A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (Em = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT

AKAP95 regulates splicing through scaffolding RNAs and RNA processing factors

YesAlternative splicing of pre-mRNAs significantly contributes to the complexity of gene expression in higher organisms, but the regulation of the splice site selection remains incompletely understood. We have previously demonstrated that a chromatin-associated protein, AKAP95 (AKAP8), has a remarkable activity in enhancing chromatin transcription. In this study, we have shown that AKAP95 physically interacts with many factors involved in transcription and RNA processing, and functionally regulates pre-mRNA splicing. AKAP95 directly promotes splicing in vitro and the inclusion of a specific exon of an endogenous gene FAM126A. The N-terminal YG-rich domain of AKAP95 is important for its binding to RNA processing factors including selective groups of hnRNP proteins, and its zinc finger domains are critical for pre-mRNA binding. Genome-wide binding assays revealed that AKAP95 bound preferentially to proximal intronic regions on a large number of pre-mRNAs in human transcriptome, and AKAP95 depletion predominantly resulted in reduced inclusion of many exons. AKAP95 also selectively coordinates with hnRNP H/F and U proteins in regulating alternative splicing events. We have further shown that AKAP95 directly interacts with itself. Taken together, our results establish AKAP95 as a novel and mostly positive regulator of premRNA splicing and a possible integrator of transcription and splicing regulation, and support a model that AKAP95 facilitates the splice site communication by looping out introns through both RNA-binding and protein-protein interaction.This work was supported by a UAB start-up fund to H.J

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components

DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers

DIS3 is the catalytic subunit of the exosome, a protein complex involved in the 3’ to 5’ degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two proteincoding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino-acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 as well as the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared to isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared to isoform 2 whereas Acute Myeloid Leukemia (AML) and chronic myelomonocytic leukaemia (CMML) patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers

The epigenetic landscape of renal cancer

This is an accepted manuscript of an article published by Nature in Nature Reviews: Nephrology on 28/11/2016, available online: https://doi.org/10.1038/nrneph.2016.168 The accepted version of the publication may differ from the final published version.The majority of kidney cancers are associated with mutations in the von Hippel-Lindau gene and a small proportion are associated with infrequent mutations in other well characterized tumour-suppressor genes. In the past 15 years, efforts to uncover other key genes involved in renal cancer have identified many genes that are dysregulated or silenced via epigenetic mechanisms, mainly through methylation of promoter CpG islands or dysregulation of specific microRNAs. In addition, the advent of next-generation sequencing has led to the identification of several novel genes that are mutated in renal cancer, such as PBRM1, BAP1 and SETD2, which are all involved in histone modification and nucleosome and chromatin remodelling. In this Review, we discuss how altered DNA methylation, microRNA dysregulation and mutations in histone-modifying enzymes disrupt cellular pathways in renal cancers

Zastosowanie olejów roślinnych w modyfikowanych borem węzłach ślizgowych

In the article, the results of the conducted research were discussed, and the aim was the evaluation of the characteristics of the operation of sliding pairs when vegetable oils are used for lubrication. Particularly, friction pairs with hard layers containing boron were taken into account. For comparison, two of the most common vegetable oils were used during the tests. Rapeseed oil was chosen, since it is most frequently applied in Europe, soybean oil was chosen due to its occurrence in North American areas. The oils are also applied in industry for commercial aims as oil bases for lubricants. The products based on vegetable oils can constitute an alternative for those of mineral origin. However, vegetable oils are characterized by different properties than mineral oils. The most essential issue is the fact that biolubricants are free of sulphurcontaining compounds that can have an unfavourable influence on the health of human beings. The wear measurements were conducted on a block-on-ing test stand T-05. In the sliding pair lubricated with vegetable oils, steel specimen interacted with a counterpart made of bearing alloy. In the second variant of a friction pair, a steel ring specimen with a boronized surface layer was used. The changes of the surface roughness of the specimen and counterpart during operation were evaluated. In the comparative evaluation, the best results were obtained choosing the sample with the hard layer lubricated with soybean oil. In this case, the observed wear of the surface of sliding pairs was the lowest. The lowest friction force and the lowest temperature of the elements of the friction pair also corresponded to this. The application of the TiB2 layer allows reducing the wear of both the sample with the layer and the counterpart with the bearing alloy layer.W artykule omówiono wyniki przeprowadzonych badań, których celem była ocena charakterystyki pracy węzłów ślizgowych, do których smarowania zastosowano oleje roślinne. Podczas testów w celu porównania wykorzystano dwa najbardziej rozpowszechnione oleje roślinne. Wybrano jako najczęściej znajdujący zastosowanie na terenach europejskich olej rzepakowy oraz wykorzystywany w warunkach amerykańskich olej sojowy. Oleje te znajdują zastosowanie również w przemyśle do celów komercyjnych jako bazy olejów smarowych. Produkty oparte na olejach roślinnych stanowić mogą alternatywę dla tych pochodzenia mineralnego. Oleje roślinne charakteryzują się jednak odmiennymi właściwościami niż oleje mineralne. Najbardziej istotną kwestią jest to, iż nie zawierają one składników mogących niekorzystnie oddziaływać na zdrowie istot żywych. Pomiary sił tarcia i zużycia węzła smarowanego tymi olejami wykonano na testerze typu klocek--rolka T-05. W smarowanym olejami roślinnymi węźle współpracowała pierścieniowa próbka stalowa z przeciwpróbką pokrytą stopem łożyskowym. W drugim wariancie pary ciernej zastosowano pierścieniową próbkę stalową, na której zastosowano borowanie w postaci warstwy TiB2. Ocenie poddano zużycie współpracujących elementów oraz zmiany chropowatości ich powierzchni w trakcie pracy. W badaniach porównawczych najlepsze rezultaty uzyskano dla próbek z naniesioną powłoką TiB2 smarowanych olejem sojowym. W tym przypadku zużycie elementów pary ciernej było najmniejsze. Odpowiadały temu również najmniejsze wartości siły tarcia i temperatury podczas pracy

Wpływ oleju silnikowego na tarcie i zużycie warstw powierzchniowych z borem

The aim of the present work is to determine the influence of surface layers with boron and engine oil on the processes of friction and wear in friction pairs. The ring samples with a borided surface layer cooperated under test conditions with counterparts made with CuPb30 bearing alloy. During the tests, the friction pairs were lubricated with 15W/40 Lotos mineral oil and 5W/40 Lotos synthetic oil. The friction pairs lubricated by Lotos synthetic oil a generate stronger friction force and higher temperature in the contact area of friction pairs, as compared to the pairs lubricated by Lotos mineral oil. Lubrication of the friction pairs by mineral oil in the start-up phase causes faster stabilization of the friction conditions than in the case of lubrication by synthetic oil. The wear of bearing alloy was lower when lubricated by Lotos mineral oil than by Lotos synthetic oil. The process of friction in the contact area of the friction pair leads to the destruction of the lubricant and the reduction of its operational properties, especially at high temperatures.Celem pracy jest określenie wpływu warstwy wierzchniej z borem i oleju silnikowego na proces tarcia i zużycia w parach ciernych. Próbki pierścieniowe z borowaną warstwą powierzchni współpracowały w warunkach testowych z przeciwpróbkami wykonanymi ze stopu łożyskowego CuPb30. W trakcie testów pary cierne smarowano olejem mineralnym Lotos 15W/40 i olejem syntetyzującym Lotos 5W/40. Pary cierne smarowane olejem Lotos syntetyczny wytwarzają większe siły tarcia i wyższe temperatury w obszarze styku par ciernych w porównaniu z parami smarowanymi olejem Lotos mineralny. Smarowanie pary ciernej olejem Lotos mineralny w fazie rozruchu powoduje szybszą stabilizację warunków tarcia w obszarze styku niż w przypadku smarowania pary ciernej olejem syntetycznym. Zużycie stopu łożyskowego w warunkach smarowania olejem Lotos mineralny było niższe niż przy smarowaniu olejem Lotos syntetic. Proces tarcia w obszarze styku pary ciernej prowadzi do destrukcji czynnika smarnego i zmniejszenia jego właściwości eksploatacyjnych szczególnie w wyższych temperaturach

Ocena właściwości tribologicznych uszczelnień pracujących w kontakcie z olejami roślinnymi

The article describes the results of the measurement to evaluate the operation characteristic of o-ring seals in case of the operation with vegetable oils. The measurements were made on the model of a sealing node in hydraulic cylinder, where the round cross section seal cooperated with the piston rod. The changes of the friction force during rod movement resulting from the friction between the seal and the rod were analysed. The tests were carried out with two vegetable oils mostly used in industry: rape oil and soybean oil. The products based on vegetable oils can be an alternative to products made on the basis of petroleum. However, vegetable oils are characterized by other properties than mineral oils. The physical and chemical properties of vegetable oils depend on the composition of the fatty acid mixture. The observation of the influence of the occurrence of oxidative gelation on friction force was also conducted. In case of vegetable oils, oxypolymerization tendencies of these fluids are a very significant factor, which can influence the characteristics of friction nodes operation.W artykule omówiono wyniki przeprowadzonych badań, których celem była ocena charakterystyki pracy uszczelnienia w ruchu posuwisto-zwrotnym działającego w kontakcie z olejami roślinnymi. Pomiary wykonano na modelu węzła uszczelniającego w cylindrze hydraulicznym. Badaną parą uszczelniającą było tłoczysko i pierścień uszczelniający typu o-ring. Analizie poddano zmiany siły tarcia podczas roboczego suwu tłoczyska oraz wpływ na ich wartość prędkości przesuwu tłoczyska. Podczas testów w celu porównania wykorzystano dwa szeroko rozpowszechnione oleje roślinne: olej rzepakowy i olej sojowy. Produkty oparte na olejach roślinnych stanowić mogą alternatywę dla tych pochodzenia mineralnego. Oleje roślinne charakteryzują się jednak odmiennymi właściwościami w porównaniu z olejami mineralnymi. W ich przypadku obserwuje się tendencję do przebiegających z czasem reakcji polimeryzacji prowadzących do zmian lepkości i żelowania oleju. Wpływ tych zjawisk na prace węzła uszczelniającego również był przedmiotem obserwacji