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Schiff bases - investigation of complexes with transition metal ions of 3d group
Get PDFReakcijom para-nitrofenilhidrazina s para metoksiacetofenonom, odnosno s para nitrobenzaldehidom u razrijeđenoj octenoj kiselini, u molarnom omjeru 1 : 1, priređeni su novi derivati hidrazina, C13H10N4O4 i C15H15N3O3. Reakcijom kondenzacije para nitrobenzaldehida i para nitroanilina u alkoholnom mediju u molarnom omjeru 1 : 1 pripravljena je Schiffova baza, C13H9N3O4. Struktura pripravljenih spojeva okarakterizirana je 1D i 2D tehnikama spektroskopije NMR u DMSO-d6. Metodom kontinuirane varijacije istražen je stehiometrijski omjer vezanja cinkova(II) i niklova(II) nitrata heksahidrata sa sintetiziranim ligandima u otopini DMSO-d6. Priprava istih kompleksa provedena je i mehanokemijskom reakcijom bez otapala, u omjeru metal : ligand = 1 : 1. Kompleksi su okarakterizirani 1H NMR spektroskopijom.Novel hydrazine derivatives, C13H10N4O4 and C15H15N3O3, were synthesized by diluting acetic acid and para-nitrophenylhydrazine with para methoxyacetophenone and para nitrobenzaldehyde with a molar ratio of 1:1. The Schiff base, C13H9N3O4, was synthesized by the condensation of para nitrobenzaldehyde with para nitroaniline in alcohol medium in a molar ratio of 1:1. The molecules were characterized by one- and twodimensional NMR spectroscopy. Using the continuous variation method (Job plot) the stoichiometric binding ratio of zinc(II) and nickel(II) nitrate hexahydrate with the synthesized ligands were investigated in DMSO-d6 solutions. The synthesis of the same complexes was also carried out by mechanochemical reaction without solvent, in a molar ratio metal : ligand = 1:1. The synthesized compounds were characterized by 1H NMR spectroscopy
MULTIFUNKCIONALNO MOLEKULSKO PREPOZNAVANJE SEKUNDARNE STRUKTURE DNA/RNA MOLEKULSKIM SENZORIMA
No full textBy simple synthetic procedures were prepared various novel conjugates of cyanine
dyes linked to amino acid side chains (Cy-aa), designed for easy incorporation into
any position within peptide backbone. The proof of principle was confirmed by the
first peptide-bond coupling with pyrene-fluorophore, to give peptidic cyanine-pyrene
(Cy-aa-Pyr) conjugates.
In general, all new conjugates revealed micromolar affinities toward ds-DNA or
ds-RNA, accompanied by moderate thermal stabilization of polynucleotide double
helix. However, conjugates showed recognition of various DNA or RNA secondary
structures by two highly sensitive methods, fluorescence and CD spectropolarimetry.
Detailed analysis of all results revealed several most important structural variations,
which can be used for the fine tuning of DNA/RNA selectivity. The length of a linker
between cyanine and amino acid is proportional to aggregation ability of a cyanine
dye within polynucleotide binding site, at variance to addition of fluorine or chlorine
on cyanine core, which hampered aggregation. Furthermore, fluorine or chlorine on
cyanine switched the selectivity of fluorescence response in favour of GC-DNA, in
respect to non-halogenated conjugates being fluorescently selective toward AU-RNA.
Change of cyanine, from thiazole orange (TO) into oxazole yellow (YO), induced an
Abstract Doctoral dissertation
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inversion of the induced CD sign only for GC-DNA, as a consequence of different aggregation pattern of Cy-aa within the DNA binding site. Two newly prepared cyanine-pyrene (Cy-aa-Pyr) conjugates proved the concept of linking two FRET-paired chromophores. The FRET-system was fluorescently silent in a free solution but selectively switched-on upon binding to the target (DNA, RNA, protein). Namely, the efficiency of FRET was dependent on DNA or RNA secondary structure, as well as on the structure of Cy-aa-Pyr conjugate. Moreover, FRET was only observed for protein (BSA) binding of one conjugate linked over quinoline moiety of cyanine, while its Cy-aa-Pyr analogue linked over benzothiazole remained FRET silent. Both Cy-aa-Pyr revealed strong fluorescence changes of pyrene for protein (BSA) binding and strong emission of cyanine only for DNA/RNA binding, thus behaving as double-headed fluoro-probes. Some of new conjugates were applicable also in in vitro staining experiments due to their low cytotoxicity and showed specific staining of mitochondria within living cells.Rad ne sadrži sažetak na drugom jeziku
The effect of Sirtuin 3 on rotenon-induced oxidative stress in MCF-7 breast cancer cells
Get PDFVisoke razine reaktivnih kisikovih vrsta (ROS) u stanici izazivaju oksidativni stres koji može dovesti do genomskog oštećenja i nestabilnosti te nekontrolirane proliferacije ili smrti stanica. Stanična linija raka dojke MCF-7 okarakterizirana je slabom genskom ekspresijom te izostankom proteinske ekspresije Sirtuina 3 (Sirt3), mitohondrijske NAD+ ovisne deacetilaze koja kontrolira brojne biološke aktivnosti, a između ostalog i štiti od ROS-a. Stoga se u ovom radu istraživala uloga Sirt3 u MCF-7 stanicama tretiranima rotenonom, inhibitorom mitohondrijskog lanca transporta elektrona i supstancom koja potiče oksidativni stres. Za tu svrhu korištene su transfecirane MCF-7 stanice koje nemaju ili pak imaju stabilnu ekspresiju proteina Sirt3, a istražen je njegov utjecaj na stvaranje ROS-a, starenje, metabolizam, ekspresiju određenih proteina te je istražena uloga u rotenonom-izazvanom oštećenju proteina, lipida i DNA. Za analizu navedenih parametara korištene su sljedeće metode: fluorescencijska analiza količine ROS-a, analiza senescencije, MTT test, Western blot, te metoda lipidne peroksidacije i karbonilacije proteina. Dobiveni rezultati su jasno pokazali da rotenon u MCF-7 stanicama izaziva oksidativni stres na svim analiziranim razinama, te da je Sirt3 stanice zaštitio od oksidativnog stresa na način da je smanjio razinu ROS-a, senescenciju i oštećenje lipida u odnosu na kontrolne stanice. Ipak, Sirt3 nije djelovao zaštitno na proteine i DNA, niti je u značajnoj mjeri spriječio staničnu smrt, što implicira da je aktivirao staničnu zaštitu, ali ne u dovoljnoj mjeri. Nadalje, stanice sa Sirt3 imaju veće oštećenja DNA i lipida što ukazuje da Sirt3 sam ili u kombinaciji s rotenonskim tretmanom ne održava niti poboljšava tumorske karakteristike MCF-7 stanica. Štoviše, s obzirom na faktore u kojima stanice sa ekspresijom Sirt3 pokazuju veću osjetljivost na stres izazvan rotenonom, ovi rezultati upućuju da bi Sirt3 mogao imati ulogu inhibitora rasta tumora, odnosno tumor-supresorsku ulogu u MCF-7 stanicama.In the cell, high levels of reactive oxygen species (ROS) cause oxidative stress that can lead to genomic damage and instability and uncontrolled proliferation or cell death. MCF-7 breast cancer cell line is characterized by low gene expression and lack of protein expression of Sirt3, a mitochondrial NAD+ dependent deacetylase that controls many biological activities and, among other things, protects against ROS. Therefore, the role of Sirt3 in MCF-7 cells treated with rotenone, an inhibitor of the mitochondrial electron transport chain, and a causative agent of oxidative stress, was investigated in this thesis. For this purpose, transfected MCF-7 cells with or without stable expression of Sirt3 protein were used, and its effect on ROS generation, aging, metabolism, expression of certain proteins, and role in rotenone-induced damage to proteins, lipids and DNA was explored. The following methods were used to analyze these parameters: fluorescence analysis of ROS with MitosoxRed dye, senescence analysis with SA-β-gal staining, MTT assay, Western blot, and determination of lipid peroxidation and protein carbonylation. The results clearly showed that rotenone in MCF-7 cells induces oxidative stress at all analyzed levels, and that in several cell parameters, Sirt3 protected against oxidative stress (decreased ROS levels, senescence, and lipid damage relative to control cells). However, Sirt3 failed to protect proteins and DNA from damage, and to prevent the cell death, implying that it activated several protective cellular systems, but not sufficiently to fully protect cells. Furthermore, cells with overexpressed Sirt3 showed more DNA and lipid damage, which implies that Sirt3 alone or in combination with rotenone treatment does not maintain or improve the tumor characteristics of the MCF-7 cells. Moreover, given the factors in which Sirt3-overexpressing cells show greater sensitivity towards stress, the possibility to consider Sirt3 as a tumor suppressor in MCF-7 cells is not excluded
Transition metal complexes as synthetic proteases: study of the regioselectivity of peptide bond cleavage by complex compounds of Pt (II) and Pd (II)
No full textSelektivno presijecanje peptidne okosnice jedna je od najvećih prepreka prilikom analize proteina. Peptidna veza je stabilizirana rezonantnom strukturom i kao takva izuzetno nepodložna hidrolizi. Dostupni reagensi za cijepanje često djeluju neselektivno, neki su i otrovni te zahtijevaju visok utrošak i ograničene uvjete reakcije. Ovi razlozi potiču razvoj novih i učinkovitijih proteaza.
Hidroliza peptidne veze potpomognuta metalnim ionima je obećavajuća alternativa enzimskom cijepanju proteina s perspektivnim primjenama u biokemiji i bioinženjerstvu. Mnogi ioni i kompleksi metala testirani su na takvu reaktivnost na većem broju supstrata, od dipeptida i oligopeptida do proteina. Među takvim, visoku učinkovitost pokazali su prijelazni metali.
U ovom radu ispitana je regioselektivnost presijecanja peptidne veze u sekvenciji goveđeg hemoglobina i humanog monoklonskog antitijela IgG1 različitim kompleksima platine(II) i paladija(II) kako bi odredili na koji način ovi metalni ioni i sterički faktori usmjeravaju proteolizu.
Selektivna hidroliza peptidne veze postignuta je tijekom 12-satne inkubacije, pri pH 2.5 i temperaturi 40 ᵒC te primjenom kompleksa u suvišku 10:1 spram mjesta vezanja na supstratu. Nastali peptidni fragmenti određeni su tandemskom spektrometrijom masa na temelju specifičnog fragmentacijskog uzorka prekursorskog iona. Pritom je kompleks platine(II) s bidentatno koordiniranim etilendiaminom pokazao veću selektivnost naspram analoga paladija(II). Za spoj [Pt(en)(H2O)2]2+ identificirano je 17 fragmenata u sekvenciji hemoglobina, odnosno 10 fragmenata za IgG1. U slučaju kompleksa [Pd(en)(H2O)2]2+, određen je slijed 34 peptida u sekvenciji hemoglobina te 11 peptida u sekvenciji IgG1.
Mjesta presijecanja, utvrđena su analizom aminokiselina u neposrednoj blizini, a to su: l-metionin, l-cistein te l-histidin. U manjoj mjeri hidroliza goveđeg hemoglobina dogodila se u blizini l-asparaginske kiseline (hemoglobin) te nepolarnih l-glicina i l-triptofana (IgG1). Kada je ispitivanje provedeno sa složenim, dinuklearnim spojevima tipa [{M(en)(H2O)}2(-L)]4+, premoštenim aromatskim pirazinom i piridazinom, učinkovitost i prinos hidrolize bili su gotovo jednaki. Naši rezultati ukazuju na mogućnost primjene spojeva platine(II) i paladija(II) za proteolizu, prikazuju uvjete potrebne za reakciju te selektivnost proteolize kao temelj primjene ovih spojeva u biokemijskoj praksi.Selective cleavage of the peptide backbone is one of the major obstacles in protein analysis. Peptide bond is stabilized by a resonant structure, and therefore extremely insusceptible to hydrolysis. Accessible cleavage reagents are often nonselective, some are toxic, require high fold molar excess and harsh conditions. Aforementioned reasons prompted the development of new, more efficient chemical proteases.
Metal ion-assisted peptide bond hydrolysis is a favourable alternative for enzymatic cleavage of proteins with promising applications in biochemistry and bioengineering. Many metal ions and their complexes have been tested for such reactivity on a number of substrates, from dipeptides and oligopeptides to proteins. Among such, high efficiency was demonstrated by transition metal ions.
In this study, we investigated the regioselectivity of peptide bond cleavage by various platinum(II) and palladium(II) complexes to determine how these metal ions, and steric factors direct proteolysis of bovine hemoglobin and human monoclonal antibody IgG1.
Selective hydrolysis was accomplished by applying 10-fold molar excess of reagents, during 12-hour incubation, at pH 2.5 and 40 ᵒC. Product fragments were determined, based on the specific fragmentation pattern of their precursor ion, by tandem mass spectrometry. Thereat, platinum(II) complex with bidentate-coordinated ethylenediamine showed greater selectivity than the palladium(II) analog. For [Pt(en)(H2O)2]2+ 17 fragments were identified after cleaving hemoglobin sequence, apropos to 10 fragments for IgG1. In respect to [Pd(en)(H2O)2]2+, we assigned 34 product fragments to hemoglobin polypeptide chain, and 11 fragments to the sequence of IgG1.
Regioselectivity of cleavage, determined by amino acids in the immediate vicinity, included side chains of l-methionine, l-cysteine and l-histidine. To a lesser extent, hydrolysis of bovine hemoglobin occurred near l-aspartic
acid (hemoglobin) and non-polar l-glycine and l-tryptophan (IgG1). When we examined efficiency of more complex, dinuclear compounds bridged with aromatic pyrazine or pyridazine, here shown as [{M(en)(H2O)}2(-L)]4+, the efficacy and yield of hydrolysis were almost equal. Our results indicate the possibility of using platinum(II) and palladium(II) compounds for proteolysis, show reaction conditions in which hydrolysis occurs, and demonstrate selectivity of proteolysis as the basis for the application of these compounds in biochemical practice
Definiranje regije kritične za agregaciju proteina TRIOBP-1 povezanog sa shizofrenijom
No full textSchizophrenia, major depressive disorder and bipolar depressive disorder are categorized as chronic mental illnesses due to their lifelong and recurring nature. The complexity of their biological and environmental background has made it difficult to determine the causes of such disorders as well as developing successful treatment therapies. The recent discovery of several proteins found aggregating in brain samples taken from patients suffering from chronic mental illness indicates that disrupted proteostasis may be one of the mechanisms contributing to the development of these disorders.
Trio and F-actin-binding protein 1 (TRIOBP-1) was one of the proteins found aggregating in brain tissue of schizophrenia patients. Further study of this protein suggested that its aggregation propensity relies on a 25-amino acids long “linker region” positioned in the central domain (amino acids, aa 324-348), in between the first two coiled-coils. Here, we used plasmid constructs containing different lengths of linker region of TRIOBP-1 and over-expressed them in mammalian cells. We confirmed that the proposed linker region is responsible for the aggregation of TRIOBP-1 and refined it to an even shorter sequence. We narrowed it down to an 8-amino acid long sequence (aa 333-340), however, the following amino acids (aa 341-348) also retained some of the TRIOBP-1’s aggregation propensity. We propose that this refined aggregating region should be the focus of future research in understanding the molecular mechanisms of TRIOBP-1’s disrupted proteostasis, giving us insight into the pathology of chronic mental illness.Shizofrenija, klinička depresija i bipolarni poremećaj kategorizirani su kao kronične mentalne bolesti zbog njihove cjeloživotne i ponavljajuće naravi. Složenost njihove biološke i okolišne pozadine otežala je utvrđivanje uzroka takvih poremećaja kao i razvoj uspješnih terapija liječenja. Nedavno otkriće nekoliko agregirajućih proteina u uzorcima mozga uzetih od pacijenata koji pate od kroničnih mentalnih bolesti ukazuju na to da poremećena proteostaza može biti jedan od mehanizama koja doprinosi razvoju tih poremećaja.
Trio i F-aktin-vezujući protein 1 (TRIOBP-1) jedan je od agregirajućih proteina pronađenih u moždanom tkivu pacijenata sa shizofrenijom. Daljnje proučavanje ovog proteina sugeriralo je da se njegova sklonost agregaciji oslanja na "povezujuću regiju" sačinjenu od 25 aminokiselina koja se nalazi u središnjoj domeni (aminokiseline, ak 324-348), između prva dva namotaja sastavljena od α-heliksa. U ovome radu smo koristili konstrukte plazmida koji su sadržavali dijelove povezujuće regije TRIOBP-1 proteina i eksprimirali ih u stanicama sisavaca. Potvrdili smo da je predložena povezujuća regija odgovorna za agregaciju TRIOBP-1 te smo je dalje definirali na kraći slijed. Utvrdili smo niz od 8 aminokiselina (ak 333-340) važnih za agregaciju, no i aminokiseline koje slijede (ak 341-348) također zadržavaju dio agregirajućih sklonosti TRIOBP-1. Predlažemo da ova definirana agregirajuća regija bude fokus budućih istraživanja u razumijevanju molekularnih mehanizama poremećene proteostaze TRIOBP-1 proteina, s ciljem dobivanja uvida u patologiju kroničnih mentalnih bolesti
Dokazi povezanosti šizofrenije i poremećaja izazvanih glutenskom prehranom
No full textGluten-related disorders affect all functional systems of the human body.
So far more than twenty-five diseases and syndromes have been linked to
gluten-related nutrition, including mental illnesses such as depression and
anxiety. The main aim of this thesis was a review of literature linking
schizophrenic symptoms, including schizophrenia itself, to a spectrum of
gluten-related disorders. Because of the rarity of the target diseases, the
main approach to linking the two are clinical cases and biological studies,
through population-wide studies is not available.
The human body is not capable of fully digesting gluten. In the process of
partial break-down of gluten, gliadin exorphins are made. Individuals
suffering from gluten-related disorders, if not on gluten-free diet, have
increased permeability of the intestinal wall. This syndrome is also called
Leaky gut syndrome. Increased permeability is caused by the protein
zonulin whose levels increase after gluten ingestion in individuals suffering
from gluten-related disorders. In healthy individuals this autoimmune
reaction goes unnoticed or is not present. Symptoms following the reaction
are chronic, and last, as long the gluten is present in the body what can be
from couple of days to a couple of years. Most common symptoms are
malnutrition, low energy levels, fatigue, bloating, stomach pains.
This literature review focuses on gluten-related disorders with non-specific
neurological symptoms such as depressive symptoms, hallucinations,
apathy, lack of concentration, “foggy mind”, headaches, irritability, sleep
abnormalities or behavioural alternations. A possible explanation behind
those is the theory of gliadin exorphins, toxic peptides, that by passing
through the body from the gastrointestinal tract to the brain are causing
non-specific neurological symptoms. Even though there are some possible
mechanisms explaining this neurological manifestation of gluten-related
disorders, more research is needed to form definite conclusions.Poremećaji izazvani glutenskom prehranom utječu na sve funkcionalne
sustave ljudskog tijela. Do sada je prepoznato više od dvadeset i pet bolesti
i sindroma koji su povezani s glutenskom prehranom, uključujući mentalne
bolesti poput depresije i anksioznosti. Glavni je cilj ovog rada pregled
literature koja prikazuje simptome šizofrenije i povezuje šizofreniju sa
spektrom poremećaja izazvanih glutenskom prehranom. Zbog rijetkosti
ovih bolesti, osnovne su metode dokazivanje njihove povezanosti klinički
slučajevi i laboratorijska istraživanja, dok populacijske studije nisu
dostupne.
Ljudsko tijelo nije sposobno u potpunosti probaviti gluten. U procesu
djelomične razgradnje glutena stvaraju se gliadinski egzorfini. Ako
pojedinci koji pate od poremećaja izazvanih glutenskom prehranom nisu na
dijeti bez glutena, oni imaju povećanu propusnost crijevne stijenke. Ovaj
se sindrom naziva i Sindromom propusnog crijeva. Povećana propusnost
uzrokovana je proteinom zonulinom, čija se razina povećava nakon unosa
glutena kod osoba koje pate od poremećaja izazvanih glutenskom
prehranom. Kod zdravih pojedinaca ova autoimuna reakcija prolazi
nezapaženo ili je nema. Simptomi koji se javljaju nakon reakcije kronični
su i traju toliko dugo koliko je gluten prisutan u tijelu – od nekoliko dana
do nekoliko godina. Najčešći simptomi su pothranjenost, niska razina
energije, umor, natečenost, bolovi u želucu.
Ovaj pregled literature usredotočuje se na poremećaje izazvane
glutenskom prehranom s nespecifičnim neurološkim simptomima kao što
su depresivni simptomi, halucinacije, apatija, nedostatak koncentracije,
„maglovit um”, glavobolja, razdražljivost, poremećaji spavanja ili promjene
ponašanja. Mogući su uzročnici gliadinski egzorfini, toksični peptidi koji
prolaskom kroz tijelo iz gastrointestinalnog trakta u mozak uzrokuju
nespecifične neurološke simptome.
Iako postoje neki mogući mehanizmi koji objašnjavaju ovu neurološku
manifestaciju poremećaja izazvanih glutenskom prehranom, za donošenje
čvrstih zaključaka potrebno je više istraživanja
Neuroinflamacija u amiotrofičnoj lateralnoj sklerozi (ALS) - genetski dokazi i terapije
No full textALS is a progressive neurodegenerative disease. It is characterized by motoneuron degradation which leads to muscle atrophy and death due to respiratory failure. The only drugs approved for ALS treatment are riluzole and edaravone, a benzothiazole modulating glutamate uptake and an antioxidant, respectively. Research conducted in the last decades identified neuroinflammation as one of the main ALS hallmarks. It is characterized by microglial activation, infiltration of T cells and astrocytosis. T cells are present in negligible numbers in the healthy central nervous system (CNS) but cross the blood-brain-barrier at the site of injury and are thought to activate microglia and promote motor neuron damage. Microglial and astroglial activation furthers this process. Mutations found in genes encoding for SOD1 (superoxide dismutase), FUS (RNA-binding protein FUS), TDP-43 (TAR DNA-binding protein 43) and C9orf72 (chromosome 9, open-reading frame 72) have been found to indirectly or directly modulate the immune system. Neuroinflammation is thought to be an aftermath of disrupted autophagy and/or hyperactivation of the NF-κB pathway, which in turn result in an increased release of pro-inflammatory cytokines from microglia. All these findings open a possibility that immunomodulatory therapies could be a promising approach in ALS. Therefore, some of the therapies focus on immune response manipulation as a means of decreasing the inflammation and blocking the disease progression. This thesis will present major genetic mutations found in ALS patients and explain their connection with the immune system, supporting the hypothesis that immunomodulation is a promising therapeutic approach.Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest. Karakterizirana je degeneracijom motornih neurona koja vodi do atrofije mišića i smrti uslijed respiratornog zastoja. Jedini lijekovi odobreni za tretman ALS-a su riluzole, benzotiazol koji modulira unos glutamata, te antioksidans edaravone. Istraživanja provedena u posljednjim desetljećima su identificirala neuroinflamaciju kao jednu od glavnih odrednica ALS-a. Karakterizirana je aktivacijom mikroglija, infiltracijom T stanica i astrocita. U zdravom središnjem živčanom sustavu prisutan je vrlo mali broj T stanica, ali prelaze krvno-moždanu barijeru na mjestima ozlijede i smatra se da doprinose oštećenju neurona i aktivaciji mikroglije. Aktivacija mikroglija i astrocita dodatno pospješuje ovaj proces. Mutacije pronađene u genima koji kodiraju za SOD1 (superoksid dismutazu), FUN (RNA-vezujući protein FUS), TARDBP (TAR DNA-vezujući protein 43, TDP-43) i C9orf72 (kromosom 9, otvoreni okvir čitanja 72) su direktno ili indirektno povezane s modulacijom imunog sustava. Mehanizmi koji dovode do neuroinflamacije uključuju poremećenu autofagiju i pretjeranu aktivaciju NF-κB signalnog puta, koji dovode to pretjeranog lučenja pro-upalnih citokina. Sva ova otkrića otvaraju mogućnost da bi imunomodulacijske terapije bile dobar pristup liječenju ALS-a. Upravo zbog toga, neke terapije se fokusiraju na manipulaciju imunim sustavom kako bi smanjile upalu i spriječile progresiju bolesti. Ovaj završni rad će predstaviti genetičke mutacije pronađene u ALS pacijentima i objasniti njihovu vezu s imunom sustavom, podupirući hipotezu da je imunomodulacija obećavajući terapijski pristup
Važnost DISC1 proteina u procesu neurorazvoja
No full textDisrupted In Schizophrenia 1 (DISC1) je važan protein uključen u razne biološke puteve koji definiraju proces neurorazvoja. Početno je otkriven kao disfunkcionalni gen posljedično kromosomskoj translokaciji koja je poistovjećena sa mentalnim bolestima, te su mnoge biološke studije pokazale DISC1 protein kao visoko-profilni rizični faktor za razvoj shizofrenije i srodnih poremećaja. DISC1 je pronađen u jezgri, mitohondrijima, kao i u citoskeletnim strukturama poput centrosoma. Njegov pleiotropni učinak izražen je u mnogim funkcijama, substaničnim lokacijama te u širokom rasponu proteina s kojima je u interakciji, a od kojih većina također sudjeluje u signalnim putovima i procesima koji utječu na više neurorazvojne funkcije kao što su migracija neurona, proliferacija neuronskih stanica i odrasla neurogeneza. Najvažniji proteini uključeni u ove procese, a koji su u interakciji s DISC1 su NDEL1, NDE1, LIS1, PCM1 i GSK3β. Ta činjenica podupire neurorazvojnu hipotezu nastanka psihopatologije takvih poremećaja, predlažući da naslijeđeni ili rano dobiveni moždani defekt dolazi do izražaja tek u adolescentnoj dobi. Takva zajednička sagledavanja sugeriraju da shizofrenija i srodne bolesti mogu predstavljati krajnju točku više različitih patogenih putova.The Disrupted In Schizophrenia 1 (DISC1) is an important protein involved in many biological pathways that define the process of neurodevelopment. Having originally been identified as a gene disrupted by a chromosomal translocation that segregated with major mental illness, many biological studies have since indicated DISC1 as one of the highest profile risk factors for schizophrenia and other related illnesses. DISC1 is found at the nucleus, the mitochondria and cytoskeleton-associated structures, as the centrosome. It displays a pleiotropic character with many suggested functions, subcellular locations and a broad range of interacting partners, majority of which take part in signaling pathways and processes that affect higher neurodevelopmental brain functions as well, such as neuronal migration, neuronal cell proliferation or adult neurogenesis. Major DISC1 binding partners involved in these processes include nuclear distribution protein nude-like 1 (NDEL1), nuclear distribution protein nude homolog 1 (NDE1), lissencephaly protein 1 (LIS1), pericentriolar material 1 (PCM1) and glycogen synthase kinase 3β (GSK3β). This supports a neurodevelopmental hypothesis to underlie the psychopathology of these disorders, proposing that a brain defect is inherited or sustained early in life, but is not fully expressed until adolescence. Together these findings suggest that schizophrenia and related illnesses may represent the end point of multiple different pathogenic pathways
Quality of minced meat from Croatian market
No full textCilj ovog rada bio je ispitati kvalitetu mljevenog mesa dostupnog na hrvatskom tržištu, uključujući svinjsko i goveđe mljeveno meso te ocijeniti sukladnost s obzirom na propisane zahtjeve za mljeveno meso definirane Uredbom EU br. 1169/2011. Korišteni su komercijalno dostupni uzorci, podijeljeni u 3 kategorije ovisno o vrsti mesa od kojeg se mljeveno meso sastoji. Za potrebe istraživanja, analizirano je ukupno 42 uzorka mljevenog mesa od toga: 12 uzoraka u kategoriji goveđeg mljevenog mesa, 13 u kategoriji svinjskog mljevenog mesa, a 17 uzoraka miješanog mljevenog mesa (svinjsko i goveđe u omjeru 60:40). Parametri kvalitete mljevenog mesa ispitani su primjenom standardnih akreditiranih metoda: udio kolagena (spektrofotometrijska metoda HRN ISO 3496:1999), udio bjelančevina (titracijska metoda po Kjeldahl-u HRN ISO 937:1999) i udio masti (gravimetrijska metoda po Soxhlet-u HRN ISO 1443:1999).
Analizom dobivenih rezultata utvrđeno je da devet uzoraka ne udovoljava zahtjevima propisanim u Pravilniku. Radi se o uzorcima čiji je omjer kolagena i bjelančevina te udio ukupne masti prelazio graničnu vrijednost. Sljedeći rezultate analize moglo bi se zaključiti da mesna industrija u manjem postotku zavarava potrošače u vidu vjerodostojnosti
informacija koje su istaknute na deklaracijama mesnih proizvoda. Mljeveno meso koje u svom sastavu sadrži meso slabije kvalitete je upravo meso bogato vezivnim tkivom i količinom masti iznad dozvoljene granice što je u radu i utvrđeno analizama količine hidroksiprolina i ukupne masti.Food quality is a fundamental aspect of food that is affected by consumer demands and depends on the quality of the raw materials, the choice of technological processing process and the quality of the packaging material for the final product. Consciousness of food production as well as its quality is nowadays diminished, so the food industry in many ways tries to fool the consumer to maximise profit. The most common form of consumer malpractice is selling a product under a false name - counterfeit so it is the aim of such an act to conceal the poor quality of the final product, which means that the authenticity of the food in view of the credibility of the information that has been highlighted is substantially impaired. One example of food counterfeit is the inappropriate composition of minced meat with respect to the labelling of the packaged product.
The aim of this paper was to examine the quality of minced meat available on the Croatian market, including pork and beef minced meat and to evaluate compliance with the requirements for minced meat as defined in the EU Regulation no. 1169/2011. Commercially available samples were used, divided into 3 categories depending on the type of meat from which the minced meat was produced. For the purpose of the research, a total of 42 samples of minced meat were analyzed, of which: 12 samples in the beef minced meat category, 13 in the pork minced meat category, and 17 mixed minced meat samples (pork and beef meat ratio 60:40). Meat quality parameters were tested using standard accredited methods: collagen content (spectrophotometric method HRN ISO 3496: 1999), protein content (Kjeldahl titration method HRN ISO 937: 1999) and fat content (Soxhlet gravimetric method HRN ISO 1443: 1999).
An analysis of the obtained results found that nine samples did not meet the requirements from the Ordinance. For all of them collagen to protein ratio and proportion of total fat exceed the limit value. Following the results of the analysis, it could be concluded that the meat industry is
partially misleading consumers in terms of the credibility of the information on minced meat declarations. Minced meat, which in its composition contains meat of lower quality, is precisely meat rich in connective tissue and fat content above the permitted limit, which is founded in the study by analyzing the amount of hydroxyproline and total fat
Optimization of immunoflorescence for detection of CD63 protein: impact of different cell˙s permeabilization and immobilization compounds
No full textCD63 je ubikvitarno izražen glikoprotein iz obitelji tetraspanina. U većini stanica glavni udio ovog proteina nalazi se u kasnim endosomima, lizosomima i sekretornim vezikulama. Pronađen je i u Weibel-Palade tijelima endotelnih stanica, gustim granulama trombocita te je detektiran i u podskupini premelanosoma. CD63 je transmembranski protein koji se sastoji od 4 transmembranskih hidrofobnih domena, 2 izvanstanične domene nejednakih veličina – male (engl. small extracellular loop, SEL) i velike izvanstanične petlje (engl. large extracellular loop, LEL) - i 3 unutarstanične regije uključujući N- i C- terminalne repove proteina.
U ovom radu ispitivani su optimalni uvjeti za detekciju proteina CD63 novoproizvedenim monoklonskim protutijelom hCD63.01 na ljudskim staničnim linijama MRC-5 (ljudski plućni fibroblast) metodom imunofluorescencije. Ispitani su različiti uvjeti i reagensi za permeabilizaciju stanica: alkohol metanol i deterdženti (saponin, Triton X-100, Tween 20) u različitim koncentracijama, dvije koncentracije otopine za blokiranje nespecifičnog vezivanja, te utjecaj različitih razrjeđenja hCD63.01. Zaključno, rezultatima prikupljenim kvantifikacijom imunofluorescencijskog signala, koja je provedena na slikama dobivenim fluorescentnim mikroskopom, utvrđeno je da je permeabilizacija 0,5% Tweenom 20 najpogodnija u usporedbi s drugim reagensima za permeabilizaciju. Preporučena koncentracija otopine za blokiranje je 5%, a razrjeđenje hCD63.01 je 1:200. Potrebno je provesti daljnja ispitivanja metodama imunoprecipitacije, western blota, kolokalizacije markerima organela i konfokalnom mikroskopijom kako bi se utvrdilo da hCD63.01 uistinu veže protein CD63 te da se ustanovi točna lokalizacija detektiranog proteina u stanicama.CD63 is a ubiquitously expressed glycoprotein from the tetraspanin family. In most cells, the major pool of this protein is found in late endosomes, lysosomes, and secretory vesicles. It was also found in Weibel-Palade bodies of endothelial cells, dense platelet granules, and was detected in the premellanosome subset. CD63 is a transmembrane protein consisting of 4 transmembrane hydrophobic domains, 2 non-cellular domains of unequal size - small extracellular loop (SEL); large extracellular loop (LEL) - and 3 intracellular regions including N- and C- terminal tails of the protein.
In this study, optimal conditions for the detection of CD63 protein by the newly produced monoclonal antibody hCD63.01 on human cell lines MRC-5 (human lung fibroblast) were investigated by immunofluorescence method. Different conditions and reagents for cell permeabilization were compared: alcohol methanol and detergents (saponin, Triton X-100, Tween 20) at different concentrations, two concentrations of the blocking buffer, and the effect of different dilutions of hCD63.01. In conclusion, the results obtained by quantification of the immunofluorescence signal, which was carried out on the images obtained by fluorescence microscopy, determined that permeabilization with 0.5% Tween 20 was the most appropriate when compared with other permeabilization reagents. The recommended concentration of the blocking solution is 5% and the dilution of hCD63.01 is 1: 200. Further studies including immunoprescipitation, western blot and double immunofluorence with markers for cellular organells are needed to to determine if hCD63.01 indeed binds the CD63 protein and to determine the exact localization of the detected protein in the cells