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Roba koja se uvozi, izvozi, provozi, premješta ili se nalazi u nekom carinskom postupku podliježe fitosanitarnoj kontroli. Nadzor bilja, biljnih proizvoda i drugih nadziranih predmeta u prometu preko državne granice i sprječavanje unošenja i širenja štetnih organizama jedni su od glavnih ciljeva i razloga djelovanja fitosanitarne kontrole na graničnim prijelazima. Fitosanitarnim pregledom provjerava se da li su bilje, biljni proizvodi i drugi nadzirani predmeti zaraženi štetnim organizmima i da li udovoljavaju posebnim fitosanitarnim zahtjevima. Fitosanitarni pregled je provjera isprava, identiteta i zdravstvenoga stanja bilja, biljnih proizvoda i drugih nadziranih predmeta s namjerom da se utvrdi jesu li u i na bilju prisutni štetni organizmi, a čije se unošenje na prostor Republike Hrvatske i Europske unije nastoji spriječiti prilikom ulazne kontrole na graničnim prijelazima. Fitosanitarni nadzor i kontrolu biljaka, biljnih proizvoda i drugih nadziranih predmeta obavljaju fitosanitarni inspektori koji djeluju u sklopu Ministarstva poljoprivrede Republike Hrvatske.

Metastaziranje je proces širenja stanica primarnog tumora u druge dijelove organizma. Primarni tumor je obično izlječiv, međutim, udaljene metastaze koje nastaju od njega su najčešći uzrok smrti u oboljelih od tumora. Metastaziranje je složeni proces, sastavljen od 4 glavna događaja: (1) nastanak primarnog tumora, (2) migracija stanica primarnog tumora u krvotok i njihovo širenje kroz tijelo, (3) zaustavljanje stanica tumora u nekom udaljenom organu i izlazak iz krvotoka te, (4) stvaranje novih krvnih žila oko sekundarnog tumora (slike 1 i 2). U Laboratoriju za molekularnu onkologiju bavimo se proučavanjem procesa metastaziranja istraživanjem dva gena/proteina: Toll-like receptor 3 (TLR3) i nonmetastatic protein 23 (nm23). Naši rezultati pokazali su da aktivacija TLR3 može potaknuti migraciju stanica tumora ždrijela te aktivirati druge proteine uključene u proces metastaziranja. Nm23-H1 je prvi otkriveni supresor metastaziranja - može inhibirati nastanak metastaza, a da ne utječe na rast i razvoj primarnog tumora. Naši rezultati pokazuju da Nm23-H1 može smanjiti migraciju stanica karcinoma jezika.

The dual role of Toll-like receptors in metastatic cell line

Uloga gena/proteina TLR3 u stanicama primarnog karcinoma i metastaza čovjeka

The dual role of TLR3 gene/protein in human primary carcinoma and metastatic cells

Oxidative stress results in excessive production of reactive oxygen species (ROS), leading to lipid peroxidation and to formation of reactive aldehydes. Lipid-derived aldehydes are relatively stable and may diffuse to targets away from the initial oxidative injury. It was previously showed that oxidative stress may be involved in the transcriptional regulation of cytomegalovirus (CMV) immediate early promoter, involved in viral reactivation from latency. In the present paper we have used plasmid containing YFP gene under the control of CMV-1 promoter and monitored the influence of hydrogen peroxide and reactive aldehydes, 4-hydroxy-2-nonenal (HNE) and acrolein, on CMV-1 promoter activation. HNE had the strongest effect enhancing on CMV-1 promoter activation three fold within 4 as well as 24 hours after 24 M HNE treatment. The results presented indicate that HNE generated during oxidative stress might have strong impact on immune suppressed individuals with CMV infection activating the virus.

The expression and functionality of TLR3 in different human tumor cell lines

The expression and functionality of TLR3 in different human cell lines

Ekspresija i funkcionalnost TLR3 u različitim humanim tumorskim staničnim linijama

The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: Δ F508, 58.33% ; G542X, 3.12% ; N1303K, 2.08% ; R1162X ; 621 + 1G → T ; G85E ; Y569C ; E585X ; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (Δ F508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.

MECP2 gene mutation screening in Rett syndrome patients from Croatia

Analiza mutacija gena za cističnu fibrozu i analiza povezanosti haplotipova

The neurobiology of posttraumatic stress disorder (PTSD) involves alterations in multiple neuroendocrine and neurotransmitter systems. Platelet monoamine oxidase (MAO-B) has been associated with susceptibility to various psychiatric disorders, personality traits and behaviours. Platelet MAO-B activity and MAO-B intron 13 polymorphism (a G/A substitution) were determined in male war veterans (n=106) with DSM-IV diagnosed current and chronic PTSD, divided into subgroups of PTSD patients with (n=28) or without (n=78) psychotic features, combat exposed veterans (n=41) who did not develop PTSD, and healthy control men (n=242). Two-way ANOVAs revealed a significant effect of diagnosis and smoking, a significant effect of smoking, no significant effect of genotype, and no significant interaction between genotype, smoking or diagnosis, on platelet MAO-B activity. One-way ANOVAs showed significantly lower platelet MAO-B activity in smokers than in nonsmokers. After controlling for smoking, veterans with psychotic PTSD had significantly higher platelet MAO-B activity than veterans with or without PTSD, or healthy subjects. The results were obtained on peripheral biochemical marker, i.e. platelet MAO activity. The MAO-B intron 13 polymorphism was not functional, and did not affect platelet MAO-B activity. The aallele frequencies of the MAO-B genotype were similarly distributed among healthy controls and veterans with or without PTSD and/or psychotic symptoms. The results suggest that platelet MAO-B activity, controlled for smoking status, might be used as a peripheral marker of the psychotic symptoms in PTSD.

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids (PUFAs) in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces delta9/delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of PUFAs, changed the yeast’ s sensitivity to oxidative stress induced by hydrogen peroxide. This difference in sensitivity was followed by the formation of 4-hydroxy-2-nonenal (HNE), one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. HNE-protein adducts were also present at a significant, albeit lower, level under physiological conditions in the desaturase-expressing strain, in the absence of exogenous stressors. These data demonstrate that a yeast strain conditionally expressing delta12 desaturase provides a novel and attractive system to study physiological and pathophysiological processes induced by PUFA peroxidation, which are highly relevant to disease processes, such as cancer, in humans.

Učestalost 35delG mutacije gena GJB2 u oboljelih od nesindromskoe gluhoće u Hrvatskoj

Sequence analysis of CFTR coding region in Croatian population

Expression level of TLR3 and TLR9 genes in different tumor cell lines.

Dosadašnja istraživanja lipidne peroksidacije kod Saccharomyces cerevisiae su uključivala egzogeno dodavanje višestruko nezasićenih masnih kiselina u podlogu kako bi se oksidansima mogla izazvati lipidna peroksidacija budući da Saccharomyces cerevisiae prirodno nema višestruko nezasićenih masnih kiselina u sastavu lipida. Konstrukcijom transgenog Saccharomyces cerevisiae koji u sebi nosi plazmid s genom za desaturazu, enzim koji uvodi dvostruke veze i tako stvara višestruko nezasićene masne kiseline, nastao je testni sustav u kojem dolazi do endogene lipidne peroksidacije. U ovom istraživanju pokušali smo odrediti može li se transgeni kvasac koristiti kao modelni organizam koji bi omogućio ispitivanje učinaka oksidacijskog stresa na više eukariotske organizmime u kojima prirodno dolazi do lipidne peroksidacije. Svrha istraživanja bila je utvrditi koliko je transgeni S. cerevisiae osjetljiv na vodikov peroksid, koji je reaktivan kisikov spoj (ROS) i posrednik u nizu staničnih bioloških procesa vezanih uz oksidacijski stres te dolazi li do prilagodbe na oksidacijski stres i povećanog stvaranja glutationa koji je glavna molekula u obrani organizma od toksičnih produkata lipidne peroksidacije i nekih ROS. Prvo je utvrđeno da se gen za desaturazu eksprimira te da je desaturaza aktivna i proizvodi višestruko nezasićene masne kiseline (PUFA). Daljnim istraživanjem osjetljivosti na oksidanse, utvrđena je povećana osjetljivost transgenog S. cerevisiae u odnosu na divlji tip kad je desaturaza inducirana neposredno prije pokusa. Međutim, kada je desaturaza bila uzastopno inducirana tijekom dva tjedna prije izvođenja pokusa, primijećena je veća vijabilnost stanica transgenog S. cerevisiae u odnosu na divlji tip nakon tretmana vodikovim peroksidom. Pretpostavljamo da je došlo do prilagodbe transgenog S. cerevisiae u kojem zbog desaturaze dolazi do lipidne peroksidacije pa je obrana stanice od oksidansa neprekidno aktivna. To je potvrđeno mjerenjem razine glutationa u stanici jer su rezultati pokazali da je razina glutationa u stanici transgenog S. cerevisiae povišena za gotovo 50% u odnosu na sve kontrole.

Oxidative stress is defined as a shift in oxidation-reduction balance toward oxidation, causing lipid peroxidation of biomembranes. To provide an in vitro test system for analysis of physiological consequences of lipid peroxidation, yeast, Saccharomyces cerevisiae, was genetically modified with w6-desaturase. Desaturase introduces double bonds to saturated fatty acids, so they become susceptible to lipid peroxidation. Thus modified yeast can develop endogenous oxidative stress resembling the analogous stress in mammalian cells. Yeast strains were grown on media with either glucose (controls) or galactose (inducer of desaturase expression). Before testing sensitivity to oxidants, real time PCR was performed to see if desaturase is expressed, and gas chromatography to see if it is active. Influence of short-term and long-term desaturase induction to oxidative stress sensitivity was examined by treatment with H2O2 or 4-hydroxynonenal (HNE) and plating them on agar as spots. Experiments showed that the desaturase was expressed and active only in modified strain grown on galactose, as expected. Short-term desaturase expression made transgenic strain more sensitive to oxidant challenge than wild type. On the other hand, long-term expression made desaturase strain less sensitive than wild type. These observations could be due to cumulative effect of of endogenous and exogenous oxidative stress in short-time expression, while long-term expression of desaturase induces adaptation mechanisms. Hence, the cellular adjustment to low level of endogenous oxidative stress with consequential lipid peroxidation may lead to increased resistance to harmful effects of oxidative stress.

Oxidative stress is defined as a shift in oxidation-reduction balance toward oxidation. One of the consequences of oxidative stress is lipid peroxidation. To provide an in vitro test system for analysis of physiological consequences of lipid peroxidation, yeast, Saccharomyces cerevisiae, was genetically modified with desaturase inserted pYES2 plasmid. Desaturase introduces double bonds to saturated fatty acids, so they become susceptible to lipid peroxidation (caused by oxidative stress). Thus modified yeast can develop endogenous oxidative stress resembling similar stress in mammalian cells. Hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE) were used as mediators of oxidative stress to see sensitivity of modified strain. Yeast were grown on media with either glucose or with galactose, respectively, to induce desaturase expression linked to GAL1 promotor on pYES2. They were treated with H2O2 (in concentrations 0, 7, 1, 1, 5 and 2 mM) or with HNE (50, 100 and 200 μ M) for 2 hours and afterwards plated on agar in small volume as spots. Further, we wanted to see the antioxidant defense of the cell. Major antioxidant defense in cells against toxic lipid peroxidation product HNE and several ROS is tripeptide glutathione (GSH), so measurements of GSH levels were done. Finally, to see if HNE entered the cells, HNE immunoelectronmicroscopy was performed with genuine monoclonal antibodies against HNE-histidine conjugates. Transgenic strain was more sensitive than the wild type to H2O2 and HNE. This can be explained by cumulative endogenous oxidative stress of desaturase-modified strain combined with endogenous oxidative stress. Modified strain showed increase in GSH level compared to level when grown on glucose and to wild type level grown on galactose. However, electronmicroscopy has shown that neither wild nor transgenic type of yeast had detectable HNE-protein adducts unless they were treated by HNE. These results indicate that endogenous oxidative stress increased glutathione level as a defense mechanism, but due to presence of endogenous oxidative stress further challenge made these cells more susceptible to hydrogen peroxide and HNE, mediators of oxidative stress.

Stobadine (STB), novel pyridoindole derivative which scavenges hydroxyl, superoxide, alkoxyl and peroxyl radicals, and quench singlet oxygen has been proved neuroprotective in many experimental models. Since, in clinic preventive administration is not possible, the aim of our study was to determine if STB acts as neuroprotectant after onset of ischaemic brain damage and consequential generation of lipid peroxidation product 4-hydroxynonenal (HNE). Immunocytochemical analysis of HNE-protein conjugates, using monoclonal antibodies specific for the HNE-histidine epitope, was performed on isolated rat brain cells after ischaemic brain damage induced by occlusion of the common carotid arteries and additional in vitro incubated with STB and/or HNE. STB decreased formation of HNE-protein conjugates in brain cells which were isolated from "normo-oxigenated" brain area but not in the brain cells isolated from ischaemic brain. When stobadine was added to cell cultures of human neuroblastoma IMR32, prior or after HNE, cell-ELISA method showed decrease in HNE positivity. This indicates that STB can be effective early after onset of oxidative stress, when damage of brain cells might be reversible.