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Inhibition of development of Shiga toxin-converting bacteriophages by either treatment with citrate or amino acid starvation
Get PDFObjectives: Shiga toxin–producing Escherichia coli (STEC) are pathogenic strains, whose virulence depends on induction of Shiga toxin–converting prophages and their subsequent lytic development. We explored which factors or conditions could inhibit development of these phages, potentially decreasing virulence of STEC. Materials and Methods: Lytic development of Shiga toxin–converting bacteriophages was monitored after mitomycin C-provoked prophage induction under various conditions. Phage DNA replication efficiency was assessed by measurement of DNA amount in cells using quantitative polymerase chain reaction. Results: We demonstrated that the use of citrate delayed Shiga toxin–converting phage development after prophage induction. This effect was independent on efficiency of prophage induction and phage DNA replication. However, an excess of glucose reversed the effect of citrate. Amino acid starvation prevented the phage development in bacteria both able and unable to induce the stringent response. Conclusions: Lytic development of Shiga toxin–converting bacteriophages can be inhibited by either the presence of citrate or amino acid starvation. We suggest that the inhibition caused by the latter condition may be due to a block in prophage induction or phage DNA replication or both. Applications: Our findings may facilitate development of procedures for treatment of STEC-infected patients
The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast
Get PDFRot1 is an essential yeast protein originally shown to be implicated in such diverse processes
such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies.
More recently also a role as a molecular chaperone has been discovered. Here we report that
Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the
oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in
the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards
compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo
Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no
effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3Δ
cells was found to be decreased to 45% compared to wild-type membranes, and model
glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an
underglycosylation pattern. By affinity chromatography a physical interaction between Rot1
and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation
process, as glycosylation of distinct glycoproteins of this type were affected as
well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper
glycosylation.
Keywords: ROT1/ N‐glycosylation/ O‐glycosylation/ oligosaccharyltransferase/ dolichol/
Saccharomyces cerevisiae
Downloaded from http://glycob.oxfordjournals.org/ at Instytut Biochemii i Biofizyki PAN on April 17, 201
Genetic structure of declining chinstrap penguin (Pygoscelis antarcticus) populations from South Shetland Islands (Antarctica)
Get PDFSeabirds and their response to climate perturbations are important bioindicators of changes in Antarctic ecosystems. During 30 years of observations of two chinstrap penguin (Pygoscelis antarcticus) colonies, one on King George Island and the other on Penguin Island (South Shetland Islands, Antarctica), the size of the breeding populations decreased by 84 and 41 %, respectively. We applied analyses of amplified fragment length polymorphisms to study the genetic structure of the two populations and to evaluate the influence of the sudden population decrease. Our data indicate that there were only weak genetic differences between the populations, which were not strong enough to support the hypothesis of population differentiation. Weak genetic differences observed between the two populations seem not to be determined by selection processes. We hypothesize that the very low level of between-population genetic structure can be explained by some extent of genetic drift, which is largely compensated by gene flow. Moreover, the two populations seem to remain in a stationary state. Our results support the hypothesis of limited natal philopatry in chinstrap penguins. The observed decrease in population size is probably caused by migration or a rise in juvenile mortality due to the increasing krill limitation of the marine food web. However, detailed research is required to address this issue
Trimetyloaminowe pochodne poli-cis i poli-trans liniowych oligomerów izoprenowych, sposób ich wytwarzania oraz ich zastosowanie
No full textExperimental Relocation of the Mitochondrial ATP9 Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution
Get PDFOnly a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential
functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has
both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of
only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes
naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully
relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in
Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of
chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced
hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional
oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock
response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates
the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the
nucleus during the evolution of multicellular organisms
Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm.
Get PDFIntracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell's intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH
Atmospheric pressure photoionization mass spectrometry as a valuable method for the identification of polyisoprenoid alcohols
Get PDFRATIONALE: The aim of this study was to determine whether Atmospheric Pressure Photoionization (APPI) was
better suited for the mass spectrometric (MS) analysis of polyisoprenoid alcohols than the commonly used Electrospray
Ionization (ESI) method. The APPI method should make possible the use of non-polar solvents without any of the
additives required by ESI, together with improved detection limits.
METHODS: The liquid chromatography (LC)/APPI-MS and LC/ESI-MS spectra of polyisoprenoid alcohol standards
were acquired in both positive and negative ion mode, in methanol and hexane, using a triple quadrupole/linear ion trap
tandem mass spectrometer equipped with both an ESI and an APPI ion source.
RESULTS: In the positive ion mode peaks corresponding to [M +H � H2O]+ and [M +H]+ ions were observed in the
APPI-MS spectra of polyprenols and dolichols, respectively. In the negative ion mode peaks corresponding to
[M +O2]�
• and [M+ Cl]� ions were observed for both classes of polyisoprenoid alcohols. The detection limit of
polyisoprenoid alcohols was established at the level of 10 pg.
CONCLUSIONS: APPI turned out to be a method of choice for the identification and quantitation of polyisoprenoid
alcohols by MS using both polar and non-polar solvents. APPI also enabled greater differentiation of polyprenols and
dolichols occurring together in natural samples and gave much better TIC chromatograms without the need for the post-column salt addition required by ESI
Certain protein transducing agents convert translocated proteins into cell killers.
Get PDFThe majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution
Odpowiedź roślin na czynniki biotyczne
Get PDFWzrost i rozwój roślin zależy także od interakcji z ożywionymi elementami środowiska - innymi roślinami, mikroorganizmami, zwierzętami. Relacje te mogą mieć różny charakter, niektóre są dobroczynne inne zaś niekorzystne dla przebiegu procesów życiowych roślin. W tej części podręcznika skoncentrujemy się na odpowiedzi roślin na wybrane stresogenne bodźce biotyczne: patogeny, szkodniki oraz rośliny pasożytnicze.
W odróżnieniu od stresów abiotycznych, kiedy odpowiedź rośliny zależy wyłącznie od natężenia i czasu trwania bodźca, oddziaływanie z czynnikami biotycznymi ma charakter bardzo dynamiczny i na jego wynik wpływ ma zarówno stan fizjologiczny roślin, funkcjonowanie preformowanych i indukowanych mechanizmów defensywnych, jak również uruchamianie przez atakujące organizmy systemów przeciwobronnych.
Jak dotąd, najpełniej scharakteryzowano oddziaływania roślin z czynnikami chorobotwórczymi (patogenami). Dlatego też pojęcia, koncepcje i hipotezy, sformułowane dla potrzeb tej dziedziny, stały się pierwowzorem dla opisu oddziaływań z innymi organizmami.
Mimo, że w środowisku żyje wiele potencjalnych patogenów, ich kontakt z roślinami rzadko prowadzi do rozwoju choroby. Dzieje się tak, ponieważ większość roślin jest odporna na większość roślinnych patogenów. Najstarszą a zarazem najpowszechniejszą formą odporności jest niegościnność (ang. non-host resistance). Termin ten opisuje przypadki, gdy mikroorganizm nie jest w stanie skolonizować żadnej z roślin w obrębie danego gatunku. Odporność ta może być warunkowana, zarówno istnieniem specyficznych barier (typ budowy organów roślinnych, zawartość szkodliwych lub odstraszających związków chemicznych), jak również działaniem indukowanych mechanizmów obronnych. Podobną odpowiedź obserwuje się podczas ataku szkodników - nicieni i owadów, a także podczas inwazji przez pasożytnicze rośliny, i choć natura tych procesów może się różnić, wszystkie opisywane są wspólnym pojęciem - niegościnność. Niektóre organizmy patogenne wykształciły, na drodze ewolucji, mechanizmy pozwalające przełamać u niektórych roślin ten typ odporności. Mówimy o nich, że są przystosowane lub zaadaptowane do danej rośliny, która nazywana jest wtedy gospodarzem. Często zasiedlenie gospodarza przez intruza związane jest z produkcją specyficznych czynników zwanych efektorami. Dlatego też niektóre rośliny wykształciły inny typ odporności, skierowany swoiście przeciw konkretnym rasom, odmianom, izolatom lub biotypom intruzów, a oparty na rozpoznaniu efektorów, stąd nazywany odpornością indukowaną przez efektory, ETI (ang. effector triggered immunity).
Jedną z bardziej skutecznych strategii obronnych uruchamianych w różnych typach odpowiedzi i skierowaną wobec różnych organizmów jest reakcja nadwrażliwości (HR, ang. hypersensitive response). Polega ona na obumieraniu komórek lokalnie w miejscu inwazji, co prowadzi do zablokowania ataku
