Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana

A routinely used protocol is described here including protoplast isolation and PEG-mediated plasmid DNA transformation followed by LSM analysis. We have isolated protoplasts from suspension cultures of wild-type Arabidopsis thaliana Col-0, than protoplasts were transformed with different constructs of AtCRK5 gene tagged with GFP. The protoplast isolation and PEG-mediated transformation process took 6-8 hours. Localization studies could be carried out by LSM microscopy in 0-24 hrs followed transformation. Using this method, we could have localized both 35S::cCRK5-GFP and g::gCRK5-GFP fusion proteins in plasma membrane of cell suspension originated protoplasts, while an N-terminal myristoylation site masked version of 35S::cCRK5-GFP and the N-terminal GFP tagged 35S::GFP-cCRK5 fusion proteins could be found in cell nuclei. Mislocalization of the two last fusion proteins is in good agreement with the fact that the N-terminal myristoylation sites of these proteins were impaired. As a conclusion, the Arabidopsis suspension derived protoplast system is very quick tool for identification of protein localization and to provide possibility to obtain preliminarily information on possible protein localization prior to plant regeneration

A fehérjedegradáció szerepének vizsgálata cukor/stressz jelátviteli folyamatok során Arabidopsis thaliana modellnövényben = The role of protein degradation during sugar/stress signaling in Arabidopsis thaliana

Pályázatunk során kapcsolatot kerestünk az ubiquitin-függö fehérjedegradáció útvonala és a cukor/abiotikus stresszfolyamatok között Arabidopsis-ban a PRL1 (cukor szignálutat reguláló fehérje) és az UFD1 (ubiquitin fusion degradation 1) fehérjék tanulmányozásával. Génexpressziós vizsgálatokkal kimutattuk, hogy az Ufd1 a növény minden részében közel egyforma erősséggel kifejeződik, míg a Prl1 leginkább az apikális merisztémákban expresszálódik. Sem cukor, sem hormonkezelésre nem változott a két gén expressziója, azonban biotikus stresszválaszt indukáló syringolin, illetve hő és hidegstressz hatására az Ufd1 génexpressziója megnövekedett, amely összefüggésbe hozható az UFD1 chaperon-szerű funkciójával az ERAD folyamatában. Az UFD1-HA fehérjét immunlokalizációval mind a sejtmagban, mind a citoszólban detektáltuk, a HA-PRL1 viszont kizárólag a sejtmagban volt látható. Biokémiai vizsgálataink azt mutatták, hogy a PRL1 maga is proteoszóma-függő módon degradálódik és kölcsönhatásban van a spliceosome AtCdc5 komponensével, amely tovább asszociálódik a fehérjedegradációs rendszer különbözö elemeihez.. Az Arabidopsis UFD1 fehérjekomplex tisztításával kimutattuk, hogy in vivo kötődik a 26S proteoszómához és az AtCdc48 ATPázhoz, azonban a PRL1-el közvetlen kölcsönhatást nem sikerült kimutatnunk. Eredményeink alapján azt feltételezzük, hogy az UFD1 és a PRL1 különböző szubsztrátokon keresztül más-más sejtfolyamatban szabályozzák a fehérjedegradációs utakat. | The aim of the present work was to connect the ubiquitin proteasome system (UPS) and the sugar/abiotic stress signalling by characterization of Prl1 (novel regulator in sugar signalling) and Ufd1 (ubiquitin fusion degradation 1) in Arabidopsis. In plants the function of UFD1 protein is completly undisolved. Our gene expression data showed that Ufd1 was almost equally present in all tissues of Arabidopsis, while the Prl1 gene mostly was expressed in the meristematic tissues of root and shoot. Neither hormon nor sugar treatments affected the expression level of Ufd1 and Prl1, however Ufd1 was upregulated by heat and cold stress and by syringolin causing biotic stress, which coincides with the chaperon-like role of UFD1 in the ERAD pathway. The UFD1 protein was detected by immunolocalization studies in both cytoplasm and nucleus, while HA-PRL1 was exclusively present in the nucleus. We showed that the degradation of PRL1 was proteasome dependent. In addition PRL1 was associated with the spliceosome component AtCDC5 protein, which interacts with various elements of the protein degradation system, indicating a potential role for PRL1 in mRNA splicing and UPS. Biochemical purification of UFD1 complex prooved that UFD1 interacts with the proteasome and the AtCdc48 ATPase, but there is no direct interaction between UFD1 and PRL1. Our data suggest that in Arabidopsis PRL1 and UFD1 regulates protein degradation in different cellular processes

Gene expression patterns of insulin-like growth factor 1, insulin-like growth factor 2 and insulin-like growth factor binding protein 3 in human placenta from pregnancies with intrauterine growth restriction

Introduction: In this study, we compared insulin-like growth factor (IGF)-gene expression patterns and characteristics of glucose and insulin metabolism in human placenta from pregnancies with or without intrauterine growth restriction (IUGR). Materials and methods: We compared 101 human placentas from intrauterine growth restriction pregnancies to those of 140 normal pregnancies treated at our department in a one-year period. We have also assessed the serum glucose and insulin levels of the IUGR and control groups. Several possible predicting factors of IUGR were also investigated. Results: Risk for IUGR was suggested by gestational weight gain and gestational increase in maternal body mass index (BMI) as well as maternal birthweight. In pregnancies without IUGR, umbilical cord glucose and insulin levels were significantly higher than in pregnancies with IUGR. In placentas from pregnancies with IUGR an overexpression of the IGF-2 and the insulin-like growth factor binding protein (IGFBP)-3 genes was found. In placentas from pregnancies with male fetal gender we found a significant overexpression of the IGF-2 gene. Discussion: Gestational weight gain and BMI increase seem to predict the development of IUGR. Insulin and carbohydrate metabolism are also impaired in IUGR fetuses. In the placentas from pregnancies with IUGR, IGF-2 is overexpressed reflecting its physiological role in optimizing energy distribution in a low-energy environment

Eco-epidemiology of Borrelia miyamotoi and Lyme borreliosis spirochetes in a popular hunting and recreational forest area in Hungary

BACKGROUND: Borrelia miyamotoi, the newly discovered human pathogenic relapsing fever spirochete, and Borrelia burgdorferi sensu lato are maintained in natural rodent populations. The aim of this study was to investigate the natural cycle of B. miyamotoi and B. burgdorferi s.l. in a forest habitat with intensive hunting, forestry work and recreational activity in Southern Hungary. METHODS: We collected rodents with modified Sherman-traps during 2010–2013 and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all ectoparasites were removed and stored. Samples were screened for pathogens with multiplex quantitative real-time polymerase chain reaction (qPCR) targeting a part of flagellin gene, then analysed with conventional PCRs and sequencing. RESULTS: 177 spleen and 348 skin samples of six rodent species were individually analysed. Prevalence in rodent tissue samples was 0.2 % (skin) and 0.5 % (spleen) for B. miyamotoi and 6.6 % (skin) and 2.2 % (spleen) for B. burgdorferi s.l. Relapsing fever spirochetes were detected in Apodemus flavicollis males, B. burgdorferi s.l. in Apodemus spp. and Myodes glareolus samples. Borrelia miyamotoi was detected in one questing Ixodes ricinus nymph and B. burgdorferi s.l in nymphs and adults. In the ticks removed from rodents DNA amplification of both pathogens was successful from I. ricinus larvae (B. miyamotoi 5.6 %, B. burgdorferi s.l. 11.1 %) and one out of five nymphs while from Ixodes acuminatus larvae, and nymph only B. burgdorferi s.l. DNA was amplified. Sequencing revealed B. lusitaniae in a questing I. ricinus nymph and altogether 17 B. afzelii were identified in other samples. Two Dermacentor marginatus engorged larva pools originating from uninfected hosts were also infected with B. afzelii. CONCLUSIONS: This is the first report of B. miyamotoi occurrence in a natural population of A. flavicollis as well as in Hungary. We provide new data about circulation of B. burgdorferi s.l. in rodent and tick communities including the role of I. acuminatus ticks in the endophilic pathogen cycle. Our results highlight the possible risk of infection with relapsing fever and Lyme borreliosis spirochetes in forest habitats especially in the high-risk groups of hunters, forestry workers and hikers

Méhen belül felszívódó magzati mellékvesevérzés ultrahang-diagnózisa = Ultrasound diagnosis of fetal adrenal hemorrhage

Absztrakt: Az újszülöttkorban igazolt mellékvesevérzések előfordulási gyakorisága az ultrahang-diagnosztika fejlődésével emelkedett az elmúlt években. A méhen belül kialakuló esetek ritkán kerülnek felismerésre. A mellékvese cysticus elváltozásainak differenciáldiagnózisa sokszor csak a megszületés után lehetséges. Esettanulmányunkban a 33. terhességi héten a magzati felhas bal oldalán kialakult és ultrahangvizsgálattal észlelt 4 × 3 cm-es cysticus elváltozás nyomon követését mutatjuk be. A terhesség alatt elvégzett képalkotó vizsgálatok (ultrahang, illetve mágneses rezonancia) mellékvesevérzést igazoltak. A 37. terhességi hétre a vérzés nyom nélkül felszívódott. Három héttel később, a 4150 grammos magzat tervezett császármetszés útján, jó állapotban jött világra. A megszületés után végzett endokrinológiai és ellenőrző ultrahangvizsgálatok eltérést nem találtak. Ez a tanulmány az első publikált eset a szakirodalomban, mely igazolja, hogy magzati mellékvesevérzés kialakulhat a méhen belül, és rövid idő alatt spontán felszívódhat. Esetünk felhívja a figyelmet arra, hogy a mellékvesevérzés a szülési traumától függetlenül is előfordulhat az újszülöttnél. Feltételezhető az is, hogy a várandósság alatti mellékvesevérzés előfordulási gyakorisága nagyobb az eddig az újszülöttkorban leírt esetekből következtetettnél. Orv Hetil. 2019; 160(52): 2073–2078. | Abstract: The confirmed incidence of new-onset adrenal gland hemorrhage has increased with the development of ultrasound diagnostics in recent years. Intrauterine developed cases are rarely recognized. Differential diagnosis of cystic lesions of the adrenal gland is often only possible after birth. In our case study, we report the ultrasonographic diagnosis and follow-up of a cystic lesion measuring 4 × 3 cm in the left fetal epigastrium in the 33rd gestational week. During pregnancy, multimodal imaging methods (both ultrasound and magnetic resonance) have confirmed the diagnosis of hemorrhage in the left adrenal gland. In the 37th gestational week, the hematoma completely resolved. At term, a 4150 gram neonate was delivered in good condition by an elective cesarean section. Postnatal endocrinological and follow-up ultrasound examinations did not find any disorder. This study is the first published case report in the literature that proves that fetal adrenal hemorrhage can intrauterin spontaneously absorb within a short period of time. Our case draws attention to the fact that adrenal bleeding may occur in the newborn regardless of birth trauma. It can also be assumed that the incidence of adrenal bleeding during pregnancy is higher than that reported in neonatal cases. Orv Hetil. 2019; 160(52): 2073–2078

A natriureticus peptidcsalád élettani jellemzői és klinikai szerepe = Physiology and clinical importance of the natriuretic peptide system

Az elmúlt három évtizedben a natriureticus peptidcsalád számos tagját izolálták. Működésük, élettani szerepük újabb részletei váltak világossá. A natriureticus peptidek polipeptidprekurzorokból jönnek létre és endogén rendszerként a szervezet folyadékhomeosztázisának szabályozásában tevékenyek az idegrendszerrel és más hormonokkal együtt. Ennek az egyensúlynak a sérülése az endothel működésének és a bal kamra funkciójának a zavarához vezet, súlyos szövődményeket okozva. Számos cardiovascularis kórképben a diagnózis felállítása és a prognózis mellett terápiás eszközként is szerepet játszanak. Újabban praeeclampsiában is leírták a BNP plazmaszintjének emelkedését. További vizsgálatok segíthetnek a natriureticus peptidek szerteágazó működésének megértéséhez és klinikai felhasználásához. Orv. Hetil., 2011, 152, 1025–1034. | In the last three decades many members of the natriuretic peptide family was isolated. The function and physiological role of these peptides are pleiotropic. All natriuretic peptides are synthesized from polypeptide precursors. Together with the sympathetic nervous system and other hormones they play key roles, like an endogenous system in the regulation of the body fluid homeostasis and blood pressure. Changes in this balance lead to dysfunction in the endothel and left ventricle, which can cause severe complications. In many cardiovascular diseases natriuretic peptides serve not only as marker for diagnosis and prognosis but they have therapeutic importance. In the last years the potential use of the elevated BNP levels for diagnosis of pre-eclampsia was examined. In our review we discuss the current understanding of molecular biology, biochemistry and clinical relevance of natriuretic peptides. Orv. Hetil., 2011, 152, 1025–1034