The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.     

Influence of tree transpiration on mass water balance of mixed mountain forests of the West Carpathians

Brief information about water balance of the Carpathian temperate forest ecosystem are presented in the paper. Experimental research was done in a mature mixed fir-spruce-beech stand in the research plot “Pol’ana-Hukavský grúň” (850 m a.s.l.) in the south-eastern part of Pol’ana Mts. in the Biosphere Reserve UNESCO in Central Slovakia. Individual parameters of water budget have been continuously monitored. The water consumption of the model beech trees, as well as approximate model beech stand transpiration was estimated on the basis of sap flow measurements and up-scaling through dendrometrical approach. Sap flow of model beech trees was estimated by direct, non-destructive and continuous measurements by tree-trunk heat balance method with internal heating and sensing of temperature. These values were compared with potential evapotranspiration according to Türc. Precipitation parameters (rain and snow precipitation, through-fall precipitation, stem-flow, fog/snow precipitation and infiltration) have been measured simultaneously. Results of mass water balance and the portion of the tree transpiration within the individual water flows are presented. Evapotranspiration of beech-fir forest ecosystem in the middle mountain region (850 m a.s.l.) includes: transpiration (35% of precipitation total), interception (21%), evaporation (8%). There are differences between tree species in mass of transpirated water. Transpiration of spruce and fir reaches two-thirds of beech transpiration. Fog precipitation contribution to the water balance of beech-fir stand is 5%. Concurrently fog precipitation lowers the interception losses of vertical precipitation.     

Functional features of a single chromosome arm in wheat (1AL) determined from its structure

Bread wheat (Triticum aestivum L.) is one of the most important crops globally and a high priority for genetic improvement, but its large and complex genome has been seen as intractable to whole genome sequencing. Isolation of individual wheat chromosome arms has facilitated large-scale sequence analyses. However, so far there is no such survey of sequences from the A genome of wheat. Greater understanding of an A chromosome could facilitate wheat improvement and future sequencing of the entire genome. We have constructed BAC library from the long arm of T. aestivum chromosome 1A (1AL) and obtained BAC end sequences from 7,470 clones encompassing the arm. We obtained 13,445 (89.99%) useful sequences with a cumulative length of 7.57 Mb, representing 1.43% of 1AL and about 0.14% of the entire A genome. The GC content of the sequences was 44.7%, and 90% of the chromosome was estimated to comprise repeat sequences, while just over 1% encoded expressed genes. From the sequence data, we identified a large number of sites suitable for development of molecular markers (362 SSR and 6,948 ISBP) which will have utility for mapping this chromosome and for marker assisted breeding. From 44 putative ISBP markers tested 23 (52.3%) were found to be useful. The BAC end sequence data also enabled the identification of genes and syntenic blocks specific to chromosome 1AL, suggesting regions of particular functional interest and targets for future research.     

Necrosis ofAesculus hippocastanum L.

V práci trvající t?i léta byla sledována choroba jírovce madalu (Aesculus hippocastanum L.), u nás i jinde v st?ední Evropě v?eobecně roz?í?ená. Hlavním pracovi?těm byl zámecký park v Lu?anech u P?e?tic (Hlávkova nadace). P?íznaky a jejich vývoj byly pozorovány také na mnohých jiných místech. Podle t?ídění L. Bose (Wageningen) pat?í tyto symptomy do skupin barevné změny (II), Nekrosa (IV), Deformace (VII) a ?áste?ně i Redukce r?stu (plod?). Jde o chorobu, kterou p?ed 60 lety popisovalSorauer aThomas jako abiosu. Poněvad? některé zjevy poukazovaly na chorobu virového p?vodu, byly podniknuty diagnostické zkou?ky. Serologická byla negativní pro chemismus madalových list?, které nejsou vhodným materiálem pro tuto metodu. Zkou?ky roubovací a o?kovací na zdravých semená?ích byly positivní. Virová nekrosa madal? je choroba systémová, která není p?ená?ena mechanicky dotekem. Snadno se v?ak p?ená?í roubováním a o?kováním. Některé známky nasvěd?ují té? pravděpodobnosti, ?e do jisté míry m??e být p?ená?ena i semenem.     

Immunomodulatory properties of subcellular fractions of a G+ bacterium, Bacillus firmus

Mucosal immunization with non-living antigens usually requires the use of an adjuvant. The adjuvant activity of Bacillus firmus in the mucosal immunization of mice was described by our laboratory previously. In the present study, subcellular localization of B. firmus activities was followed. After mechanical disintegration, subcellular components of bacterium were fractionated by differential centrifugation and salting out. Bacterial cell walls, cytoplasmic membrane fraction, soluble cytoplasmic proteins, and ribosomal fractions were isolated. Their effect on the mouse immune system was studied. Lymphocyte proliferation and immunoglobulin formation in vitro were stimulated by bacterial cell wall (BCW), cytoplasmic membrane (CMF), and ribosomal fractions. BCW and CMF increased antibody formation after intratracheal immunization of mice with influenza A and B viruses, and increased protection against subsequent infection with influenza virus. The BCW fraction even induced intersubtypic cross-protection: Mice immunized with A/California/7/04 (H3N2) + BCW were resistant to the infection by the highly pathogenic A/PR/8/34 (H1N1) virus.