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Analysis of early and late endosomes in different maturation stages of mouse megakaryocytes
No full textU endocitozi male GTPaze Rab5 i Rab7 djeluju kao molekularni prekidači i reguliraju vezikularni transport i promet kroz membranu različitih organela. Rab5 kao marker ranih endosoma je ključan za homogenu i heterogenu fuziju membrana ranih endosoma i njihovu pokretljivost dok je Rab7 marker kasnih endosoma. Smatra se da je konverzija Rab5 u Rab7 vrlo bitna za tijek endolizosomalnog puta.
Svrha istraživanja je ispitati postoji li povezanost izmeĎu maturacije endosoma i nastanka protrombocita iz megakariocita koristeći različite metodološke pristupe. Western blot analizom smo ispitali razinu ekspresije markera ranih (Rab5), kasnih (Rab7) i reciklirajućih (Rab1) endosoma u razvojnim fazama mišjih primarnih megakariocita te mišjim trombocitima. Nadalje, imunofluorescencijom i konfokalnom mikroskopijom smo analizirali lokalizaciju ranih (Rab5, EEA1) i kasnih (Rab7, LAMP1) endosoma s obzirom na stupanj zrelosti megakariocita. Tretirali smo megakariocite sa inhibitorima zakiseljavanja lumena organela koji zaustavljaju konverziju ranih u kasne endosome te smo ispitali učinak na formiranje protrombocita i morfologiju megakariocita. Naposljetku, transficirali smo Dami stanice sa Rab5 i njezinom konstitutivno aktivnom Q79L ili dominantno negativnom N133L mutantom kako bi provjerili postoje li razlike u fenotipu te utvrdili promjene u zrelosti megakariocita (ekspresija markera CD42c i veličina transficiranih stanica). Naši glavni rezultati su pokazali da je inhibicija konverzije ranih u kasne endosome rezultira smanjenim stvaranjem protrombocita te morfološkim promjenama megakariocita. Transfekcijom zrelih Dami stanica sa Rab5 (wt i konstitutivno aktivni) potvrdili smo prisutnost funkcionalnih ranih endosoma te je konstitutivno aktivni Rab5 uzrokovao veći stupanj maturacije u usporedbi sa drugim genotipovima i kontrolom.
Rezultati ukazuju na povezanost endocitoze i procesa nastanka prekursora trombocita, a daljnja istraživanja mogu doprinijeti definiranju generalnog mehanizma tog procesa.In endocytosis small GTPases Rab5 and Rab7 act like molecular switches and regulate vesicular transport and membrane trafficking. Rab5 represents a marker of early endosomes which is essential for homotypic and heterotypic fusion as well for endosome motility while Rab7 represents a marker of late endosomes. Conversion from Rab5 to Rab7 is important for the course of an endolysosomal path.
Purpose of this thesis was to investigate correlation between endosome maturation and proplatelet formation. We used Western blot analysis to examine the level of expression of early (Rab5), late (Rab7) and recycling (Rab11) endosomes in different maturation stages of mouse megakaryocytes and platelets. By immunofluorescence and confocal microscopy, we analyzed the localization of early (Rab5, EEA1) and late (Rab7, LAMP1) endosomes considering the maturation stage of megakaryocytes. In addition, we treated megakaryocytes with inhibitors that cause rapid alkalinization of acidic organelles and block conversion from early to late endosomes to investigate impact on proplatelet formation and megakaryocyte morphology. Lastly, we transfected Dami megakaryocytic cell line with Rab5 and its constitutively active Q79L or dominantly negative N133L mutant to examine differences in phenotype and to determine possible changes in megakaryocyte maturity (expression of marker CD42c and size of transfected cells). Our key results have shown that inhibition of conversion from early to late endosome resulted in reduced proplatelet formation and morphological changes in megakaryocytes. Transfection of mature Dami cells with Rab5 (wt and constitutively active) confirmed a presence of functional early endosomes. Also, constitutively active Rab5 maturation of Dami cells into higher stages in comparison with other genotypes and control.
Our results point out interconnection of endocytosis and process of platelet precursors assembly. Future experiments will have a crucial contribution in defining the general mechanism of this processes
Damage Index in 110 Systemic Lupus Erythematosus Patients
No full textObjective: To determine the prevalence of Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index among systemic lupus erythematosus (SLE) patients and to investigate the prevalence of each component of SLICC/ACR damage index.
Methods: We performed a cross-sectional study on 110 SLE patients from the Division of Rheumatology and Clinical Immunology, University Hospital Centre Rijeka, Croatia. Damage in SLE patients was assessed by SLICC/ACR damage index.
Results: The most frequently observed components of SLICC/ACR damage index were osteoporosis with fracture and cranial or peripheral neuropathy. After that followed cataract and pleural fibrosis. Malignancy was recorded as the fifth most represented component of SLICC/ACR damage index. Hormone-sensitive cancers were more common than hematologic cancers.
Conclusion: In our group of SLE patients musculoskeletal system was the most frequently observed organ system of SLICC/ACR damage index. Osteoporosis with fracture was the most common manifestation of musculoskeletal system and the main characteristic of SLICC/ACR damage index
The NDE1 genomic locus can affect treatment of psychiatric illness through gene expression changes related to microRNA-484
Get PDFGenetic studies of familial schizophrenia in Finland have observed significant associations with a group of biologically related genes, DISC1, NDE1, NDEL1, PDE4B and PDE4D, the ‘DISC1 network’. Here, we use gene expression and psychoactive medication use data to study their biological consequences and potential treatment implications. Gene expression levels were determined in 64 individuals from 18 families, while prescription medication information has been collected over a 10-year period for 931 affected individuals. We demonstrate that the NDE1 SNP rs2242549 associates with significant changes in gene expression for 2908 probes (2542 genes), of which 794 probes (719 genes) were replicable. A significant number of the genes altered were predicted targets of microRNA-484 (p = 3.0 x10-8), located on a non-coding exon of NDE1. Variants within the NDE1 locus also displayed significant genotype by gender interaction to early cessation of psychoactive medications metabolized by CYP2C19. Furthermore, we demonstrate that miR-484 can affect the expression of CYP2C19 in a cell culture system. Thus, variation at the NDE1 locus may alter risk of mental illness, in part through modification of miR-484, and such modification alters treatment response to specific psychoactive medications, leading to the potential for use of this locus in targeting treatment
The interaction of schizophrenia-related proteins DISC1 and NDEL1, in light of the newly identified domain structure of DISC1
Get PDFDISC1 and NDEL1 are both key proteins in cortical neurodevelopment, which are each also implicated in the pathogenesis of mental illness. That the two proteins interact with each other in a functionally important manner is well established, but two distinct binding domains for NDEL1 on DISC1 have been proposed. A partial domain structure for DISC1 has recently been described, consisting of 4 structured regions referred to as “D,” “I,” “S” and “C” respectively, with one of the NDEL1 binding sites lying in the “C” region of DISC1. In light of this domain structure, it can be deduced that this site is the likely location at which NDEL1 binds, although the other proposed site (which lies in the DISC1 “I” and “S” regions) may indirectly impact on DISC1-NDEL1 interactions through determination of the oligomeric state of DISC1
Detection and quantification of pesticides in Eisenia Fetida by ultra performance liquid chromatography
No full textPesticidi su prirodne ili sintetske molekule napravljene za očuvanje usjeva, hrane i zaštite od bolesti koje prenose nametnici. Veliki problem je nakupljanje pesticida u životinjama, biljkama i ljudima koje dovodi do zagađenja okoliša te šteti ljudskom zdravlju. Diamidni insekticid klorantraniliprol i triazolni fungicid propikonazol pesticidi su kojim se bavi ovaj rad.
U ovom radu opisana je standardizirana metoda za detekciju i kvantifikaciju metaboliziranih pesticida iz gujavica (Eisenia fetida) UPLC-om. Istom metodom dokazano je kako gujavice izložene propikonazolu dio tog pesticida metaboliziraju. Analizom dobivenih rezultata utvrđeno je kako gujavice metaboliziraju između 26% i 39% početne koncentracije propikonazola. Koncentracije klorantraniliprola bile su ispod razine detektabilnosti UPLC-a.
Prednosti su ove metode što je jednostavna i jeftina te laka analiza malih uzoraka. Samim time potrebno je daljnje usavršavanje i istraživanje kako bi ona pronašla svoju primjenu u industriji, zdravstvu i zaštiti okoliša.Pesticides are natural or synthetic molecules made for preserving crops, food and protecting from parasitic diseases. A major problem is accumulation of pesticides in animals, plants and humans which leads to environmental pollution and brings harm to human health. Diamidic insecticide chlorantraniliprole and triazolic fungicide propiconazole are pesticides of interest in this research.
We have described a standardized method for the detection and quantification of metabolized pesticides from earthworms (Eisenia fetida) with UPLC. It was proven that earthworms exposed to propiconazole partially metabolize that pesticide. By analyzing these results, it was confirmed that earthworms metabolize between 26-39% of initial concentration of propiconazole. The concentrations of chlorantraniliprole were below levels of detectability of UPLC.
The advantages of this method are simplicity and cost-effectiveness so it can easily produce great results on small samples. With further research and optimization this method could be applied in various industries, healthcare and environmental protection
Analiza fosfoinozitidnih fosfataza u transkriptomu i proteomu ljudskih i mišjih trombocita
No full textFosfoinozitidi (PI) su lipidi sastavljeni od više podjedinica: inozitolnog prstena na koji se veže citidin difosfat diacilglicerol (CDP-DAG) čime nastaje fosfatidilinozitol (PtdIns) – prekursor svih PI. Nadalje, PtdIns se može fosforilirati na D3, D4 ili D5 pozicijama inozitolnog prstena čime nastaju različiti PI. U konverziji između različitih fosforilacijskih stanja PI sudjeluju PI kinaze i PI fosfataze, a ovaj proces se događa na endosomima, mreži Golgijevog aparata ili na plazma membrani (PM). Vrsta PI određena je djelovanjem PI kinaza i fosfataza prisutnih u stanici, što određuje njegovu lokalizaciju i funkciju.
Ovaj rad analizira PI fosfataze eksprimirane u trombocitima. PI fosfataze podijeljene su na dvije obitelji s obzirom na način katalitičkog mehanizma – sa i bez konzerviranog katalitičkog motiva CX5R u aktivnom mjestu. Prvu grupu bez karakterističnog motiva, čine PI 5-fosfataze, a drugoj grupi pripadaju ostale obitelji fosfataza: PTEN obitelj, miotubularinska obitelj, Sac1 obitelj i obitelj 4-fosfataza. Nedostatak neke od ovih fosfataza može dovesti do defekata u metabolizmu PI, promjena u signalizaciji, te posljedično nastanka nekih bolesti.
U ovom radu istražene su razine različitih PI fosfataza u transkriptomu i proteomu ljudskih i mišjih trombocita na temelju dostupnih baza podataka, te su utvrđene sličnosti i razlike između trombocita ovih dviju vrsta
Osjetljivost netuberkuloznih mikobakterija na dezinficijense
No full textNetuberkulozne mikobakterije (NTM) predstavljaju heterogenu skupinu okolišnih bakterija koje se obično izoliraju iz različitih vodenih medija i zemlje. Premda posjeduju niski patogeni potencijal u ljudi mogu uzrokovati različite infekcije. Izvor infekcije za čovjeka može biti voda za piće, rezervari s vodom, kontaminirani tuševi, aerosol ili prašina te morska voda. Infekcije netuberkuloznim mikobakterijama u ljudi se u 80 – 90% slučajeva manifestiraju kao infekcije dišnog sustava.
U radu je ispitana osjetljivost netuberkulozne mikobakterije M. intracellulare (ATCC 13950) na dezinficijense koji se široko primjenjuju u medicinskoj praksi. Ispitivana je djelotvornost dezinficijensa / antiseptika Descocida - N (2,5%), Manorapida i 70% - tnog etilnog alkohola.
Subkultivacija ispitivanog soja izvođena je na obogaćenoj Middlebrook 7H10 podlozi u aerobnim atmosferskim uvjetima, na temperaturi 37˚C kroz 3 tjedna.
Suspenzijskim testom nije dokazan porast ispitivanog soja u subkulturama na Middelbrook 7H10 podlozi nakon djelovanja 70 % -nog etanola i manorapida u kontaktnom vremenu od 1, 5 i 30 min, čime je potvrđen mikrobicidni učinak navedenih dezinficijensa na M. intracellulare.
Descocid – N nije ostvario mikrobicidno djelovanje na M. intracellulare nakon kontaktnog vremena od 1, 5 i 30 minuta. Descocid - N primjenjen u 2,5% koncentraciji reducira broj bakterija u suspenziji ovisno o kontaktnom vremenu.Nontuberculous Mycobacteria (NTM) is a heterogeneous group of environmental bacteria most commonly isolated from water and soil. Even though NTM have low pathogenic potential, they are still capable of causing numerous diseases in humans.
Potential sources of infection consist of drinking water, water tanks, sea water, contaminated showerheads, aerosols and dust. NTM infections in 80-90% of cases manifest as pulmonary infections (2,5).
The main goal of this paper was to test the susceptibility of nontuberculous mycobacteria M. intracellulare (ATCC 13950) to disinfectants widely used in medicinal practice. The disinfectants / antiseptics whose effectiveness was tested were Descocid - N (2,5%), Manorapid and 70% ethyl alcohol. The subcultivation of the tested strain was carried out on an enriched Middlebrook 7H10 growth medium in aerobic atmospheric conditions, at the temperature 37°C for 3 weeks.
The suspension test did not show any growth of the tested strain on the Middlebrook 7H10 growth medium after the application of 70% ethyl alcohol and Manorapid solution. Therefore, the microbicidal effect of these disinfectants on M. intracellulare within contact times of 1, 5 and 30 minutes was confirmed.
Descocid – N did not have aforementioned microbicidal effect on M. intracellulare with regardless of the contact times (1, 5 and 30 minutes). Although, 2,5% solution of Descocid - N did reduce the number of bacteria in the suspension dependent on contact time
Formation of neuronal networks and the role of guidance molecules netrin-1 and semaphorin 3a
No full textThe development of the central nervous system (CNS) begins before birth and it continues to change and restructure throughout adult life. CNS development relies on inductive factors and signaling molecules secreted by nearby cells that control differentiation of neuronal cells. To study the effect of such molecules on neurons, the mammalian hipppocampus became a well-established and among most used in vitro models.
This study is focused on the effect of guidance molecules on neuronal growth cones, the major motile structures guiding both axon and dendrite navigation during the first stages of hippocampal neuronal development in vitro. At this stage, chemotropic guidance of molecular cues can be observed; for instance, netrin molecules function as chemoattractants for hippocampal neurons. On the other hand, semaphorins play part in repulsion of cells. The combination of repulsive and attractive cues allow neurons to reach their targets. Techinques traditionally used in observing molecular guidance are the growth cone collapse assay and growth cone turning assay. In addition, immunofluorescence assays combined with specific neuronal markers can be performed to discriminate between hippocampal neurons and other cell population such as astrocytes, oligodendrocytes, microglia or other non-neuronal cells that are present in the culture. Moreover, the expression of guidance molecules receptors can be investigated.
Beside well known, some newly developed techniques will be described, such as localized delivery of guidance molecules by optically manipulated liposomes. Each technique offers different approach and potential to study if, and how, can a certain molecule act as a chemoattractant or chemorepellant during axon guidance, pathfinding and formation of functional neuronal network.Razvoj centralnog živčanog sustava započinje u fetalnom stadiju, a nastavlja se se mijenjati i razvijati kroz život. Razvitak živčanog sustava ovisi o induktivnim faktorima i signalnim molekulama koje izlučuju stanice u bliskoj udaljenosti, kontrolirajući diferencijaciju neurona. Kako bi se mogao istraživati utjecaj takvih molekula, hipokampus sisavaca postao je dobro istražen i među najčešće korištenih modela „in vitro“.
Ovaj se rad zasniva na proučavanju utjecaja netrina i semaforina na čunjiće rasta, glavne pokretljive strukture koje usmjeravaju navigaciju aksona i dendrita tijekom prvih faza razvoja neurona „in vitro“. U toj se fazi može promatrati učinak spomenutih molekula (engl. guidance molecules) na kemotropno usmjeravanje; primjerice, molekule netrina djeluju kemoatraktivno na hipokampalne neurone. Semaforini uzrokuju odbijanje među stanicama. Tehnike koje se koriste pri određivanju takvih učinaka su eseji kolapsa čunjića rasta (engl. Growth cone collaps assay) i eseji preokretanja čunjića (engl. Growth cone turning assay). Također, kombinacija imunoflorescencije i specifičnih neuronskih markera, omogućava razlikovanje između hipokamplanih neurona i ostalih stanica poput astrocita, oligodendrocita, mikroglije i ostalih stanica koje se nalaze u kulturi.
Osim već poznatih tehnika, u ovom će se radu opisati i novija metoda poput lokaliziranog dostavljanja utjecajnih molekula pomoću otpički manipuliranih liposoma. Svaka metoda pruža drugačiji pristup i potencijal pri istraživanju može li, i kako, određena molekula djelovati kao kemoatraktivna ili kemorepelentna molekula pri usmjeravanju aksona, pronalaženju cilja te stvaranju funkcionalnih neuronskih mreža
Alzheimerova bolest: molekularne osnove i terapijski pristup
No full textAlzheimerova bolest (AB) je kronična neurodegenerativna bolest i najčešći oblik senilne
demencije. Najveći poznati rizični čimbenik bolesti je starenje, pa većina pacijenata oboljelih
od Alzheimerove bolesti ima 65 godina starosti ili više. No, ova bolest se ne smatra normalnom
posljedicom starenja, već je rezultat složenih interakcija između više čimbenika uključujući
dob, genetiku, okoliš, način života i koegzistirajuća medicinska stanja. Alzheimerovu bolest
karakterizira progresivno opadanje kognitivnih funkcija, koje obično započinje problemima s
kratkotrajnom memorijom. Bolest se javlja u dva oblika, nasljednom i sporadičnom. Dvije
karakteristične patološke promjene u mozgu koje se javljaju u oba oblika bolesti su odlaganje
amiloidnog β peptida u izvanstanične senilne plakove i nakupljanje abnormalnog
hiperfosforiliranog tau proteina u unutarstanične neurofibrilarne čvorove. Neurodegenerativni
proces AB-i karakterizira sinaptičko oštećenje koje je praćeno gubitkom neurona u cerebralnom
korteksu i određenim subkortikalnim regijama mozga. Oligomerni oblici agregiranog Aβ
toksično djeluju na staničnu i sinaptičku funkciju uzrokujući gubitak neurona i opadanje
kognitivnih funkcija. Ne postoji tretman koji zaustavlja ili preokreće progresiju bolesti, iako
neki mogu privremeno poboljšati simptome. Odobrena terapija AB-i uključuje tri inhibitora
acetilkolinesteraze (donepezil, galantamin i rivastigmin) i jedan antagonist NMDA receptora
(memantin). Tijekom razvoja bolesti oštećuju se kolinergički neuroni, te se inhibicijom enzima
koji razgrađuje acetilkolin nastoji povisiti njegova koncetracija u mozgu. Također, tijekom
bolesti dolazi i do prekomjerne stimulacije NMDA receptora glutamatom što uzrokuje
eksitotoksičnost (oštećenje živčanih stanica zbog prekomjerne stimulacije neurotransmitera), te
se za blokiranje tog učinka koristi antagonist NMDA receptora. Ovi lijekovi privremeno
ublažavaju probleme s pamćenjem i razmišljanjem, ali je njihov klinički učinak skroman.
Tijekom proteklog desetljeća, fokus otkrića novih terapija stavlja se na lijekove koji mijenjaju
tijek bolesti. Cilj takvih lijekova je usporiti progresiju neurodegenerativnog procesa
inhibiranjem kritičnih događaja u patofiziologiji bolesti, koji uključuju taloženje izvanstaničnih
senilnih plakova i unutarstaničnih neurofibrilarnih čvorova. Međutim, nakon desetljeća
istraživanja, Alzheimerova bolest je i dalje neizlječiva i time se smatra jednim od glavnih
ljudskih zdravstvenih izazova.Alzheimer's is a chronic neurodegenerative disease and the most common form of senile
dementia. The greatest known risk factor of the disease is aging, so most patients with the
disease are 65 years of age or older. However, it is not considered a normal part of aging, but
the result of complex interactions among many factors including age, genetics, environment,
lifestyle, and coexisting medical conditions. Alzheimer's disease is characterized by a
progressive decline in cognitive function, which usually begins with problems with short-term
memory. The disease occurs in two forms, hereditary and sporadic. Two characteristic
pathological changes in the brain that occur in both forms of disease are the accumulation of
amyloid β peptide in extracellular senile plaques and the accumulation of abnormal
hyperphosphorylated tau protein in the intracellular neurofibrillary tangles. The
neurodegenerative process of the disease is characterized by a synaptic damage which is
accompanied by the loss of neurons in the cerebral cortex and certain subcortical regions of the
brain. Aggregated oligomeric forms of Aβ have toxic effects to the cellular and synaptic
function, causing loss of neurons and decline in cognitive function. There is no treatment that
stops or reverses the progression of the disease, although some may temporarily improve the
symptoms. Approved therapy includes three inhibitors of acetylcholinesterase (donepezil,
galantamine and rivastigmine) and one NMDA receptor antagonist (memantin). During the
development of the disease cholinergic neurons are damaged, and the inhibition of the enzyme
disintegrating acetylcholine aims to increase its concentration in the brain. Likewise, during the
disease, excessive stimulation of NMDA receptor by glutamate causes excitotoxicity (nerve
cells are damaged by excessive stimulation by neurotransmitters), therefore NMDA receptor
antagonist is used to block this effect. These medications temporarily reduce memory and
thinking problems, but their clinical effect is modest. Over the past decade, the focus of
discovery of new therapies is on medicines that change the course of the disease. The aim of
such drugs is to slow down the progression of the neurodegenerative process by inhibiting
critical events in the pathophysiology of the disease, including the deposition of extracellular
senile plaques and intracellular neurofibrillary tangles. However, after decades of research,
Alzheimer's disease is still incurable and is considered to be one of the major human health
challenges
Analysis of gamma secretase inhibitors as potential drugs for Alzheimer's disease on SH-SY5Y cells
No full textZnačaj: Alzheimerova bolest je smrtonosan neurodegenerativni poremećaj za
kojeg još uvijek nije pronađen učinkovit lijek. Zbog stalnog porasta incidencije i
jako skupe zdravstvene skrbi oboljelih, Alzheimerova bolest predstavlja jedan od
najvećih izazova za farmaceutsku industriju 21. stoljeća. Postoji više različitih
teorija o patogenezi bolesti, a do danas najbolje potvrđena je amiloidna hipoteza.
Prema njoj neurodegenerativni procesi rezultat su poremećaja u metabolizmu
amiloidnih proteina koji nastaju zbog promjena u djelovanju mebranske proteaze
γ-sekretaze. Stoga, γ-sekretaza predstavlja glavni put u razvoju potencijalnih
lijekova za Alzheimerovu bolest.
Rezultati: Prvo smo optimizirali Sandwich ELISA-u za detekciju unutarstaničnog
Aβ1-40 proteina u stanicama ljudskog neuroblastoma SH-SY5Y koje nisu
genetički modificirane. Pripremljeni protokol koristili smo za ispitivanje
potencijalnih lijekova za Alzheimerovu bolest koje razvija farmaceutska tvrtka
Jiva Pharma. Svi JIVA spojevi pokazali su inhibiciju aktivnosti γ-sekretaze u
koncentracijama od 10 μM i 100 μM. Najveća inhibicija zapažena je kod spojeva
JIVA0028 i JIVA0029, koji su smanjili aktivnost enzima za više od 80%. Najveću
razliku inhibicije između dvije ispitivane koncentracije pokazao je spoj JIVA0041,
dok kod spoja JIVA0026 nije uočena značajna razlika. Niti jedan ispitivani spoj
nije utjecao na morfologiju stanica niti je u danim koncentracijama bio letalan za
stanice.
Zaključak: Svi JIVA spojevi predstavljaju potencijalne lijekove za Alzheimerovu
bolest. Međutim, najzanimljiviji su spojevi JIVA0028 i JIVA0029 koji su inhibirali
aktivnost γ-sekretaze gotovo jednako kao jedan od najpoznatijih inhibitora γ-
sekretaze semagacestat. Time predstavljaju obećavajuće strukture koje se mogu
dalje modificirati kako bi se dobili novi, još potentniji spojevi.Significance: Alzheimer's disease is a fatal neurodegenerative disorder that
currently has no effective treatment. Alzheimer's disease is one of the biggest
challenges for the pharmaceutical industry of the 21st century as the most
expensive disease in the current health care system in the industrialised world.
There are several different theories on the pathogenesis of the disease, to this
date the amyloid hypothesis appears to be the most credible. According to
amyloid hypothesis, the neurodegenerative processes are the result of disorders
in the metabolism of amyloid proteins that occur due to changes in the function
of the membrane embedded protease, γ-secretase. Therefore, γ-secretase is
currently the main target in the development of potential drug candidates for
Alzheimer's disease.
Results: We first optimized the Sandwich ELISA for the detection of intracellular
Aβ1-40 protein in non-genetically modified human neuroblastoma cells SH-SY5Y.
The created protocol was used to test potential drugs for Alzheimer's disease that
are developed by the pharmaceutical company Jiva Pharma. All JIVA compounds
showed inhibition of γ-secretase activity at concentrations of 10 μM and 100 μM.
The highest inhibition was observed in JIVA0028 and JIVA0029, which reduced
enzyme activity by more than 80%. The highest difference in inhibition between
the two concentrations was shown in JIVA0041, while no significant difference
was observed with JIVA0026. No JIVA compound had any effect on the
morphology of the cells or was lethal for the cells at given concentrations.
Conclusion: All JIVA compounds represent potential drugs for Alzheimer's
disease. The most interesting compounds are JIVA0028 and JIVA0029 who
showed inhibition of γ-secretase activity almost as good as semagacestat, one of
the best known inhibitors of γ-secretase. Thus, their structures can be further
modified to obtain new, more potent compounds