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The role of circadian clock genes in melanoma pathogenesis
No full textCirkadijalni ritam je fundamentalna fiziološka osobina svakog ţivog bića, kako onih jednostaničnih tako i sloţenih organizama. Brojne funkcije ţivotinjskog, pa tako i našeg organizma, podlijeţu ritmičkim promjenama. Cirkadijalni ritam je biološki ritam koji je usklaĎen sa dnevno-noćnim promjenama u okolišu te traje otprilike dvadeset i četiri sata. Ove dnevno-noćne promjene direktno utječu na fiziološke i morfološke funkcije unutar pojedinog organizma, a regulirani su vanjskim signalima poput svjetla i hrane. Ti signali sinkroniziraju dogaĎaje unutar organizma putem autoregulativnih povratnih sprega. U srţi cirkadijalnog ritma nalaze se satni geni koji kontorliraju širok spektar dnevno-noćnih ritmova koji su u pozadini fizioloških procesa i ponašanja. Deregulacija ovih ritmova kod sisavaca povezana je sa različitim patološkim stanjima, uključujući karcinogenezu. Zbog promjene stila ţivota, unazad nekoliko godina dolazi do povećanja broja oboljelih od melanoma. Melanom je maligni tumor koji se pojavljuje unutar postojećeg madeţa ili pak na potpuno zdravoj koţi. Nastaje malignom preobrazbom pigmentalnih stanica melanocita te se i dalje smatra kako je izlaganje jakom ultraljubičastom zračenju, izuzev obiteljsku anamnezu, glavni rizik za razvoj tumora. Smatra se kako deregulacija cirkadijalnog ritma ili njegova ponovna sinkronizacija imaju veliku ulogu u daljnjoj progresiju bolesti. Cilj ovog rada je osvrnuti se na utjecaj i povezanost poremećaja cirkadijalnog ritma i uloge satnih gena u razvoju melanoma. Brojnim eksperimentima i istraţivanjima provedenim na ţivotinjama kao i na ljudima dokazano je da je funkcija satnih gena itekako isprepletena sa razvojem tumora te je manipulacija njihove ekspresije potencijalna terapija u liječenju oboljelih od raka.Circadian rhythm is a fundamental physiological feature of essentially all living organisms, including simple cell structures as well as complex organisms. Numerous behavioral and physiological processes in the orgranism underlie rhytmical changes. Circadian rhythm refers to biological rhythm that is synchronized with day-night changes in environment and lasts approximately twenty-four hours. These daily day and night changes directly affect physiological and morphological functions in individual organisms and are refered as external signals such as light and food. On the molecular level, circadian rhythm is governed by so-called core clock genes which control a wide range of circadian rhythms in physiological processes and behavior. Deregulation of these rhythms in mammals is associated with different pathological conditions including carcinogenesis. Due to changes in everyday lifestyle in last years, there have been increased number of malignant melanoma cases. Melanoma is malignant tumor which appears in existing naevus or even on healthy skin. It starts as malignant transformation of pigment cells, melanocytes, and it is assumed that the exposure to intense ultraviolet radiation, along with family anamnesis, still remains the main reason for tumor development. It is considered that deregulation of circadian rhythm or its resynchronization plays a big role in the progression of the disease. The aim of this thesis is to elaborate the topic of the influence and correlation of the circadian rhythm perturbation and clock gene involvement in melanoma pathogenesis. Growing body of evidence points out that clock genes are involved in tumor development and the regulation of of their expression might be used as potential therapy in treatment of cancer patients
In silico analysis and antiproliferative effect of novel pyrimidine-2,4-dione-1,2,3-triazole hybrids
No full textCilj rada je predviđanje mogućih bioloških meta hibrida 1,2,3-trizola metodama molekulskog
modeliranja te utvrđivanje mjesta i načina vezivanja aktivnih spojeva u odabranoj meti in silico
metodama. U radu je ispitana antiproliferativna aktivnost osam derivata hibrida 1,2,3-triazola
na pet različitih tumorskih staničnih linija, i svi su pokazali antiproliferativni učinak in vitro sa
značajnom citostatskom aktivnošću. Između N,N-1,3-bis-(1,2,3-triazol)-5-bromouracil hibrida
značajnu citostatsku aktivnost imaju p-fluor(1), o-fluor(3) i p-klorfenil(2) supstituirani 1,2,3-
triazoli koji su pokazali jaki inhibitorni utjecaj na rast stanica ispitanih na staničnim linijama
HepG2 i HeLa. Inhibicija proliferacije zapažena je pri mikromolarnoj koncentraciji ispitanih
spojeva, i bila je jača od inhibicije proliferacije stanica istih staničnih linija inkubiranih pod
istim uvjetima s 5-fluoruracilom kao referentnom supstancom. Kod ispitanih spojeva nije
uočena mitohondrijska toksičnost. Osim metoda stanične biologije u radu je rabljen program
PASS za ispitivane spojeve kako bi se odredila moguća biološka meta. Detekcija biološke mete
zasniva se na strukturi ispitivanih spojeva te na strukturi bioloških makromolekula iz
proteinskih baza. Analiza je pokazala da je za snažno antitumorsko djelovanje spojeva 1, 2 i 3
vrlo vjerojatno odgovorna inhibicija tirozin kinaze Wee1. Uporabom programa Autodock Vina
spojevi 2 i 3 uklopljeni su u kristalnu strukturu kinaze Wee1 koja je preuzeta iz proteinske baze
podataka [PDB: 5v5y]. Analiza molekulskog uklapanja pokazala je da se spojevi 2 i 3 vežu u
ATP vezno mjesto kinaze Wee1 jednako kao i spoj MK-1775 koji je snažni i specifični inhibitor
kinaze Wee1 (IC50 = 5,2 nM). Citostatski učinci spojeva 2 i 3 mogu se pripisati inhibiciji kinaze
Wee1, "čuvara" zastoja G2-M kontrolne točke staničnog ciklusa. Osim toga, za spojeve 2, 3 i
MK-1775 metodom Western blota ispitan je učinak na fosforilaciju proteina, mogućih supstrata
kinaze Wee1. Tako je u konačnici dokazano da se ukupna fosforilacija izoliranih proteina nakon
stimulacije perifernih mononuklearnih stanica znatno smanjuje u prisutnosti specifičnog
inhibitora MK-1775 kao i ispitivanih spojeva 2 i 3, što jasno ukazuje na moguću inhibiciju
kinaze Wee1. S obzirom da je ciklin ovisna kinaza Cdk1 supstrat za kinazu Wee1, u radu se
molekulskim modeliranjem analizirao utjecaj enzimske aktivnosti kinaze Wee1 na fosforilaciju
kinaze Cdk1.The aim of this work is to use molecular modeling methods to predict the possible biological
target of the 1,2,3-triazole hybrid molecules and using in silico methods to determine the site
and the way of binding of active compounds to the selected target. Antiproliferative activity of
eight hybrid 1,2,3-triazole derivatives were tested on five different tumor cell lines, and all of
them have shown antiproliferative effect in vitro, with significant cytostatic activity. Between
N, N-1,3-bis(1,2,3-triazole)-5-bromouracil hybrids p-fluoro(1), o-fluoro(3) and pchlorophenyl(
2) substituted 1,2,3-triazoles which showed a strong inhibitory effect on HepG2
and HeLa cell line growth. Inhibition of cell proliferation was observed in the presence of
micromolar concentration of tested compounds. The inhibition of the cell proliferation was even
stronger if compared with the inhibition of the same cells under the same conditions in the
presence of the reference substance of 5-fluorouracil. No mitochondrial toxicity was observed
for compounds tested. In addition to above mentioned methods, the PASS analysis was used to
make predictions of possible biological targets for tested compounds. The selection of the
biological target(s) was based on the structure of compounds and on known structure of
biological macromolecules from protein bases. PASS analysis predicted that strong antitumor
activity of compounds 1, 2, and 3 is due the inhibition of the Wee1 tyrosine kinase. Using the
Autodock Vina program and in silico screening, compounds 2 and 3 were docked to Wee1
kinase crystal structure downloaded from the protein database [PDB: 5v5y]. Molecular fit
analysis has shown that compounds 2 and 3 specifically bind to the pocket of ATP binding site
of Wee1 kinase, and that the binding domain of the tested compounds is the same as the binding
site of specific inhibitor of Wee1 kinase MK-1775 (IC50 = 5,2 nM). Cytostatic effects of
compounds 2 and 3 can be attributed to the inhibition of the G2-M control of the cell cycle,
which is a control point of Wee1 kinase. In addition, compounds 2, 3, and MK-1775 have shown
decreased phosphorylation of the isolated proteins by Western blot after the stimulation of
peripheral mononuclear cells. Considering that the cyclin-dependent kinase Cdk1 is one of the
major substrates for Wee1 kinase, molecular modeling and in silico docking has been
undertaken to better explain the influence of Wee1 kinase activity on phosphorylation of Cdk1
kinase
Y chromosome microdeletions in infertile men
Get PDFY kromosom svojom strukturom i genima koje sadržava, s naglaskom na gene iz područja AZF (faktora azoospermije) regije, ima ključnu ulogu u kontroli i provođenju normalnog procesa spermatogeneze. U slučaju delecija u navedenoj regiji dolazi do poremećaja u procesu spermatogeneze. Najteži poremećaj koji iz toga proizlazi je neplodnost muškarca. Ona se javlja u najviše zabilježenih slučajeva, iako postoje mnogi uzroci tog poremećaja. Također, postoje značajne varijacije u prikazu učestalosti mikrodelecija, kako u svjetskoj tako i u pojedinim populacijama. Razlog tome je različita veličina ispitivane populacije te razlike u genetičkoj pozadini i okolišnim faktorima kojima su bili izloženi pojedini sudionici istraživanja. Nakon utvrđivanja mikrodelecija u muškaraca, provode se dodatni testovi kako bi se utvrdilo može li se par usmjeriti na neku od mogućih metoda potpomognute oplodnje. Najčešće metode korištene pri liječenju takvih parova su ICSI (engl. intracytoplasmic sperm injection) te IVF (engl. in vitro fertilization). Kod tih metoda dolazi do direktnog uvođenja spermija u jajnu stanicu ili puštanja spermija da u in vitro okolišu sam prodre u jajnu stanicu i oplodi ju. Obje metode imaju velike prednosti jer omogućuju oplodnju i začetak trudnoće kod parova koji prije nisu imali tu priliku. Međutim, metode imaju i značajne nedostatke, koje uključuju 100% prijenos mikrodelecija s oca na sina. Zbog tih razloga, ovaj rad objedinjuje brojna istraživanja mikrodelecija Y kromosoma s ciljem poticanja promišljanja o poboljšavanju metoda medicinski pomognute oplodnje te potencijalnog ukazivanja na neke specifičnosti koje se u pojedinačnim radovima ne bi uvidjele.The Y chromosome plays a key role in controlling and conducting the normal process of spermatogenesis with its structure and the genes it contains, expecialy with the genes of the AZF (azoospermia factor) region. If the deletions appear in the mentioned region they lead to disorders in the process of spermatogenesis. The most severe disorder, resulting from this,
is the infertility of a man. Microdeletions are the most common cause of male infertility, althought there are many causes of this disorder. There are also significant variations in the frequency of microdeletions, both in the world and in specific populations. The reason for this is the different size of the examined population, the difference in the genetic background and the environmental factors that were exposed to each participant in the research. After determining microdeletions in males, additional tests are carried out to determine whether a couple can be directed to some of the possible assisted fertilization methods. The most commonly used methods in treating this type of couples are ICSI (intracytoplasmic sperm injection) and IVF (in vitro fertilization). In these methods direct injection of sperm into the oocite or the penetration of sperm into the oocite in the in vitro environment is preformed. Both methods have great advantages because they allow fertilization and the induction of pregnancy in couples who did not have the chance before to concive a child. However, the methods also have significant disadvantages, which include 100% microdeletion transfer from the father to the son. For these reasons, this paper combines numerous studies of the Y chromosome microdeletions with the goal of encouraging thoughts on improving the assisted fertilization methods and potentially pointing to some specificities that would not be seen in the individual papers
Synergistic potential of Juniperus communis and Helichrysum italicum essential oils against nontuberculous mycobacteria
Get PDFObjective. The present study evaluated the possible synergistic antimycobacterial interactions of Juniperus communis (J. communis) and Helichrysum italicum (H. italicum) essential oils (EO). Methods. Antimycobacterial potential was tested against Mycobacterium avium and Mycobacterium intracellulare using broth and water dilution method and checkerboard synergy method. Antiadhesion and antibiofilm effect of EOs was evaluated on biotic (HeLa cells) and abiotic surface (polystyrene). To evaluate the possible mechanisms of action, cellular leakage of proteins and DNA was tested and structural changes were visualized with a transmission electron microscope. Results. MIC, MBC and MEC were 1.6 mg/ml for J. communis EO and 3.2 mg/ml for H. italicum EO against both mycobacteria. All combinations of EOs in checkerboard synergy method produced fractional inhibitory concentration index values ranging from 0.501 to 1.5, corresponding to synergistic, additive or indifferent effects. Mycobacterium avium showed a greater tendency to create biofilm but these EO at subinhibitory concentrations (sMIC) effectively blocked the adhesion and the establishment of biofilm. The exposure of both mycobacteria to MICs and sMICs lead to significant morphological changes: acquired a swollen form, ghost-like cell, disorganized cytoplasm detached from the cell wall. OD value of supernatant for both mycobacteria exposed to EOs have confirmed that there is a leakage of cellular material. Conclusion. The leakage of the cellular material is noticeably higher in sMIC, which is probably due to cell wall damage. sMIC of both EOs have an additive or synergistic effect, reducing MICs, limiting adhesion and preventing the formation of biofilms
The Effects of Chlorpromazine on the Expression of the Tetraspanin Protein CD81 in Neural Cell Lines
Get PDFU ovom radu ispitan je utjecaj antipsihotika klorpromazina na izražaj proteina CD81 u humanim stanicama porijekla iz središnjeg živčanog sustava. Klorpromazin je neuroleptik koji djeluje kao snažan antagonist dopaminskih receptora, koji sudjeluju u procesima poput učenja, pamćenja, motivacije, užitka i dr. Povrh toga, klorpromazin utječe na mnoge druge membranske i citosolne proteine što rezultira smanjenom komunikacijom između neurona i glija stanica, prvenstveno astrocita. Međutim, dosad nije opisano utječe li klorpromazin i na specifičan oblik međustanične komunikacije koji uključuje izvanstanične vezikule (IV-e). IV-e su nanostrukture omeđene membranom koje sadrže proteine, nukleinske kiseline, lipide i druge molekule. Izlučuju ih sve vrste stanica i njima sekretorne stanice mogu utjecati na fenotip primateljskih stanica. CD81 je član obitelji tetraspanina, transmembranskih proteina izraženih u svim tkivima, pa tako i u stanicama središnjeg živčanog sustava. Osim na plazmatskim membranama, CD81 prisutan je i na citosolnim membranskim odjeljcima te je prepoznat kao marker izvanstaničnih vezikula. U živčanom je sustavu CD81 uključen u interakcije neurona i astrocita te u odgovor astrocita i mikroglija-stanica na ozljedu i bolest.
U ovom radu stanične linije humanog glioblastoma (U-87 MG) i neuroblastoma (SH-SY5Y) tretirane su rastućim koncentracijama klorpromazina (4, 8, 16 i 32 µg/ml) tijekom 24 sata. Analiza protočnom citometrijom pokazala je da klorpromazin ima oprečno djelovanje na razine CD81 u ispitanim staničnim linijama. U stanicama U-87 MG dolazi do dozno-ovisnog povećanja CD81, dok kod SH-SY5Y stanica razina ovog proteina opada s porastom koncentracije klorpromazina. Ova saznanja prvi put povezuju klorpromazin i promjene izražaja CD81. Imajući na umu brojne uloge CD81 u interakcijama neurona i astrocita te prisutnost tog proteina na izvanstaničnim vezikulama, buduća istraživanja trebala bi podrobnije ispitati posljedice ovih promjena na procese međustanične komunikacije u živčanom sustavu.This study examined the effects of chlorpromazine on the expression of CD81 in representative human cell lines of the central nervous system origin. Chlorpromazine is a neuroleptic that acts as a strong antagonist of dopamine receptors, which participate in processes like learning, memory, motivation, pleasure etc. Also, chlorpromazine affects many other membrane and cytosolic proteins, resulting in decreased communication between neurons and glial cells, especially astrocytes. Nevertheless, it has not yet been determined if chlorpromazine affects a specific form of intercellular communication that involves extracellular vesicles (EVs). EVs are membranous nanostructures that carry proteins, nucleic acids, lipids and other molecules. All types of cells release EVs and with them secretory cells can change the phenotype of recipient cells. The CD81 molecule is a member of the tetraspanin protein family ubiquitously expressed in all tissues, including the central nervous system. Next to the expression in plasma membrane, CD81 is present in intracellular membrane compartments and it is a recognized marker of EVs. In the nervous system, this protein is important for neuron-astrocyte interactions and is implicated in the response of astrocytes and microglia to trauma and disease.
In this study, the human glioblastoma cell line U-87 MG and neuroblastoma SH-SY5Y cells were treated with increasing concentrations of chlorpromazine (4, 8, 16 and 32 µg/ml) for 24 hours. Flow cytometry analysis of the CD81 expression has shown that chlorpromazine affects the examined cell lines in different ways. U-87 MG cells responded to chlorpromazine with a dose-dependent increase of CD81 protein level, while SH-SY5Y cells showed decreased CD81 levels. These findings are the first to date that associate chlorpromazine with changes in CD81 expression. Considering the role of this protein in neuron-astrocyte interactions and its presence in EVs, future studies are necessary to examine the effects of chlorpromazine treatment on processes important for intercellular communication in the nervous system
Preparation, characterization and potential application of supramolecular gels in controlled-release drug delivery systems
No full textJedan od najkorištenijih nesteroidnih protuupalnih lijekova (NSAID), Ibuprofen (Ib), uzrokuje učestale gastrointestinalne nuspojave prilikom liječenja kroničnih bolesti poput artritisa i osteoartritisa. Razvojem novih sustava za isporuku izbjegla bi se upotreba oralnih formulacija ovog lijeka te nuspojave vezane uz njegovu dugoročnu primjenu. U posljednje vrijeme veliku ulogu u dizajniranju novih sustava za kontrolirano otpuštanje lijekova imaju supramolekulski gelovi, posebno organogelovi.
Sintetiziran je tripeptidni organogelator Z-L-Phe-L-Phe-L-Ala-NH2 s ciljem ispitivanja njegovih gelirajućih svojstava, mogućnosti skladištenja i kontroliranog otpuštanja Ib. Za tekuću fazu organogelova odabrani su esteri zasićenih masnih kiselina i biljna ulja. Odabrana otapala za geliranje su biokompatibilna te imaju svojstvo pojačane permeacije kroz kožu što je poželjno za razvoj transdermalnih sustava s kontroliranom isporukom. Ib je ugrađen u različite organogelove te je praćeno njegovo otpuštanje u prihvatne medije – fosfatne pufere različitih pH vrijednosti. Koncentracija otpuštenog Ib izračunata je preko jednadžbe Beer-Lambertova zakona, pomoću apsorbancije koja je prethodno mjerena u kiveti ili UV-Vis optičkom sondom.
Rezultati istraživanja pokazaju da su organogelovi sa Z-L-Phe-L-Phe-L-Ala-NH2 i esterima zasićenih masnih kiselina te biljnim uljima dobri sustavi za skladištenje lipofilnog Ib. Otpušanje Ib značajno ovisi o pH vrijednosti prihvatnog medija, ali i o otapalu korištenom prilikom priprave organogela. Otpuštanje Ib je bolje u blago bazičnom mediju (pH 7.4) nego u kiselom (pH 6.0). Otpuštanje Ib je praćeno UV-Vis spektroskopskim metodama koje pokazuju jednake rezultate u istim uvjetima.One of the most used nonsteroidal-antiinflammatory drugs (NSAID), Ibuprofen (Ib), causes frequent gastrointestinal side effects when treating chronic conditions such as arthritis and osteoarthritis. The development of new delivery systems will avoid the use of the oral formulations of this drug and the side effects associated with its long-term use. Lately, supramolecular gels, especially organogels, have played a major role in the design of new controlled drug release systems.
The tripeptide organogelator Z-L-Phe-L-Phe-L-Ala-NH2 was synthesized in order to test its gelling properties, storage capacity and the controlled release of Ib. The saturated fatty acid esters and vegetable oils were selected for the liquid phase of the organogels. The selected gelling solvents are biocompatible and have the property of enhanced permeation through the skin, which is desirable for the development of transdermal controlled delivery systems. Ib was incorporated into various organogels and was monitored for release into accepting media - phosphate buffers of different pH values. The concentration of released Ib was calculated using the Beer-Lambert law equation, using absorbance previously measured in a cuvette or with UV-Vis optical probe.
The results of the study show that organogels with Z-L-Phe-L-Phe-L-Ala-NH2 and saturated fatty acid esters or vegetable oils are good storage systems for lipophilic Ib. The release of Ib depends significantly on the pH value of the receiving medium, but also on the solvent used in the preparation of the organogels. The release of Ib is better in basic medium (pH 7.4) than in acidic (pH 6.0). Ibuprofen release was followed by UV-Vis spectroscopic methods showing the same results under the same conditions
Chemical proteomics in drug discovery - drug target selectivity
No full textU suvremenom procesu otkrivanja lijekova jedan od ključnih koraka je razumijevanje mehanizma djelovanja potencijalnog lijeka te njegova učinka na proteinsku metu. Među najsuvremenijim pristupima identifikacije proteinske mete nekog potencijalnog lijeka je kemijska proteomika. U novije vrijeme ova se tehnika unaprijedila upotrebom fotoafinitetnog označavanja molekula pomoću 3-(trifluormetil)-3-fenildiazirinske skupine (TPD, engl. 3-(Trifluoromethyl)-3-phenyldiazirine). Ta skupina se pokazala kao najprikladnija za ovakvu vrstu označavanja budući da molekule s tom funkcionalnošću osvijetljene UV zrakama (~360 nm) stvaraju karbensku vrstu koja je reaktivni intermedijar i može stvoriti kovalentnu vezu s proteinskom metom s kojom je u bliskom kontaktu.
Da bi se testiralo ovu metodu identifikacije proteinske mete te usporedilo s drugim afinitetnim tehnikama koje se često koriste u proteomici, a ne stvaraju kovalentnu vezu, fotoafinitetno (uvođenjem 3-(trifluormetil)-3-fenildiazirinske funkcionalnosti) su obilježena dva poznata inhibitora receptora epidermalnog faktora rasta (EGFR, engl. epidermal growth factor receptor), lapatinib i erlotinib. Uvođenjem TPD funkcionalnosti važne za foto-aktivaciju i stvaranje karbena kao reaktivne vrste rađeno je na dijelu molekule za koji se molekulskim modeliranjem pretpostavilo da neće narušiti način vezivanja molekule za receptor. Dodatna modifikacija tzv. „dual tag“ uvedena je na molekulu lapatiniba kako bi se nastali kovalentno vezani kompleks molekula-protein mogao izolirati i analizirati direktno iz staničnog lizata. U slučaju erlotiniba, ostavljena je alkinska skupina originalno prisutna na molekuli te je ovakva modifikacija služila kao kontrolna molekula. Također, sintetizirane su i molekule pogodne za afinitetno vezivanje na čvrsti polimerni nosač, a koje sadržavaju amino ili alkinsku skupinu.
Sintetizirane molekule su testirane in vitro kako bi se utvrdila njihova učinkovitost u inhibiciji biološke mete (EGFR) te su zatim molekule podvrgnute interakciji sa staničnim lizatima s ili bez ozračivanja UV lampom kod 360 nm ovisno o primijenjenoj tehnologiji kemijske proteomike. Ovako „uhvaćeni“ proteini su izolirani afinitetnom kromatografijom iz staničnog lizata te identificirani masenom spektrometrijom.
U svim je eksperimentima potvrđeno da je ovim tehnikama moguće identificirati biološku metu budući da je „uhvaćen“ protein sa svim modificiranim molekulama bio upravo EGFR. Osim ovih očekivanih rezultata koji validiraju metodu, identificirano je nekoliko novih proteina za koje se vjeruje da mogu biti proteinske mete ovih lijekova. Za konačnu potvrdu da su ti proteini moguće proteinske mete za lapatinib i erlotinib, bilo bi nužno provesti dodatne eksperimente validacije.
Ovim radom dokazano je da je tehnologija kemijske protemike koristan alat za pronalaženje nepoznate proteinske mete za neku aktivnu supstancu, a osobito s fotoafinitetnim obilježavanjem molekula koje ima prednosti u odnosu na druge afinitetne tehnologije.In the modern drug discovery process, a key step is to understand the mechanism of action of potential drug and its effect on the biological target. The cutting edge aproach to identify biological target of a potential drug is chemical proteomics, especially photoaffinity labeling of potential drugs. 3-(Trifluoromethyl)-3-phenyldiazirine group (TPD) proved to be the most appropriate way for this kind of labeling, since molecules with that functionality when illuminated with UV light (~360 nm) create a carbene which is reactive intermediate and forms a covalent bond with a biological target with which it is in close contact. In order to test this type of finding of biological targets and compare it with other affinity techniques used in proteomics, that do not produce a covalent bond between protein targets and the examined molecule, photoaffinity group was introduced to two known inhibitors of the epidermal growth factor receptor (EGFR) – lapatinib and erlotinib. The introduction of TPD functionality that is important for photoactivation and the formation of the carbene as a reactive species is done on the part of the molecule for which molecular modeling established that will not disrupt the way of binding to the receptor. Additional modification, so-called dual tag, was introduced into the lapatinib to form a covalently bound protein-molecule complex that may be isolate and analyze directly from the cell lysate. In the case of erlotinib, alkyne functionality that was originally present on the molecule was preserved and erlotinib was used as a control molecule. In addition, molecules with amino and alkyne functionality suitable for affinity binding to a solid support were also synthesized. The synthesized molecules were tested in vitro to determine their effectiveness in inhibiting the biological target (EGFR) and were subjected to the interaction with the cell lysate with or without UV irradiation at 360 nm depending on the applied chemical proteomic technology. Bound proteins were isolated from cell lysates and identified by mass spectrometry. Experiments have confirmed that these techniques can identify the biological target since the "caught" protein with all the modified molecules was EGFR. In addition to these expected results that validate the method, several new proteins were identified that may be potential biological targets for these drugs. For the final confirmation that identified proteins are possible protein targets for lapatinib and erlotinib, additional validation experiments should be done. In this work was confirmed that chemical proteomics technology is a useful tool for identification and characterization of biological target of an active substance. Especially reliable tool is the photoaffinity labeling of the molecules using 3-(Trifluoromethyl)-3-phenyldiazirine group that compared to other affinity technologies has the advantage that the protein creates a covalent bond with its biological target and remains undamaged when UV light is used
Nanotechnology in neuroscience
Get PDFNanotehnologija je široko područje znanosti koje obuhvaća različite discipline, poput medicine, robotike ili farmacije. U posljednjih 20 godina primjena nanotehnologije i nanomaterijala u znanosti doživjela je eksponencijalan rast. Izuzetno važno područje primjene nanotehnologije je u neuroznanosti, a kritični osvrti na literaturu su veoma važni zbog toga što objedinjuju najnovije podatke iz tog područja.
Nanočestice su jedan od primjera primjene nanotehnologije kojim znanstvenici proučavaju i otkrivaju nove podatke o zbivanjima unutar mozga. Na temelju novih saznanja stvaraju se lijekovi kojima bi se u budućnosti mogle tretirati zasad neizlječive neurodegenerativne bolesti. Nadalje, u istraživanju ispostave lijekova, nanočestice se koriste kao prijevozno sredstvo lijeku kako bi ga dovele na točno određeno mjesto koje treba tretirati. Jedna od velikih prednosti koje nanotehnologija pruža istraživanjima u neuroznanosti jest mogućnost prolaska nanočestica kroz krvno-moždanu barijeru te razne vrste nanočestica koje bi mogle poslužiti kao prijenosnici lijekova. One mogu biti sačinjene od jedne vrste materijala ili biti kombinacija različitih materijala za što bolju prolaznost kroz krvno-moždanu barijeru. Također, nanočestice mogu poslužiti kao podloga za rast matičnih stanica ili mogu služiti kao prijenosnici za dostavu molekula do matičnih stanica i ciljano usmjeravanje njihove diferencijacije.
Jedno od novijih otkrića u neuroznanosti jest razumijevanje važnosti neuroinflamacije u nastanku i razvoju bolesti. Neuroinflamacija je tek nedavno postala predmetom istraživanja i to kada je dokazano da igra veliku ulogu u patogenezi neurodegenerativnih bolesti. Također, uključena je u nastup sekundarne ozljede nakon moždanog udara ili traumatske ozljede mozga te se zbog toga provode mnoga istraživanja u svrhu tretiranja kronične neuroinflamacije mozga. U tu svrhu se mogu koristiti obrađene nanočestice koje omogućuju prijenos aktivne supstance do ciljanog mjesta u mozgu.
Iako je nanotehnologija procvala u zadnjih 20 godina, još uvijek postoje ozbiljni nedostaci koji predstavljaju prepreku u njenoj primjeni u kliničke svrhe. Potrebna su dodatna istraživanja kako bi nanočestice postale sigurna i učinkovita metoda za prijenos lijekova do ciljanog mjesta. Naglasak bi se trebao staviti na in vivo istraživanja u zdravim („wild type“) životinjama koje imaju netaknutu krvno-moždanu barijeru.Nanotechnology is a wide area that encompasses various disciplines, such as medicine, robotics or pharmacy. In the last 20 years the use of nanotechnology and nanomaterials has exponentially grown. Extremely important area of nanotechnology application is in neuroscience and reviews such as this are important as they gather the latest data from this area.
Nanoparticles are one of the examples of nanotechnology application by which scientists study and discover new data about events in the brain. Based on these new findings, we can begin to make drugs that would treat so far incurable neurodegenerative diseases. Next, regarding the research of drug delivery, nanoparticles are used as the vehicles for the drug meant to deliver the drug to the exact place that needs to be treated. One of the main advantages of nanotechnology in neuroscience is the ability of nanoparticles to cross the blood-brain barrier and various species of nanoparticles that could serve as drug carriers. They can be made of a single material, or of a combination of several materials for an easier blood-brain barrier crossing. Also, nanoparticles can serve as a base for stem cell growth or they can act as a carrier to deliver molecules to the stem cells and aim their differentiation to a specific lineage.
One of the recent discoveries in neuroscience is the importance of neuroinflammation in the appearance and development of the disease. Neuroinflammation just recently became the topic of research because of the evidence suggesting its key role in pathogenesis of neurodegenerative diseases. It is also involved in the development of secondary injury after the stroke or a traumatic brain injury, which is why scientists are researching means of manipulating chronic neuroinflammation. Engineered nanoparticles can be used for that purpose for they serve as a transport of active substances to the target site in the brain.
Although nanotechnology has bloomed in the last 20 years, there are still serious disadvantages that slow down its clinical applications. More research is needed to make nanoparticles safe and effective method for transport of drugs to a target site. Main point should be in vivo research on healthy (“wild type”) animals that have an intact blood-brain barrier
Effect of carvacrol on Apis mellifera honeybees
No full textPosljednja desetljeća predstavljaju velike izazove za većinu pčelara diljem svijeta. Brojevi pčelinjih zajednica kontinuirano opadaju, a jedan od glavnih uzroka jest globalna zaraza grinjom Varroa destructor. U današnje vrijeme, pčelari još uvijek najčešće koriste sintetičke akaricide u borbi protiv suzbijanja zaraze. No sa sve većom pojavom rezistencije u grinja, ali i s rastom broja istraživanja koja ukazuju na potencijalno štetne učinke koje sintetički akaricidi mogu imati na pčele i kvalitetu pčelinjih proizvoda, budi se interes za pronalaženjem alternativnih metoda. Eterična ulja i njihovi proizvodi pokazuju veliki potencijal akaricidnog djelovanja, od kojih se monoterpenski fenol karvakrol još uvijek nije istraživao na ovakav način. U sklopu ovog rada proučili smo učinak karvakrola na preživljavanje pčela, na aktivnost enzima pčelinjeg živčanog sustava acetilkolinesterazu (AChE) i aktivnost detoksifikacijskog enzima glutation S-transferazu (GST). Obje smo aktivnosti enzima u reakciji sa supstratom mjerili kolorimetrijskim metodama. Naši rezultati ukazuju na to da karvakrol masenog udjela 0,01% ne uzrokuje smrtnost u pčela, ali izaziva negativan učinak očitovan povećanjem aktivnosti AChE i GST. Doza karvakrola od 0,001%, s druge strane, nije pokazala nikakve toksične učinke. Utjecaj karvakrola na razinu aktivnosti važan je jer AChE moguća molekularna meta karvakrola, a osim klasične kolinergičke funkcije ima utjecaj i na različite uloge poput učenja, pamćenja i podizanja potomstva. Aktivnost GST govori nam o indukciji detoksifikacijskih mehanizama jer je riječ o jednom od glavnih enzima uključenih u obranu od ksenobiotika. S obzirom na postojanje dva oblika AChE, membranske i u vodi topljive AChE, odvojili smo ih pomoću nativne poliakrilamidne gel elektroforeze i istražili potencijal te metode za mjerenje aktivnosti oba oblika. Bojanje dobivenih gelova u malatnom puferu za razvijanje u prisutnosti supstrata 1 mM acetiltiokolin klorida (AChCl) daje obećavajuće rezultate.Past decades have represented great challenges for most beekeepers around the world. The number of beekeeping communities is in decline, and the global Varroa destructor infestation is one of the major causes. Beekeepers are still most commonly using synthetic acaricides to combat contagion. But the increasing presence of mite resistance, as well as growing number of studies suggesting potentially harmful effects that synthetic acaricides may have on bees and on the quality of bee products, increases the interest in alternative methods. Certain essential oils and their substances have shown great acaricidal activity, one of which is the monoterpenic phenol carvacrol that has yet to be investigated in this way. We examined the effect carvacrol has on the bee survival, on enzyme involved in transmission of nervous signal (acetylcholinesterase; AChE) and on enzyme involved in detoxification (glutathione S-transferase; GST). Both enzyme activities were measured by means of colorimetric methods. Our results suggest that 0.01% carvacrol is a sublethal dose that does not affect bee mortality, but causes a negative effect manifested by increased AChE and GST activity. 0.001% carvacrol, on the other hand, didn’t show any adverse effects. The influence that carvacrol has on the enzyme activity level is important since AChE might represent a molecular target of carvacrol, and because, besides its classical cholinergic function, it also causes effects on different roles such as learning, memory and offspring raising. Induction of GST activity implies an induction of detoxification processes considering its one the major enzymes involved in xenobiotic defense. Considering the existence of two AChE forms, the membrane-bound and water-soluble, AChE was separated by native polyacrylamide gel electrophoresis and the potential of this method for measuring the activity of both AChE forms was investigated. Staining the gels in maleate developing buffer with 1 mM of acetylthiocholine chloride promises the best results
Development and validation of monoclonal antibodies and transfected cells to monitor biological activities of PrPC (prion protein cellular)
Get PDFStanični prionski protein (engl. Prion Protein Cellular, PrPC) je sastavni dio različitih tkiva pri čemu je njegova fiziološka uloga i dalje predmet debate. Unutar stanice se može pronaći u više formi - citoplazmatskoj, transmembranskoj te formi koja je putem glikozilfosfatidilinozitolnog sidra pričvršćena na membranu, pri čemu je posljednja forma najraširenija. Pojedine forme proteina utvrđene su u gotovo svim odjeljcima stanice, što uključuje plazmatsku membranu, endoplazmatski retikulum, Golgijev aparat, endosome, pojedine vezikule te citoplazmu. Dodatnu kompleksnost u izučavanju biologije ovog proteina unose glikoforme, odnosno različite šećerne modifikacije, kojih ima nekoliko. Monoklonska protutijela, koja uspješno detektiraju različite molekule PrPC unutar stanice te omogućavaju praćenje pojedinih specifičnih (gliko)formi i određivanje njihova interaktoma, nezamjenjiv su alat u istraživanju biološke uloge PrPC. Ovim radom se utvrđivala djelotvornost novo-proizvedenih monoklonskih protutijela, u formi supernatanta, na sposobnost prepoznavanja mišjeg i humanog PrPC u metodama Western blot/imunoblot, imunofluorescentnoj mikroskopiji te protočnoj citometriji. Svi pokusi su rađeni na staničnim linijama mišjeg glioma GL261, mišjih embrionalnih fibroblasta MEF, mišjeg limfoma BW i transfektanti BW-PrP te na humanoj staničnoj liniji adenokarcinoma alveolarnih bazalnih epitelnih stanica A549, kao prikladnim staničnim modelima za ispitivanje PrPC i validacije monoklonskih protutijela na sposobnost prepoznavanja PrPC unutar stanice. Protutijela HuPrP.01, HuPrP.02 te HuPrP.04 su pokazala najveći potencijal u prepoznavanju humanog PrPC dok se u prepoznavanju mišjeg PrPC najboljim kandidatom pokazalo protutijelo MoPrP.03. Ova protutijela imaju potencijal za daljnji razvoj, koji će započeti proizvodnjom koncentriranih pripravaka poznate koncentracije protutijela putem pročišćavanja ispitanih supernatanata. Zanimljivo je kako je jedno od protutijela na humani PrPC, protutijelo 1G8, detektiralo PrPC putem imunofluorescentne mikroskopije u području membrane jezgre, gdje isti protein još do sada nije opisan. Zaključno, na temelju ukupnih rezultata prikupljenih različitim metodama, utvrdili smo koja novo-proizvedena protutijela vrijedi dalje razvijati, pročišćavati i validirati te koristiti u budućim istraživanjima bioloških uloga PrPC.PrPC is an integral part of different tissues, and its physiological role remains the subject of debate. Within the cell, this protein can be found in several forms, including a cytoplasmic form and a transmembrane form. However, the most widespread form is one that is attached to the membrane through a glycosylphosphatidylinositol anchor. Certain protein forms have been found in almost all cell compartments including the plasma membrane, endoplasmic reticulum, Golgi apparatus, endosomes and other vesicles, as well as the cytoplasm. An additional level of complexity in the study of the biology of this protein is its glycoprofile, or different sugar modifications, of which there are several. Monoclonal antibodies that successfully detect different PrPC molecules within cells also enable the monitoring of specific forms of PrPC (glyco)forms and determination of the PrPC interactome. The present study determined the efficacy of newly-produced monoclonal antibodies to recognize mouse and human PrPC in Western blotting/immunoblotting, immunofluorescence microscopy, and flow cytometry. Antibodies HuPrP.01, HuPrP.02 and HuPrP.04 showed the greatest potential for recognition of human PrPC, while the antibody MoPrP.03 showed best capability in recognition of mouse PrPC. These antibodies have the potential for further development, which will start by the production of concentrated preparations of known antibody concentrations by purifying the supernatants that were tested. Interestingly, one of the anti-HuPrP antibodies, 1G8, detected immunofluorescence signal around the nucleus membrane, where the PrPC localization has not been previously described. Based on the obtained results, we have determined which newly-produced antibodies are candidates for further development, purification and validation, and can be used in future research of the biological role of PrPC