GLIKOZILACIJA PROTEINA POSTELJICE U ANEMBRIONALNOJ TRUDNOĆI

Blighted ovum is a form of miscarriage, involving the absence or disappearance of an embryo very early in pregnancy. Due to contemporary lack of understanding of the nature of blighted ovum, our objective was to demonstrate the glycosylation patterns of placental proteins in this failure of pregnancy. Six placentas were taken from women with blighted ovum and twentyone placentas were provided from healthy women undergoing elective termination of normal pregnancies. Olygosaccharide branches were detected by Western-blot method using lectins: SNA and PHA-E, after preliminary separation of proteins by discontinuous SDS-PAG electrophoresis. Much stronger expression of GP74 has been identified in blighted ovum at the beginning of the eleventh week of gestation (with PHA-E), than in normal placenta. The same result was obtained for GP25 being stronger in blighted ovum at the end of eleventh week (with SNA). It is possible to conclude that the differences in glycoprotein changes beetwen the blighted ovum and the normal placenta, can only be found at the quantitative level, but not at the qualitative level.»Blighted ovum« (anembrionalna trudnoća) predstavlja oblik pobačaja, koji uključuje nedostatak ili nestanak zametka vrlo rano tijekom trudnoće. Dosadašnja skromna saznanja o ovom poremećaju potaknula su nas na detaljnija ispitivanja prikaza glikozilacijskih obrazaca placentarnih proteina. Promjene glikoproteinskog sastava detektirane su Western-blot metodom koristeći lektine SNA i PHA-E, koja je slijedila nakon preliminarne separacije proteina diskontinuiranom PAGE elektroforezom. Uspjeli smo identificirati mnogo jaču ekspresiju GP 74 u blighted ovum-u, nego u normalnoj placenti na početku jedanaestoga tjedna trudnoće, koristeći lektin PHA-E. Isti smo rezultat dobili i za GP25, koji je bio jačeg intenziteta u blighted ovum-u pri kraju jedanaestoga tjedna, koristeći lektin SNA. Naši rezultati navode nas na zaključak da se razlika između blighted ovum-a i normalne placente može uočiti samo na razini kvantitativnih glikoproteinskih promjena, ali ne i na kvalitativnoj razini

THE ROLE OF GLYCOPROTEINS IN THE PROCESSES OF IMPLANTATION AND PLACENTATION

Implantacija se danas smatra centralnim događajem u razvoju zametka sisavaca, a zahtijeva usklađen odnos između dvije vrste tkiva koja dolaze u interakciju: trofoblasta i sluznice maternice. Za razumijevanje molekularnih mehanizama implantacije od ključnog je značenja poznavanje zbivanja na razini međustaničnog prepoznavanja i međuodnosa stanica i međustaničnog matriksa. Glikoproteini se općenito smatraju potencijalnim medijatorima staničnih interakcija u brojnim biološkim sustavima, uključujući procese prepoznavanja i adhezije. Ovaj rad donosi sustavni ¬pregled glikoproteinskih molekula koje sudjeluju u procesima implantacije i placentacije te njihovu ulogu u navedenim događajima.The proces of implantation is considered nowadays to be the central event in the development mammalian embryo. Implantation requires very subtle interactions between two tissue types: the trophoblast of the embryo and the endometrium of the uterus. In order to better understand the molecular mechanisms of implantation, it is crucial to investigate events of intercellular recognition and interaction beetwen cells and extracellular matrix. Glycoproteins are considered to be the potential mediators of cellular interactions in a number of biological sistems, including processes of cellular recognition and adhesion. This paper reports on an extended review of glycoprotein molecules that are responsible for biological processes of implantation and placentation, and studies their roles in these events

Comparison of Glycosylation Patterns of Placental Proteins Between Normal Pregnancy and Missed Abortion

Contemporary understanding of missed abortion, as the case of spontaneous abortion where embryo is retained in uterus for four weeks or more after its death, is very poor. Aiming to improve the level of knowledge about this process, we have compared glycosylation patterns of placental proteins in normal pregnancy and missed abortion. Oligosaccharide branches were detected by Western-blot using SNA, DBA and PHA-E lectins. The comparison of samples of the same gestational age enabled identification of changes in protein glycosylation between normal and pathological placentas. Lectin DBA detects in normal placenta the glycoprotein GP 105 during the eleventh week, which is absent in missed abortion. PHA-E identifies GP 71 during fourteenth week only in normal placenta. However GP 25 recognized by SNA in missed abortion was not found in normal pregnancy at tenth week. These results indicate that abnormal placental development is associated with changes in glycoprotein structures, and that glycoconjugates might have an important role in placental development

Analysis of pulmonary surfactant by Fourier transform infrared spectroscopy after exposure to sevoflurane and isoflurane

Pulmonary surfactant, consisting primarily of phospholipids and four surfactant-specific proteins, is among the first structures that is exposed to inhalation anesthetics. Consequently, changes of pulmonary surfactant due to this exposure could cause respiratory complications after long anesthetic procedures. Fourier transform infrared (FTIR) spectroscopy was used to explore the effects of two inhalation anesthetics, sevoflurane and isoflurane, on a commercially available pulmonary surfactant. The research was primarily focused on the effect of anesthetics on the lipid component of the surfactant. Four different concentrations of anesthetics were added, and the doses were higher from the low clinical doses typically used. Recorded spectra were analyzed using principal component analysis, and the Student's t-test was performed to confirm the results. The exposure to both anesthetics induced similar changes, consistent with the increase of the anesthetic concentration. The most pronounced effect was on the hydrophilic head group of phospholipids, which is in agreement with the disruption of the hydrogen bond, caused by the anesthetics. A change in the band intensities of CH2 stretching vibrations, indicative of a disordering effect of anesthetics on the hydrophobic tails of phospholipids, was also observed. Changes induced by isoflurane appear to be more pronounced than those induced by sevoflurane. Furthermore, our results suggest that FTIR spectroscopy is a promising tool in studying anesthetic effects on pulmonary surfactant

Preživljenje fetusa određeno utjecajem 5-azacitidina na placentu

DNA methylation as a regulatory mechanism for mammalian gene expression is involved in the process of placentation as well as foetal development. 5azaC is a demethylating agent, which incorporates into DNA instead of cytosine and prevents its methylation, subsequently changes expression of genes involved in normal placental and foetal development. Single dose of 5azaC (5 mg/kg) was administered to pregnant rats at 11th, 12th and 13th day of gestation. Placentas and foetuses were isolated at day 20, weighted and histologicaly analyzed. After 5azaC administration on the 11th day of gestation, treated placentas were significantly smaller and labyrinth was significantly reduced. Consequently complete foetal resorption occurred. When administrated at day 12 of gestation, labyrinth was slightly recovered and 24% foetuses survived, while after its application at 13th day of gestation, almost normal distribution of placental layers was found and survival was 96%. These results confirmed epigenetic influence upon placental development. 5azaC caused changes in its structure, especially the reduction of labyrinth that is crucial for foetal survival. After establishment of normal placental layers distribution, 5azaC has no impact on placental morphology, neither on foetal survival. On the other hand, the influence of 5azaC remains visible in appearance of foetal malformations.Metilacija DNA kao regulatorni mehanizam ekspresije gena u sisavaca uključena je kako u procese placentacije tako i razvitka fetusa. Demetilacijsko sredstvo 5azaC ugrađuje se u DNA umjesto citozina i sprječava njegovu metilaciju, što ima za posljedicu promijenjenu ekspresiju gena uključenih u normalnu placentaciju i fetalni razvitak. Trudne ženke Fisher soja štakora tretirane su s 5 mg/kg 5azaC jedanaestog, dvanaestog i trinaestog dana gestacije. Dvadesetog dana gestacije izolirani su, izvagani i histološki analizirani kako fetusi tako i placente. Nakon primjene 5azaC jedanaestog dana gestacije, tretirane placente bile su značajno manje, a labirint reduciran, zbog čega je uslijedila potpuna resorpcija fetusa. Dvanaesti dan primjene 5azaC rezultirao je blagim povećanjem labirinta, a preživljenje fetusa bilo je 24%. Tek trinaestog dana aplikacije 5azaC uspostavila se normalna distribucija labirinta i bazalnog sloja s preživljenjem od 96%. Dobiveni rezultati potvrđuju epigenetski utjecaj na razvitak placente. Primjena 5azaC uzrokovala je promjene u strukturi placente, osobito redukciju labirinta čija je uloga očito ključna za preživljenje fetusa. Nakon uspostave normalne građe placente, 5azaC više nije imao utjecaja na njezinu morfologiju niti na preživljenje fetusa. S druge strane, njegov je teratogeni utjecaj ostao i dalje vidljiv na fetusima u obliku različitih malformacija

Definiranje kvalitete života kod bolesnica s lokaliziranim i uznapredovalim stadijem raka dojke - prvi korak prema onkološkom genetskom savjetovanju

The important goal in breast cancer treatment is to improve patient quality of life. Due to the huge economic burden, it is necessary to estimate the health state utility values for different breast cancer stages accurately. A group of 114 women filled out the EuroQol-5D-3L questionnaire at two time points. The participants were divided into three groups, as follows: group 1 including healthy high-risk individuals; group 2 including patients with localized stage breast cancer; and group 3 including patients with advanced stage breast cancer. Results were expressed either as summary health state utility score or summary visual-analog score. The EuroQol utility index score and EuroQol visual-analog score were statistically significantly higher in the group of healthy high-risk individuals. The EuroQol visual-analog score was mostly correlated with the anxiety/depression and pain/discomfort quality of life dimensions. Health state utility values for different breast cancer stages are a necessary tool to perform economic analyses in breast cancer management decision making, due to its huge economic burden. Special attention should be paid to assessment of the psychosocial aspects of the disease, as well as pain management.Važan cilj u liječenju raka dojke je poboljšanje kvalitete života bolesnica. Zbog velikog financijskog opterećenja važno je točno procijeniti parametre kvalitete života (engl. health state utility values) za različite stadije raka dojke. Skupina od 114 žena ispunila je upitnik EuroQol-5D-3L u dvije vremenske točke. Sudionice su podijeljene u 3 skupine: 1. skupina – zdrave visokorizične osobe; 2. skupina – bolesnice s lokaliziranim stadijem raka dojke i 3. skupina – bolesnice u uznapredovalom stadiju raka dojke. Rezultati su predočeni ili kao sveukupni indeks kvalitete života bolesnica (engl. summary health state utility score) ili kao rezultat na vizualno-analognoj ljestvici (engl. summary visual-analog score). EuroQol utility index score i EuroQol visual-analog score bili su statistički značajno viši u skupini zdravih visokorizičnih osoba. Utvrđeno je da EuroQol visual-analog score korelira sa sljedećim dimenzijama kvalitete života: anksioznost/depresija i bol/nelagoda. Parametri kvalitete života (engl. health state utility values) za različite stadije raka dojke važan su alat za provođenje ekonomske analize pri donošenju odluka o liječenju raka dojke, prvenstveno zbog velikog financijskog opterećenja. Osobitu pozornost trebalo bi usmjeriti na procjenu psihosocijalnih aspekata bolesti, kao i liječenju boli

Preživljavanje embrijskog tkiva štakora nakon ektopične transplantacije

Investigation of the developmental potential of embryonic cells is important in designing novel approaches in the field of regenerative medicine. The purpose of our experiments was to compare the survival of different embryonic tissues transplanted at an ectopic site in vivo. Embryo proper (9.5-day-old), neural retinas (20-day-old), lensectomized eyes (14- and 18-day-old), epiglottis (17-day-old), mandible (13- and 14-day-old), lacrimal gland (17- and 20-day-old) were microsurgically isolated from rat embryos and transplanted under the renal capsule. The embryo-proper survived in transplants for at least 60 days. Fetal rat retina survived in transplants for 180 days forming rosettes. Neural and glia cells with abundant neuropil as well as plexiform layers were found by electron microscopy. The lensectomized eye survived in transplants for 66 days. Transdifferentiation of the retina to lens cells was discovered. The epiglottis, mandible and lacrimal gland survived in transplants for at least 14 days. In the mandible, fully structured teeth developed. This study showed not only the early postimplantation embryo but also various more developed tissues and organs to be able to survive under the renal capsule for at least 14 days. This ectopic site has therefore proved to be a very convenient environment for transplantation experiments.Istraživanje razvojnoga potencijala embrijskih stanica osnova je novih pristupa terapiji u području regenerativne medicine. U našim istraživanjima proučavali smo preživljenje različitih embrijskih tkiva nakon ektopične transplantacije in vivo. Zametak u užem smislu starosti 9,5 dana, neuralna mrežnica (20 dana), lensektomirano oko (14 i 18 dana), epiglotis (17 dana), mandibula (13 i 14 dana) te suzna žlijezda (17 i 20 dana) mikrokirurški su izolirane iz štakorskih zametaka navedenih starosti i transplantirane pod bubrežnu čahuru odraslih štakora. Zametak je u transplantatu preživio 60 dana. Fetalna mrežnica preživjela je čak 180 dana stvarajući rozete u transplantatu. Elektronskom mikroskopijom u njoj su dokazane živčane i glija stanice s obilnim neuropilom i mrežastim slojem. Lensektomirano oko preživjelo je 66 dana, a u transplantatu je otkrivena transdiferencijacija mrežnice u stanice leće. Epiglotis, mandibula i suzna žlijezda preživjele su 14 dana u transplantatu. U mandibuli su se razvili dobro formirani zubi. Pokazali smo, dakle, da uz rani poslijeimplantacijski zametak štakora, različita druga diferenciranija tkiva i organi zametka mogu također preživjeti pod bubrežnom čahurom najmanje 14 dana. Ovo ektopično mjesto ponovno se je dokazalo kao vrlo pogodno za pokuse transplantacije

Ekspresija nuklearnog antigena stanične proliferacije i proteina retinoblastoma u presađenoj fetalnoj suznoj žlijezdi štakora

A model of ectopic organogenesis of the rat fetal lacrimal gland was developed to study lacrimal gland organogenesis. It was done by its transplantation under the renal capsule. In transplants, expression of the tumor suppressor retinoblastoma gene (Rb) and proliferation cell nuclear antigen (PCNA) was assessed at the protein level. Eyes of 17- and 20-day-old rat fetuses were isolated under a dissecting microscope. Lacrimal glands were found and transplanted under the renal capsule of an adult male. After 14 days, transplants were routinely prepared for immunohistochemistry. DAKO Animal Research kit (Peroxidase) was used for detection of monoclonal mouse anti-PCNA and monoclonal mouse anti-human retinoblastoma gene product. In transplants, teratoma-like structures developed that contained lacrimal gland epithelial cells and ducts as well as epidermis. PCNA signal was detected in the nuclei of excretory duct cells and in epidermal basal layer cells. Some transplant cells were found to have the ability of proliferation preserved even after a 14-day period. PCNA signal was absent in well differentiated epithelial cells of lacrimal gland. Intranuclear Rb protein expression was only detected in several scattered cells, indicating the proliferating compartment to be usually larger than the differentiating one in fetal tissues. Assessment of the PCNA and Rb gene expression could prove important for elucidation of pathologic processes of the lacrimal gland, such as tumors or dry eye syndrome.Razvijen je model ektopične organogeneze fetalne suzne žlijezde štakora za ispitivanje organogeneze suzne žlijezde. To je postignuto presađivanjem suzne žlijezde ispod bubrežne kapsule. U presatcima je procjenjivana ekspresija gena tumorske supresije retinoblastoma (Rb) i jezgrenog antigena proliferirajućih stanica (PCNA) na razini bjelančevina. Oči fetusa štakora starih 17 i 20 dana izolirane su pod disekcijskim mikroskopom. Suzne žlijezde su pronađene i presađene pod bubrežnu kapsulu odraslog mužjaka. Nakon 14 dana su presatci rutinski pripravljeni za imunohistokemijsku analizu. Za otkrivanje monoklonskog mišjeg anti-PCNA i monoklonskog mišjeg anti-humanog proizvoda gena retinoblastoma uporabljen je DAKO Animal Research test (peroksidaza). U presatcima su se razvile teratomu slične strukture koje su sadržavale epitelne stanice suzne žlijezde i kanale, te epidermis. Signal PCNA otkriven je u jezgrama stanica odvodnog kanala, te u stanicama epidermnog bazalnog sloja. Time je pokazano kako su neke stanice presatka zadržale sposobnost proliferacije i nakon 14-dnevnog razdoblja. Signal PCNA bio je odsutan u dobro diferenciranim epitelnim stanicama suzne žlijezde. Unutarstanična ekspresija proteina Rb otkrivena je samo u nekoliko razasutih stanica, što pokazuje da je proliferirajući odjeljak u fetalnim tkivima obično veći od diferencirajućeg odjeljka. Procjena ekspresije PCNA i gena Rb mogla bi biti važna za pojašnjenje patoloških procesa u suznoj žlijezdi, kao što su tumori ili sindrom suhog oka

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

Aim was to establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta. Fischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 μmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed. RESULTS: At four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P<0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared. CONCLUSION: Our findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents