Protein Translocon at the Outer Envelope of Chloroplasts

The first step of preprotein translocation across the membranes of chloroplasts is facilitated by the Toc translocon. Aim of this work was to elucidate the dynamics and the mechanism of action of this molecular machine. The central, stably associated part of the Toc translocon, the Toc core complex, consists of the pore forming Toc75 and two receptors with GTPase activity, Toc34 and Toc159. The question of Toc159 localization was addressed since controversal results on this topic were reported. In this study, membrane localization of Toc159 was confirmed, which has further implications on the mode of its action. To understand the necessity of multiple isoforms of Toc components as found in Arabidopsis thaliana, expression analysis and tissue-specific localization were conducted. Gathered data suggested the existence of several types of the complex, assembled from different types of subunits. These complexes have different preprotein specificities. Expression analysis provided further arguments for dynamic association of the intermembrane space complex with the Toc core complex. Comparison of gene expression and protein presence of translocon subunits contradicts the function of Tic20 as a general pore for stromal targeted proteins, but not as a protein conducting channel per se. For further analysis of the Toc translocon structure and function, its purification and reconstitution into proteoliposomes was reinvestigated. To this end, a technique for liposome size determination in a single spectrophotometric measurement was developed

The Molecular Concept of Protein Translocation across the Outer Membrane of Chloroplasts

Chloroplasts, plant organelles which facilitate photosynthesis, originated when photosynthetic bacteria became a part of non-photosynthetic eukaryotic cell. Chloroplast proteins synthetised in the cytosol have to be transported into the organelle, which is facilitated by the Toc complex on the outer envelope. The preprotein receptors Toc34 and Toc159 associate with the poreforming Toc75 to form the Toc core complex. Toc64 and Toc12 dynamically associate with the core complex and recruit chaperones, forming the intermembrane space complex. Describing Toc159 as integral membrane protein provides insight into its function. After receiving preproteins from Toc34, it uses GTP hydrolysis to push the precursor into the translocation channel. GTP hydrolysis by Toc34 controls its binding/handover cycle. Moreover, different isoforms of Toc components form complexes with different specificity for photosynthetic and non-photosynthetic preproteins. After initial translocation steps, a series of binding spots seems to constitute an affinity chain that guides preproteins further on their translocation pathway. This model parallels the "acid chain" described in mitochondrial import. Considering recent data, a new "big picture" of chloroplast import begins to emerge

Određivanje referentnih intervala hematoloških i biokemijskih pokazatelja u krvi ovaca pasmine lička pramenka

Haematological and blood biochemical variables are key elements for routine assessment of animal health status, with implications in animal production and food safety. However, only reliable reference intervals of haematological and biochemical blood variables can provide the basis for accurate interpretation of the obtained blood results. Haematological and biochemical blood values critically dependent on living conditions, region, husbandry, rearing location, and diet. The aim of this study was to determine the reference intervals for selected important and informative haematological and blood biochemical variables for females of the Croatian indigenous sheep breed, Lika pramenka. We used several advanced statistical methods and assessed their applicability for determination of relevant reference intervals under conditions specific to indigenous sheep breeding. The results show that the reference intervals from the available literature are orientational but not adequate for the specific challenges encountered in raising indigenous breeds in their natural habitat. This was illustrated by the large number of clinically healthy animals with blood variable values falling outside the reference intervals given in the sources available previously.Hematološki i biokemijski krvni pokazatelji ključan su element pri rutinskom procjenjivanju zdravstvenog statusa životinja, produktivnosti životinja te sigurnosti hrane. Međutim, samo pouzdani referentni intervali hematoloških i biokemijskih pokazatelja u krvi mogu pružiti osnovu za precizno tumačenje dobivenih rezultata. Hematološke i biokemijske vrijednosti u krvi uvelike ovise o uvjetima držanja, regiji u kojoj se životinje drže, načinu uzgoja, mjestu uzgoja te hranidbi životinja. Cilj ovog istraživanja bio je utvrditi pouzdane referentne intervale za odabrane važne i informativne hematološke i biokemijske pokazatelje u krvi ženskih životinja hrvatske izvorne pasmine ličke pramenke. U istraživanju su korištene napredne statističke metode te je procijenjena njihova primjenjivost za određivanje relevantnih referentnih intervala u specifičnim uvjetima ovčarstva izvornim pasminama. Rezultati pokazuju da su referentni intervali iz dostupne literature orijentacijski, ali ne i dovoljni pri specifičnim izazovima u uzgoju izvornih pasmina u njihovom prirodnom staništu. To je ilustrirano velikim brojem klinički zdravih životinja čije se vrijednosti krvnih pokazatelja nalaze izvan referentnih intervala danim u prijašnjim istraživanjima

Referentni rasponi hematoloških i biokemijskih pokazatelja u ovaca pasmine dalmatinska pramenka utvrđeni bootstrap metodom na malom uzorku.

Monitoring of animal health status in organic farming relies in part on the availability of exact reference intervals for key hematological and biochemical blood parameters. Once determined, these intervals provide a baseline for interpreting routine laboratory blood tests in order to assess nutritional status, stress or disease. In this study, reference intervals for hematological and blood biochemical parameters were determined for Dalmatian pramenka, an indigenous Croatian sheep breed. A recently developed nonparametric method based on bootstrap resampling was used for calculation of reference intervals, and its performance was compared with that of a conventional nonparametric method. Hematological and blood biochemical profi les from 114 organically reared Dalmatian pramenka sheep were constructed and used for calculation of reference intervals using the new approach. The new method enabled convenient and reliable determination of reference intervals from a relatively small sample size, without the need for transformation or fi ltering of the raw data. Separate reference ranges for male and female sheep were found to be unnecessary for most of the investigated parameters.Praćenje zdravstvenog statusa u ekološkom uzgoju dijelom se temelji na raspoloživosti preciznih referentnih raspona za ključne hematološke i biokemijske pokazatelje. Jednom utvrđeni, ovi rasponi služe kao polazište za interpretaciju rutinskih laboratorijskih pretraga krvi, dajući informacije o hranidbenom statusu životinje te izloženosti stresu ili bolesti. Ovim istraživanjem utvrđeni su referentni rasponi za ključne hematološke i biokemijske pokazatelje ovaca izvorne hrvatske pasmine dalmatinska pramenka. Pri tome je korištena razmjerno nova neparametrijska metoda temeljena na ponovljenom uzorkovanju s ponavljanjem (bootstrap), a njeni rezultati su uspoređeni s rezultatima odabrane konvencionalne neparametrijske metode. Hematološki i biokemijski profi li 114 ekološki uzgajanih ovaca pasmine dalmatinska pramenka poslužili su za računanje referentnih raspona novom metodom, koja se pokazala jednostavnom i pouzdanom čak i na malom uzorku, bez potrebe za transformacijom ili filtriranjem izvornih podataka. Određivanje zasebnih intervala za muške i ženske životinje se pokazalo nepotrebnim kod većine istraživanih parametara

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies. This work is licensed under a Creative Commons Attribution 4.0 International License

Hematological and biochemical parameters of ostriches after vaccination against Newcastle disease

Background and Purpose: Although chickens are routinely vaccinated against Newcastle disease, vaccination of ostrich is less well understood. To assess the effect of vaccination on the health of ostriches, key biochemical parameters and differential blood count were monitored after vaccination by La Sota strain of the Newcastle disease virus, which is widely used in chickens. Materials and Methods: The investigation was performed in 24 adult ostriches divided into three study groups and a control group, each comprising six ostriches. In the study groups birds were vaccinated via different routes: drinking water, oculo-nasally or by spraying. Blood samples were collected immediately before the vaccination and on days 7, 14, 21 and 28. Total erythrocyte counts, hemoglobin concentrations, hematocrit values, as well as total and differential leukocyte counts were assessed. Total albumins and globulins in serum were quantitated spectrophotometrically. Results: Erythrocyte count, hemoglobin concentration, hematocrit values and total leukocyte count were not significantly changed in any group. Only leukocyte differentiation yielded a significant decrease in eosinophiles in all groups and a significant monocyte increase in groups vaccinated via drinking water and oculo-nasally.While the lower eosinophil count could be attributed to the experimental stress, increased monocyte percentage indicates successful immunological reaction against the vaccine virus. In all groups, total serum proteins were elevated within physiological boundaries, with albumin to globulin ratio suggesting stimulation of antibody production. Conclusion: The results did not indicate any adverse health effects. Therefore, the vaccine which is already routinely used for chickens can be safely applied in ostrich

Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2'-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization

Adaptation or Malignant Transformation: The Two Faces of Epigenetically Mediated Response to Stress

Adaptive response to stress is a fundamental property of living systems. At the cellular level, many different types of stress elicit an essentially limited repertoire of adaptive responses. Epigenetic changes are the main mechanism for medium- to long-term adaptation to accumulated (intense, long-term, or repeated) stress. We propose the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis which assumes that the unspecific adaptive stress response grows stronger with the increasing stress level, epigenetically activating response gene clusters while progressively deregulating other cellular processes. The balance between the unspecific adaptive response and the general epigenetic deregulation is critical because a strong response can lead to pathology, particularly to malignant transformation. The main idea of our hypothesis is the continuum traversed by a cell subjected to accumulated stress, which lies between an unspecific adaptive response and pathological deregulation—the two extremes sharing the same underlying cause, which is a manifestation of a unified epigenetically mediated adaptive response to stress. The evolutionary potential of epigenetic regulation in multigenerational adaptation is speculatively discussed in the light of neo-Lamarckism. Finally, an approach to testing the proposed hypothesis is presented, relying on either the publicly available datasets or on conducting new experiments

Indigenous sheep breeds in organic livestock production in karst areas of Croatia

Organic sheep breeding in Croatia is based on indigenous breeds, which are well adapted to their environment. This practice eliminates most of the problems usually encountered when imported foreign breeds have to be adjusted to the new conditions. Karst areas, encompassing about 50% of Croatian territory, are the natural habitat to eight of nine indigenous sheep breeds. These areas are nearly free from pollution, which makes them ideal for organic production. These facts were the foundations for the onset of the projects “Organic lamb production in Croatia” and “Standardisation of some health parameters of Croatian indigenous sheep breeds”. Their goal is to promote and support the development of organic sheep production in Croatian karst areas. In the course of two years, we monitored housing conditions, feeding regime and general health status of animals from five organic flocks. We took blood and faeces samples to determine normal ranges of key physiological and biochemical parameters, since no such previous data existed. They will provide the basis for health monitoring as an aid to veterinarians and sheep breeders in organic production. The development of organic livestock production in the karst areas of Croatia is aimed at accelerating the economic growth of this underdeveloped region by providing a framework for creating a competitive product, while preserving the traditional way of life and protecting the environment