Egyszikű és kétszikű fajok szomatikus embriogenezisének összehasonlító vizsgálata biorekatorban, különös tekintettel a tenyésztés fizikai környezetére = Comparative study of vitro somatic embryogenesis of monocotyledonous and dicotyledonous plant in bioreactors with special reference to their physical conditions of cultivation

Triticum aestivum, Triticum monococcum and Fragaria x ananassa éretlen embrió és levél eredetű embriogén in vitro kultúráit és sejtszuszpenziós kultúráit hoztuk létre az egyes fajtákra optimalizált táptalajokon. A különböző búzafajták esetében, a fermentációs léptéknövelési rendszert optimalizáltuk a táptalaj pH értékeinek módosításával, míg a Fragaria esetében egy erősen regeneratív rendszert hoztunk létre. Az elért eredmények vonatkozásában, egy relatíve egyszerű és hatékony tenyésztési módszert fejlesztettünk ki rázatott lombikos és bioreaktoros kísérletekben, annak érdekében, hogy tanulmányozni tudjuk azokat a stressz reakciókat, melyek a tápközegek pH értékeinek periódikus változásai okoznak az átoltások során. Minden egyes 5,8 pH értéknél történő átoltás után, a tápközeg pH értéke nagyon gyorsan 4,4-re esik le, mind a rázatott lombikos, mind pedig a bioreaktoros kísérletekben. Ennek a pH esésnek a döntő szakasza már az oltást követő első órában megfigyelhető. Ezután a pH stabilizálódik egy valamivel magasabb szinten. Az ilyen pH fluktuáció által keltett stressz megszüntethető, ha az átoltás előtt a tápközeg pH értékét 4.6-ra állítjuk be, amely nagyobb hatásfokú sejtszaporodást eredményez. | Summary Triticum aestivum, Triticum monococcum and Fragaria x ananassa immature embryo and leaf originated highly embryogenic in vitro cultures and cell suspension cultures were initiated on optimalised culture media in each species. In case of the different wheat species the fermentation scaling up system were optimalised by modifying the pH conditions of the culture media, meanwhile in case of Fragaria highly regenerative system were established. According to the obtained results, a relatively simple and effective culture systems were developed. in shaking flasks and in bioreactor experiments to study the stress reactions caused by the periodic changes of medium pH during subculturing. After each subculture inoculation at pH 5.8 the medium pH rapidly drops to 4.4 both in shaking flask and bioreactor studies. The most part of this pH decline occurs already in the first hours after inoculation . After this pH stabilizes on a somewhat higher pH value. This stress condition caused by pH fluctuation can be eliminated by decreasing the pH of the medium to 4.6 before inoculation which results in higher cell production efficiency

Plasztisz-differenciálódás: a szerkezet és a működés összefüggései = Plastid differentiation: connection between structure and function

A plasztisz-differenciálódás folyamatában a struktúra és a funkció közötti összefüggéseket vizsgáltuk különböző szerveződési szinteken. Sötétben nevelt csíranövényeket, szerv- és szövettenyészetet, szövet-homogenátumokat, membrán-preparátumokat, intakt növényeket és a természetből begyűjtött rügyeket és egyéb szerveket használtunk. A folyamat indikátoraként a NADPH:protoklorofillid oxidoreduktáz (POR) enzim, illetve a pigmentek komplexeit használtuk. Különböző fluoreszcencia spektroszkópiai módszereket és elektronmikroszkópos vizsgálatokat végeztünk. Megállapítottuk, hogy a POR monomer, dimer és oligomer komplexei is flash fotoaktívak, és dinamikusan egymásba alakulnak. A nem levél eredetű szervekben proplasztisz jellegű etioplasztiszok vannak, amelyekben a POR- és nem kötött pigment-monomerek dominálnak, valamint NADPH hiány jellemző. Emiatt a monomer dominanciájú szervek természetes fényen fotodegradálódnak. A makrodomén szerveződés együtt jár a prolamelláris test kialakulásával, fejlett etioplasztiszok képződésével, növeli a fotoredukció hatékonyságát, és véd a fotodegradáció ellen. A plasztisz-differenciálódást a citokinin szint és a N-ellátottság szabályozza. Proplasztiszok, etioplasztiszok és átmeneti állapotú plasztiszok természetes körülmények között is kialakulnak, egyes kiválasztásra differenciálódott szövetekben a kloroplasztisz speciálisan módosul. Eredményeink alapján a klorofill bioszintézis befejező lépését és a plasztisz-differenciálódást leíró reakciósémákat módosítani kell. | Relationships between the structure and function were studied in the process of plastid differentiation, on different organizational levels. Dark-grown seedlings, organ- and tissue cultures, tissue homogenates, membrane preparations, intact plants, buds and other organs collected from the nature were used. Different complexes of the NADPH:protochlorophyllide oxidoreductase (POR) enzyme and of pigments were used as indicators of the differentiation process. Various fluorescence spectroscopic measurements and electron microscopy studies were done. We have proved that the monomer, dimer and oligomer complexes of POR are flash-photoactive and they transform into each other dinamically. In non-leaf organs, proplastid type etioplasts can be found in which monomers of POR and of non-bound pigments are dominant and NADPH shortage is characteristic. Therefore, these organs undergo photodegradation at natural light conditions. The macrodomain organization proceeds together with the formation of prolamellar bodies, i.e. of developed etioplasts, it increases the efficiency of photoreduction and provides protection against photodegradation. Plastid differentiation is regulated by citokinin concentrations and nitrogen supply. Proplastids, etioplasts and plastids of transitional developmental stages can be found in the nature. In certain secretion tissues, the chloroplasts are specifically modified. On the basis of our results, the reaction schemes describing the terminal steps of chlorophyll biosynthesis and plastid differentiation should be modified

In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses. In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group) were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively. The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology. Keywords: Autoantibody tumor biomarker, Cancer immunology, Colorectal cancer, Immunomics, Protein microarra

GC-MS identification and GC-FID quantitation of terpenoids in Ononidis spinosae Radix

A GC-MS method has been developed for the qualitative analysis of sterol and triterpene (terpenoid) constituents of Ononis spinosa L. (spiny restharrow) root without derivatization. β-Sitosterol, campesterol, stigmasterol, stigmastan-3,5-diene sterol compounds and the triterpene derivatives β-amyrin and α-onocerin were identified. A validated GC-FID quantitative method was developed for measuring β-sitosterol, the main sterol component, in various extracts of this plant, obtained with organic solvents and by supercritical fluid extraction. The extracts were cleaned by saponifying and then the β-sitosterol was quantified in the non-saponifiable fractions by GC-FID with an internal standard. In addition, the relative concentrations of the other terpenoids were also determined. The β-sitosterol content in the non-saponifiable solvent extraction fractions was 0.19-5.5%, that of the supercritical fractions were 4.8-9.2%, depending on the experimental conditions. The hexane and the pilot scale SFE extracts were considered as main sources of terpenoids (71.8%; 93.3%, respectively). © 2008 Vieweg+Teubner | GWV Fachverlage GmbH