bi se spriječila njihova agregacija te olakšala administracija.
Dokazano je da se hidrofobnost porfirina povećava s duljinom alkilnih lanaca te da se na isti način povećava i njihova akumulacija u tumorske stanice. Smatra se da bi prisutnost epoksidne jedinice na dugom alkilnom lancu porfirina mogla povećati njegovu protutumorsku aktivnost budući da je prethodno pokazano kako epoksidirane masne kiseline te neki njihovi metaboliti posjeduju protutumorsku aktivnost.
U ovom radu provedena je optimizacija sinteze N-metiliranog tripiridilporfirina konjugiranog s dugim alkilnim lancem (18C) koji sadrži polarnu skupinu. Uspoređena je učinkovitost novosintetiziranog porfirina te prethodno sintetiziranog N-metiliranog tripiridilporfirina konjugiranog s dugim alkilnim lancem (18C) za primjenu u PDT-u na ljudskim epitelijalnim stanicama adenokarcinoma dojke (MDA-MB-231). Novosintetizirani spoj, iako je djelotvoran, manje je učinkovit od prethodno sintetiziranog. Međutim, on ne posjeduje tamnu toksičnost na MDA-MB-231 stanice, za razliku od spoja koji ne posjeduje polarnu skupinu na alkilnom lancu.Photodynamic therapy (PDT) is clinically approved, minimally invasive therapeutic procedure characterized by cytotoxic activity against tumor cells and various pathogens. It is based on dynamic interactions between the photosensitizer (PS), light of a certain wavelength and molecular oxygen, which lead to the destruction of the target tissue.
One of the key components of PDT is PS selection and design. Most of the PSs used in PDT are based on porphyrin structure due to their favorable photophysical properties. Amphiphilic porphyrins are important for clinical application since they enable passive tumor targeting, which has previously been proven to be more effective than active targeting. Their hydrophobic parts enable them to enter and accumulate in cells, while hydrophilic parts make them soluble in water, which is important to prevent their aggregation and facilitate administration.
It has been proven that hydrophobicity of porphyrins increases with the length of the alkyl chains and as a consequence their accumulation in tumor cells also increases. It is thought that the presence of an epoxy unit on the long alkyl chain of porphyrin could increase its antitumor activity since it has previously been shown that epoxidized fatty acids and some of their metabolites possess antitumor activity.
In this thesis, the optimization of synthesis of N-methylated tripyridylporphyrin conjugated with long alkyl chain (18C) containing a polar group was performed. The effectivness of newly synthesized porphyrin and previously synthesized N-methylated tripyridylporphyrin conjugated with a long alkyl chain (18C) for use in PDT on human epithelial breast adenocarcinoma cells (MDA-MB-231) was compared. The newly synthesized compound, although effective, is less effective than previously synthesized one. However, it does not possess dark toxicity on MDA-MB-231 cells, unlike the compound that does not posess a polar group on the alkyl chain
NMDA receptor in depression
No full textDepresija je jedan od najčešćih mentalnih poremećaja i predstavlja vodeći uzrok invaliditeta u svijetu. Karakteriziraju ju intenzivna tuga, gubitak interesa za uobičajene aktivnosti, osjećaj bespomoćnosti i bezvrijednosti, umanjen apetit, gubitak energije i drugi simptomi. Procjenjuje se da preko 300 milijuna ljudi u svijetu pati od depresije. Neki od faktora koji doprinose razvoju depresije su genetički faktori, stres i traumatski događaji, te neravnoteža neurotransmitera u mozgu (adrenalin, noradrenalin, dopamin, glutamat, GABA, acetilkolin). Neurotransmiteri imaju ulogu posrednika u prijenosu signala između neurona vežući se za specifične receptore.
NMDA (N-metil-D-aspartat) ekscitatorni je receptor glutamata. Kada se glutamat veže za NMDA receptor dolazi do prijenosa živčanih signala. NMDA receptor otkrili su Richard Morris i Jeff Watkins, a njegovo otkriće bilo je ključno je za razumijevanje neurotransmisije i funkcioniranje mozga.
Istraživanja su pokazala da je disfunkcija NMDA receptora povezana s razvojem i manifestacijom depresije zbog nedovoljne neurotransmisije glutamata. Zbog toga neki antidepresivi za metu imaju NMDA receptor. Najpoznatiji takav lijek je ketamin koji djeluje kao antagonist NMDA. Nedavno su razvijene i druge vrste antagonista NMDA receptora kao što esketamin (S-ketamin) koji je enantiomer ketamina. Imaju izrazito brz antidepresivan učinak, ali privremen. Vrlo su dobra alternativa za rezistentnu terapiju, ali se javlja problem toksičnosti i ovisnosti.Depression is one of the most common mental disorders and is a leading cause of disability worldwide. It is characterized by intense sadness, loss of interest in usual activities, feelings of helplessness and worthlessness, decreased appetite, loss of energy, and more. It is estimated that over 264 million people worldwide suffer from depression. Some factors contributing to the development of depression include genetic factors, stress, traumatic events, and imbalance of neurotransmitters in the brain (such as adrenaline, noradrenaline, dopamine, glutamate, GABA, and acetylcholine). Neurotransmitters play a role as mediators in signal transmission between neurons by binding to specific receptors.
NMDA (N-methyl-d-aspartate) is an excitatory receptor for glutamate. When glutamate binds to the NMDA receptor, nerve signals are transmitted. Richard Morris and John Watkins discovered it, and its discovery was crucial for understanding neurotransmission processes and brain functioning.
Research has shown that dysfunction of NMDA receptors is associated with the development and manifestation of depression due to insufficient glutamate neurotransmission. Therefore, some antidepressants target NMDA receptors. The most well-known medication is ketamine, which acts as an NMDA antagonist. Other types of NMDA receptor antagonists have been developed, such as esketamine (S-ketamine), which is the enantiomer of ketamine. They have a rapid and pronounced antidepressant effect but are temporary. They serve as a good alternative for treatment-resistant depression, but toxicity and addiction issues may arise
EH domena EHD3 proteina je potrebna, ali ne i dovoljna, za njegovu agregaciju
No full textSchizophrenia is a serious mental illness characterised by impaired
perception of reality and changes in behaviour. The molecular background of
schizophrenia is not yet fully known. In addition to genetic and
environmental factors, protein aggregation is being researched as one of the
possible causes of schizophrenia. Protein aggregation occurs when, affected
by endogenous and external stresses, cellular mechanisms get disrupted,
resulting in protein misfolding and protein aggregates accumulating. EHdomain containing 3 (EHD3) is a protein that has been found to aggregate in
the brains of schizophrenia patients. EHD3 is a protein involved in endocytic
trafficking and recycling as well as D1 receptor internalization. Full length
EHD3 has been shown to aggregate in SH-SY5Y cells, while two constructs
lacking the C-terminus of the protein, EHD3 amino acids 1-434 and 1-399,
did not aggregate. This implied that amino acids 435-535, representing its
EH domain, were required for aggregation. Also, full length EHD3 has been
shown to induce EHD3 1-399 to aggregation when co-expressed. In this
thesis we aimed to determine if the EH domain aggregates independently
and if it would co-aggregate when co-expressed with full length EHD3. When
expressed individually, EHD3 435-535 did not aggregate, implying that the
EH domain is not sufficient for EHD3 aggregation by itself. When coexpressed with full length EHD3, EHD3 1-399 and EHD3 1-434 the EH
domain also did not aggregate, which might imply that while the EH domain
is necessary for the initialization of EHD3 aggregation, other structural
regions like the helical and/or the G domain interact and recruit other
molecules that make up the aggregates. Future research is needed to
determine which EHD3 regions interact with the EH domain causing the
protein to aggregate and co-aggregate in the cell.Šizofrenija je ozbiljna mentalna bolest koju karakteriziraju narušena
percepcija stvarnosti i promjene u ponašanju pojedinca. Molekularni
mehanizmi šizofrenije još uvijek nisu dovoljno istraženi. Uz poznate
genetske i okolišne čimbenike, agregacija proteina se istražuje kao
potencijalni uzrok šizofrenije. Do agregacije proteina dolazi kada se uslijed
različitih endogenih i mehaničkih stresova naruše stanični mehanizmi što
rezultira pogrešnim slaganjem proteina i nakupljanjem njihovih agregata.
EH-domain containing 3 (EHD3) protein jedan je od proteina koji su
identificirani kao agregirajući u mozgovima pacijenata oboljelih od
šizofrenije. To je protein koji sudjeluje u endosomalnom transportu i
recikliranju te u internalizaciji D1 receptora. Dokazano je kako EHD3
agregira u SH-SY5Y staničnoj liniji, dok konstrukti kojima nedostaje Cterminalna regija, EHD3 1-434 i EHD3 1-399 ne agregiraju, Ovo upućuje
kako su aminokiseline 435-535, koje predstavljaju EH domenu proteina,
nužne za njegovu agregaciju. Također je pokazano kako EHD3 može
inducirati agregaciju EHD3 1-399 kada se zajedno eksprimiraju u stanici. U
ovom radu nastojali smo pokazati agregira li EH domena samostalno u
stanici, te hoće li ko-agregirati s cijelim EHD3 proteinom ukoliko se zajedno
eksprimiraju. EHD3 435-535 (sadrži isključivo EH domenu) ne agregira kada
se samostalno eksprimira u stanici, niti su cijeli EHD3, EHD3 1-399 i EHD3
1-434 inducirali njegovu agregaciju. Ovo upućuje da je EH domena ključna
za inicijalizaciju agregacije EHD3 proteina, no druge strukturalne regije
proteina, poput zavojnica i G domene, su potrebne za interakciju i
regrutaciju drugih molekula koje izgrađuju agregat. Daljnja istraživanja
potrebna su kako bi odredili koje to regije stupaju u interakciju s EH
domenom rezultirajući agregacijom i ko-agregacijom EHD3 proteina
Uloga optineurina u procesu fagocitoze
No full textOptineurin is an adaptor protein implicated in multiple cellular processes, such as autophagy, vesicular trafficking, exocytosis, and inflammation. Mutations in the optineurin gene have been linked with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease impacting motor neurons. Previous research has found that phagocytosis, the process of removing extracellular particles by professional phagocytic cells, was disrupted in ALS, and research from our laboratory on cell lines has shown slightly impaired phagocytosis in optineurin deficient cells. The goal of this thesis was to research the role of optineurin in phagocytosis using primary cells. To do that, we performed a flow cytometry-based assay on primary wild type (WT) and optineurin-insufficient OPTN470T bone marrow-derived macrophages (BMDMs) generated from mice aged 3 months and 2 years and microglia generated from neonatal mice. We found no difference between the genotypes in the number of phagocytic cells or their phagocytic ability in basal condition in neither BMDM nor microglia. The same lack of difference persisted when primary cells were pre-treated with inflammatory stimuli, as well as when degradation through autophagy and lysosomes was blocked. Interestingly, we found that phagocytosis was impaired in BMDMs from older mice compared to BMDMs from younger mice, but the difference was not significant when cells were pre-treated with an inflammatory stimulus. In conclusion, this study did not confirm the role of optineurin in phagocytosis of neuronal debris by primary cells, but the difference in the level of phagocytosis between cells generated from younger and older mice opens up a new route of researchOptineurin je adaptorski protein uključen u više staničnih procesa kao što su autofagija, promet vezikula, egzocitoza i upala. Mutacije optineurina nedavno su se povezale s amiotrofičnom lateralnom sklerozom (ALS-om), neurodegerativnom bolešću koja pogađa motoričke neurone. Prijašnja istraživanja pokazala su da je fagocitoza, proces u kojem profesionalni fagociti uklanjanju izvanstanične čestice, ometena u ALS-u. Istraživanja iz našeg laboratorija na staničnim linijama pokazala su da stanice bez optineurina imaju nešto nižu razinu fagocitoze. Cilj ovog istraživanja bio je vidjeti u primarnim stanicama ima li optineurin ulogu u procesu fagocitoze. U tu svrhu proveli smo esej fagocitoze baziran na protočnoj citometriji. Za istraživanje smo koristili wild type (WT) i OPTN470T optineurin insuficijentnu mikrogliju dobivenu iz neonatalnih miševa, kaoi makrofage dobivene iz koštane srži miševa starih 3 mjeseca i 2 godine. Nismo našli razliku između ova dva genotipa u broju stanica koje fagocitiraju niti u njihovoj sposobnosti fagocitoze u bazalnom stanju. Razlike nije bio niti nakon tretmana stanica s upalnim stimulansima, kao ni kad se blokirala razgradnja autofagijom i lizosomima. Zanimljiva je bila razlika između BMDM-ova iz mladih i starih miševa koja je prestala biti značajna nakon što su stanice bile tretirane upalnim stimulusom. Zaključno, ovo istraživanje na primarnim stanicama nije potvrdilo ulogu optineurina u procesu fagocitoze neuronalnog otpada. Razlika u razini fagocitoze između primarnih stanica dobivenih iz mladih i starih miševa otvara nove mogućnosti u istraživanju ovog staničnog procesa
Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities
No full textTime-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury
The impact of glucose and glutamine on the level of phosphoglycerate-dehidrogenase in human malignant cells in vitro
No full textZloćudni tumori nastaju zbog poremećaja regulacijskih mehanizama, neophodnih za rast i dijeljenje stanice. Nerijetko su im u podlozi nastanka mutacije u različitim genima, najčešće protoonkogenima i tumorsupresorskim genima. Početak zloćudne preobrazbe povezan je s nastankom klona brzoproliferirajućih stanica, na koji se nastavljaju evolucija i selekcija klonova s najsnažnijim replikacijskim potencijalom. Zloćudni tumori posjeduju deset temeljnih obilježja, među kojima je i reprogramirani metabolizam. Metabolička aktivnost stanica zloćudnog tumora promijenjena je u odnosu na zdrave stanice od kojih je tumor nastao, a naročito se intenzivno istražuje u odnosu na metabolizam glukoze i glutamina. Za razliku od zdravih stanica koje svoje metaboličke puteve preusmjeravaju u glikolizu u anaerobnim uvjetima, stanice zloćudnih tumora sklone su metaboliziranju velikih količina glukoze glikolizom u uvjetima u kojima je opskrba kisikom optimalna. Ovaj se fenomen naziva aerobna glikoliza ili Warburgov učinak. Osim glukoze, stanice zloćudnih tumora imaju i veliku potrebu za glutaminom, koji služi kao izvor dušika u biosintetskim reakcijama. Biosinteza i iskorištavanje serina u metabolički reprogramiranim stanicama smatra se iznimno važnim za njihovu proliferaciju. Prvi enzim biosinteze serina je fosfoglicerat dehidrogenaza (PHGDH), čija je povećana ispoljenost, kao posljedica amplifikacije dijela kromosoma, dokazana u nekim vrstama zloćudnih tumora. Cilj ovog istraživanja bio je provjeriti postojanje stanično specifične ispoljenosti PHGDH na razini transkripta (RT-qPCR), proteina i u odnosu na unutarstanični smještaj proteina (Western blot), u odgovoru na dvije vrste gladovanja, u tri linije stanica podrijetlom od tumora glave i vrata, te debelog crijeva. Rezultati pokazuju da se ispoljavanje ciljnog proteina u sve tri linije stanica pojačava u gladovanju. Ova promjena može (FaDu), i ne mora (Detrot562, HT29) biti praćena povećanjem transkripcijske aktivnost gena PHGDH. Neočekivano je otkriveno da u linijama stanica podrijetlom od tumora glave i vrata (FaDu, Detroit562), PHGDH u gladovanju ulazi u jezgru, dok ovaj fenomen izostaje u stanicama podrijetlom od karcinoma debelog crijeva (HT29).The occurrence of malignant tumors is consequential to disturbed regulatory mechanisms that are needed for cellular growth and division. Mutations of protooncogenes and tumor suppressor genes are frequently associated with their genetic background. Initiation of malignant transformation relates to the onset of a highly proliferative clone, while its evolution depends on the selection of clones with the most prominent replicative potential. Reprogrammed metabolism is one of ten basic cancer hallmarks. Metabolic activity of cancer cells is changed with respect to the cells from which specific cancer originates. Currently, the most intense research efforts are focused on the metabolism of glucose and glutamine. Contrary to untransformed cells which utilize glycolytic pathways when deprived of oxygen, malignant cells utilize a high amount of glucose through the same pathway, even when sufficient oxygen is available. This phenomenon is known as aerobic glycolyisis or the Warburg effect. In addition to glucose, malignant cells need glutamine, which they use as a source of nitrogen in biosynthetic reactions. Serine metabolism is considered to be of utmost importance for the proliferation of malignant cells. The first enzyme of serine biosynthesis is phosphoglycerate dehydrogenase (PHGDH). This enzyme is highly expressed in some malignant tumors, as a consequence of chromosome amplification.
The objective of this research was to explore the cellular specific expression of PHGDH at the level of the gene transcriptional activity (RT-qPCR), and the level and localization of the protein product, respectively, in three cell lines originating from head and neck malignant tumors and colon carcinoma, exposed to two types of starvation. In all three cell lines, exposure to starvation is associated with an increased level of the PHGDH protein. This change may (FaDu) and may not (Detroit562, HT29) be related to transcriptional activity of the PHGDH gene. During starvation, PHGDH unexpectedly translocates into the nucleus of the head and neck originating cell lines (FaDu, Detroit562), while the effect is absent in colon cancer originating HT29 cells
CYP3A4
No full textGlucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11β-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRR). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concen-tration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behav-ior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11β-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is as-sociated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of bind-ing of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined us-ing the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resvera-trol could affect CYP3A activity