Journal articles: 'Substrate localisation'

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Li, Yanfang, Ki-Eun Park, and Ryan A. Cabot. "Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos." Reproduction, Fertility and Development 27, no. 2 (2015): 385. http://dx.doi.org/10.1071/rd13205.

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Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/β heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

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Neilson, D., and J. S. Thakur. "Continuous Localisation - Delocalisation Transition at Intermediate Electron Densities." Australian Journal of Physics 52, no. 5 (1999): 779. http://dx.doi.org/10.1071/ph99060.

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We find in 2D electron layers in quantum transistors that the interplay between the electron correlations and their interactions with defects in the semiconductor substrate generates a continuous localisation–delocalisation transition for intermediate electron densities (5 ≲ rs ≲ 9). We distinguish this transition from the discontinuous metal–insulator transition which is observed at lower electron densities (rs ≳ 10). The approach we use is based on the behaviour of electrons at low densities. We take into account the interactions between electrons and also their interactions with disorder. We determine a zero temperature phase diagram of localised and delocalised states as a function of electron and impurity densities. The phase boundary of the continuous transition is determined by the localisation length of the electrons.

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Matsuoka, Ken, and Takuji Kawahara. "Modulational instability and wave localisation in Toda lattice with elastic substrate effect." Wave Motion 38, no. 2 (August 2003): 117–30. http://dx.doi.org/10.1016/s0165-2125(03)00024-6.

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Rozova, Vlada S., Ayad G. Anwer, Anna E. Guller, Hamidreza Aboulkheyr Es, Zahra Khabir, Anastasiya I. Sokolova, Maxim U. Gavrilov, et al. "Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness." PLOS Computational Biology 17, no. 7 (July 23, 2021): e1009193. http://dx.doi.org/10.1371/journal.pcbi.1009193.

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Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

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Zhao, Yan, Lilas Dagher, Chao Huang, Peter Miller, and Nassir F. Marrouche. "Cardiac MRI to Manage Atrial Fibrillation." Arrhythmia & Electrophysiology Review 9, no. 4 (December 24, 2020): 189–94. http://dx.doi.org/10.15420/aer.2020.21.

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AF is the most common arrhythmia in clinical practice. In addition to the severe effect on quality of life, patients with AF are at higher risk of stroke and mortality. Recent studies have suggested that atrial and ventricular substrate play a major role in the development and maintenance of AF. Cardiac MRI has emerged as a viable tool for interrogating the underlying substrate in AF patients. Its advantage includes localisation and quantification of structural remodelling. Cardiac MRI of the atrial substrate is not only a tool for management and treatment of arrhythmia, but also to individualise the prevention of stroke and major cardiovascular events. This article provides an overview of atrial imaging using cardiac MRI and its clinical implications in the AF population.

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Land, C. S., and P. W. Hochachka. "COMPARTMENTATION OF LIVER PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE AQUATIC TURTLE PSEUDEMYS SCRIPTA ELEGANS: A REASSESSMENT." Journal of Experimental Biology 182, no. 1 (September 1, 1993): 271–73. http://dx.doi.org/10.1242/jeb.182.1.271.

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Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the conversion of oxaloacetate to pyruvate in the first step of gluconeogenesis. Since oxaloacetate is impermeable to the inner mitochondrial membrane, the localisation of PEPCK within the cell plays a major role in defining substrate preferences for gluconeogenesis. As a result, the immunochemically distinct isoenzymes of PEPCK found within the mitochondrial and the cytosolic cell fractions vary in their proportion to one another between organs and species and with prandial state.

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Collins, G., T. Mahony, A. M. Enright, A. Gieseke, D. de Beer, and V. O'Flaherty. "Determination and localisation of in situ substrate uptake by anaerobic wastewater treatment granular biofilms." Water Science and Technology 55, no. 8-9 (April 1, 2007): 369–76. http://dx.doi.org/10.2166/wst.2007.279.

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Radiotracer incubation experiments and beta microimaging, along with fluorescent in situ hybridisation (FISH), are proposed as a complementary approach to specific methanogenic activity testing and measurement of in vitro substrate utilisation rates to understand better the ecophysiology of anaerobic granular biofilms from wastewater treatment reactors.

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Herburger, Klaus, Louise M. Ryan, Zoë A. Popper, and Andreas Holzinger. "Localisation and substrate specificities of transglycanases in charophyte algae relate to development and morphology." Journal of Cell Science 131, no. 2 (August 21, 2017): jcs203208. http://dx.doi.org/10.1242/jcs.203208.

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Njeim, Mario, and Frank Bogun. "Selecting the Appropriate Ablation Strategy: the Role of Endocardial and/or Epicardial Access." Arrhythmia & Electrophysiology Review 4, no. 3 (2015): 184. http://dx.doi.org/10.15420/aer.2015.4.3.184.

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Percutaneous catheter ablation has emerged as an effective treatment modality for the management of ventricular tachycardia. Despite years of progress in this field, the role of epicardial mapping and ablation needs to be further refined. In this review, we discuss the relationship between the type of underlying heart disease and the location of the arrythmogenic substrate as it pertains to a procedural approach. We describe the contribution of preprocedural and intraprocedural diagnostic tools for the localisation of the arrhythmogenic substrate, with a special emphasis on cardiac MRI and electrophysiological mapping. In our opinion, the preferred approach to target ventricular tachycardia should depend on the patient’s underlying heart disease and the location of scar tissue, which can be best visualised using cardiac MRI.

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Preťová, A., B. Obert, and H. Y. Wetzstein. "Substrate Infiltration and Histological Fixatives Affect theIn Situ Localisation ofβ-Glucuronidase Activity in Transgenic Tissues." Acta Biotechnologica 23, no. 4 (December 2003): 383–88. http://dx.doi.org/10.1002/abio.200390049.

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Dabravolski, Siarhei A., and Stanislav V. Isayenkov. "Evolution of the Membrane Transport Protein Domain." International Journal of Molecular Sciences 23, no. 15 (July 22, 2022): 8094. http://dx.doi.org/10.3390/ijms23158094.

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Membrane transport proteins are widely present in all living organisms, however, their function, transported substrate, and mechanism of action are unknown. Here we use diverse bioinformatics tools to investigate the evolution of MTPs, analyse domain organisation and loop topology, and study the comparative alignment of modelled 3D structures. Our results suggest a high level of conservancy between MTPs from different taxa on both amino acids and structural levels, which imply some degree of functional similarities. The presence of loop/s of different lengths in various positions suggests tax-on-specific adaptation to transported substrates, intracellular localisation, accessibility for post-translation modifications, and interaction with other proteins. The comparison of modelled structures proposes close relations and a common origin for MTP and Na/H exchanger. Further, a high level of amino acid similarity and identity between archaeal and bacterial MTPs and Na/H exchangers imply conservancy of ion transporting function at least for archaeal and bacterial MTPs.

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BOUGHTON, ZOË. "Accent levelling and accent localisation in northern French: Comparing Nancy and Rennes." Journal of French Language Studies 15, no. 3 (November 2005): 235–56. http://dx.doi.org/10.1017/s0959269505002140.

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This article addresses the contention that the regional accents of northern France have become increasingly uniform (‘levelled’) in recent decades. A qualitative, micro-level analysis is carried out on the speech of two older working-class male informants, one from each of the cities of Nancy and Rennes. To contextualise the data, which are drawn from sociolinguistic interviews, previous accounts of the relevant français régionaux are summarised. Close examination of non-standard features in the present data shows that whereas the Nancy informant displays several localised traits, the Rennes speaker's accent is more typical of general colloquial and lower-class usage. While regionally marked variants are disappearing, the degree of accent levelling varies according to region, and thus according to substrate dialect.

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Bird, A. Daniel, Spencer Greatorex, David Reser, Gareth G. Lavery, and Timothy J. Cole. "Hydroxysteroid dehydrogenase HSD1L is localised to the pituitary–gonadal axis of primates." Endocrine Connections 6, no. 7 (October 2017): 489–99. http://dx.doi.org/10.1530/ec-17-0119.

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Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate.

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Grachev, Mikhail A., Arkadij A. Mustaev, Evgeny F. Zaychikov, Anton J. Lindner, and Guido R. Hartmann. "Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius." FEBS Letters 250, no. 2 (July 3, 1989): 317–22. http://dx.doi.org/10.1016/0014-5793(89)80746-x.

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Guilloteau, J. A., J. Lesavre, A. Liénard, and P. Genty. "Wastewater Treatment over Sand Columns – Treatment Yields, Localisation of the Biomass and Gas Renewal." Water Science and Technology 28, no. 10 (November 1, 1993): 109–16. http://dx.doi.org/10.2166/wst.1993.0215.

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Domestic wastewater treatment by infiltration-percolation is a process that is becoming common in France. The aim of this study is to find the depth of biologically active substrate in an infiltration basin by determining the depth of the media colonised by the biomass and by studying oxygen renewal mechanisms. The study using sand columns has allowed simultaneous comparison, on the same profile, of biomass content (ATP), gaseous composition (chromatography) and the variations of the effluent quality (carbon, nitrogen, phosphorus). Down to a depth of 30 cm, removal rates achieved in terms of conventional treatment parameters are very high (COD and SS > 90%, NH4+ ≈ 95%, total phosphorus ≈ 50%). Beyond a depth of 15 cm, the biomass content (expressed in ATP) is ten times less than at the surface, and virtually ceases to develop. Monitoring of O2 levels points to the need for drying periods in order to ensure natural ventilation of the basins. The primary settling stage must be effective in order to avoid any risk of clogging which would prevent the air from being renewed by diffusion.The length of the drying period must be almost double that of the flooding period to allow the media to recover as much of its treatment capacity as possible. This study pinpointed the depth of the biologically active substrate at arround 30 cm. The data obtained from this trial project point to the following design criteria: 1.5 m2/p.e. spread over three basins, and a drying period twice as long as the flooding period. The sand depth will depend on the plant's overall water quality objectives: around 0.50 m for the removal of carbonaceous pollution and nitrification of Kjeldahl nitrogen, a greater depth for disinfection purposes.

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Jullien, Denis, Paola Vagnarelli, William C. Earnshaw, and Yasuhisa Adachi. "Kinetochore localisation of the DNA damage response component 53BP1 during mitosis." Journal of Cell Science 115, no. 1 (January 1, 2002): 71–79. http://dx.doi.org/10.1242/jcs.115.1.71.

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53BP1 is a vertebrate BRCT motif protein, originally described as a direct interactor of p53, which has recently been shown to be implicated in the early response to DNA damage. Upon DNA damage, 53BP1 re-localises to discrete nuclear foci that are thought to represent sites of DNA lesions and becomes hyperphosphorylated. Several observations suggest that 53BP1 is a direct substrate for the ataxia telangiectasia mutated (ATM) kinase. So far, 53BP1 behaviour during mitosis has not been reported in detail. We have examined 53BP1 subcellular distribution in mitotic cells using several antibodies against 53BP1, and ectopic expression of GFP-tagged 53BP1. We found that 53BP1 significantly colocalised with CENP-E to kinetochores. 53BP1 is loaded to kinetochores in prophase, before CENP-E, and is released by mid-anaphase. By expressing various GFP-tagged 53BP1 truncations, the kinetochore binding domain has been mapped to a 380 residue portion of the protein that excludes the nuclear localisation signal and the BRCT motifs. Like many kinetochore-associated proteins involved in mitotic checkpoint signalling, more 53BP1 appears to accumulate on the kinetochores of chromosomes not aligned on the metaphase plate. Finally, we show that 53BP1 is hyperphosphorylated in mitotic cells, and undergoes an even higher level of phosphorylation in response to spindle disruption with colcemid. Our data suggest that 53BP1 may have a role in checkpoint signalling during mitosis and provide the evidence that DNA damage response machinery and mitotic checkpoint may share common molecular components.

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Peters, Daniel, Laura Kay, Jeyanthy Eswaran, Jeremy Lakey, and Meera Soundararajan. "Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism." International Journal of Molecular Sciences 19, no. 12 (December 2, 2018): 3839. http://dx.doi.org/10.3390/ijms19123839.

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Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are “atypical” Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be “relic” domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an “internal arginine finger”, allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms.

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Schregle, Richard, Stefanie Mueller, Daniel F. Legler, Jérémie Rossy, Wolfgang A. Krueger, and Marcus Groettrup. "FAT10 localises in dendritic cell aggresome-like induced structures and contributes to their disassembly." Journal of Cell Science 133, no. 14 (June 16, 2020): jcs240085. http://dx.doi.org/10.1242/jcs.240085.

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ABSTRACTDendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 (also known as UBD), which localises to aggresomes, structures that are similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here, we have investigated whether FAT10 is involved in the formation and turnover of DALIS, and whether proteins accumulating in DALIS can be modified through conjunction to FAT10 (FAT10ylated). We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs, and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing, which may provide a functional rationale as to why FAT10 is selectively induced upon DC maturation.

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Raiborg, Camilla, Bjørn Bremnes, Anja Mehlum, David J. Gillooly, Antonello D’Arrigo, Espen Stang, and Harald Stenmark. "FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes." Journal of Cell Science 114, no. 12 (June 15, 2001): 2255–63. http://dx.doi.org/10.1242/jcs.114.12.2255.

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Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.

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Steinmetz, Eva Louise, Denise Nicole Dewald, and Uwe Walldorf. "Drosophila Homeodomain-Interacting Protein Kinase (Hipk) Phosphorylates the Hippo/Warts Signalling Effector Yorkie." International Journal of Molecular Sciences 22, no. 4 (February 13, 2021): 1862. http://dx.doi.org/10.3390/ijms22041862.

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Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.

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Comparetti, Antonio, Carlo Greco, Michele Massimo Mammano, Navickas Kestutis, Orlando Santo, and Venslauskas Kestutis. "Valorisation of urban green areas for producing renewable energy and biochar as growing substrate of Sicilian aromatic and nutraceutical species in a circular economy." RIVISTA DI STUDI SULLA SOSTENIBILITA', no. 2 (January 2020): 299–314. http://dx.doi.org/10.3280/riss2019-002-s1019.

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This work is aimed at evaluating the possible building up of a gasifier for the energy valorisation of residual biomass deriving from urban areas and their surroundings. The area and the volumes of supply and economic convenience have been determined by implementing a GIS-based geographic method to the localisation and quantification of non-uniform elements of plant biomass. The pyrolysis and gasification processes of the lignocellulosic biomass obtained from pruning and maintenance operations of the urban and ornamental green include the production of a residual amount of biochar. The biochar can be used in agriculture as a soil improver and a substrate alternative to peat in the sustainable production of Sicilian aromatic and nutraceutical species grown in pots within a circular economy.

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Srivastava, Devika, Rukmini Mukherjee, Debdatto Mookherjee, and Oishee Chakrabarti. "Mahogunin-mediated regulation of Gαi localisation during mitosis and its effect on spindle positioning." Biochemistry and Cell Biology 94, no. 4 (August 2016): 359–69. http://dx.doi.org/10.1139/bcb-2015-0161.

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Mahogunin RING Finger 1 (MGRN1) is a ubiquitin E3 ligase known to affect spindle tilt in mitotic cells by regulating α-tubulin ubiquitination and polymerization. In cell culture systems we have found that expressing truncated mutants of MGRN1 leads to various other mitotic anomalies, such as lateral and angular spindle displacements. This seems to be independent of the MGRN1 ligase activity. Our experiments suggest that MGRN1 regulates the balance between the lower molecular weight monomeric Gαi and larger trimeric G-protein complex, along with its abundance in the ternary complex that regulates spindle positioning. The cytosolic isoforms of MGRN1 lead to the enrichment of monomeric Gαi in the cytosol and its subsequent recruitment at the plasma membrane. Excess Gαi at the cell cortex results in an imbalance in the assembly of the ternary complex regulating spindle positioning during mitosis. These observations seem independent of the ligase activity of MGRN1, although we cannot exclude the involvement of an intermediate player that acts as a substrate for MGRN1, and in turn, regulates Gαi.

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Schuller, Marion, and Ivan Ahel. "Beyond protein modification: the rise of non-canonical ADP-ribosylation." Biochemical Journal 479, no. 4 (February 17, 2022): 463–77. http://dx.doi.org/10.1042/bcj20210280.

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ADP-ribosylation has primarily been known as post-translational modification of proteins. As signalling strategy conserved in all domains of life, it modulates substrate activity, localisation, stability or interactions, thereby regulating a variety of cellular processes and microbial pathogenicity. Yet over the last years, there is increasing evidence of non-canonical forms of ADP-ribosylation that are catalysed by certain members of the ADP-ribosyltransferase family and go beyond traditional protein ADP-ribosylation signalling. New macromolecular targets such as nucleic acids and new ADP-ribose derivatives have been established, notably extending the repertoire of ADP-ribosylation signalling. Based on the physiological relevance known so far, non-canonical ADP-ribosylation deserves its recognition next to the traditional protein ADP-ribosylation modification and which we therefore review in the following.

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Knapp, F. F., J. Nitsch, J. Kropp, K. Reichmann, C. Winkler, and S. N. Reske. "Ergebnisse der Fettsäure-SPECT des Myokards bei der koronaren Herzerkrankung." Nuklearmedizin 25, no. 03 (1986): 90–98. http://dx.doi.org/10.1055/s-0038-1624324.

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New developments in radiopharmacology of 123l-labeled metabolic tracers and single-photon emission computerized tomography (SPECT) allow now-a-days the assessment of parameters of cardiac energy metabolism in well-defined areas of the heart muscle. This article will present a brief outline of the basic pathophysiological principles used in the application of 123l-labeled phenyl fatty acids for the evaluation of CAD. First clinical results suggest an important application of cardiac fatty acid metabolic imaging to the detection, localisation and conceivable quantitation of myocardial ischemia, myocardial infarction and assessment of tissue viability. In addition to the diagnostic applications in CAD, cardiac fatty acid metabolic imaging may provide new perspectives to pathophysiological investigations of the coupling of local flow and substrate utilisation in vivo and the effect of therapeutic interventions.

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Tominaga, K., T. Watanabe, and M. Hosoda. "Self-electro-optic effect device using Wannier-Stark localisation in an unstrained InGaAs/InAlAs superlattice grown on GaAs substrate." Electronics Letters 30, no. 10 (May 12, 1994): 782–84. http://dx.doi.org/10.1049/el:19940522.

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Salaun, Christine, Carolina Locatelli, Filip Zmuda, Juan Cabrera González, and Luke H. Chamberlain. "Accessory proteins of the zDHHC family of S-acylation enzymes." Journal of Cell Science 133, no. 22 (November 15, 2020): jcs251819. http://dx.doi.org/10.1242/jcs.251819.

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ABSTRACTAlmost two decades have passed since seminal work in Saccharomyces cerevisiae identified zinc finger DHHC domain-containing (zDHHC) enzymes as S-acyltransferases. These enzymes are ubiquitous in the eukarya domain, with 23 distinct zDHHC-encoding genes in the human genome. zDHHC enzymes mediate the bulk of S-acylation (also known as palmitoylation) reactions in cells, transferring acyl chains to cysteine thiolates, and in so-doing affecting the stability, localisation and function of several thousand proteins. Studies using purified components have shown that the minimal requirements for S-acylation are an appropriate zDHHC enzyme–substrate pair and fatty acyl-CoA. However, additional proteins including GCP16 (also known as Golga7), Golga7b, huntingtin and selenoprotein K, have been suggested to regulate the activity, stability and trafficking of certain zDHHC enzymes. In this Review, we discuss the role of these accessory proteins as essential components of the cellular S-acylation system.

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Anchan, Akshata, Panagiota Kalogirou-Baldwin, Rebecca Johnson, Dan T. Kho, Wayne Joseph, James Hucklesby, Graeme J. Finlay, Simon J. O’Carroll, Catherine E. Angel, and E. Scott Graham. "Real-Time Measurement of Melanoma Cell-Mediated Human Brain Endothelial Barrier Disruption Using Electric Cell-Substrate Impedance Sensing Technology." Biosensors 9, no. 2 (April 15, 2019): 56. http://dx.doi.org/10.3390/bios9020056.

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Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30–60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.

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Na, Z., L. Qingzhang, G. Xuejun, N. Xuemei, Y. Hongbo, and L. Chun. "Expression and localisation of glucose transporter 1 (GLUT1) in dairy goat mammary gland at different physiological stages." Canadian Journal of Animal Science 89, no. 4 (December 1, 2009): 475–80. http://dx.doi.org/10.4141/cjas09038.

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Glucose is the major energy source for mammary epithelial cells, as well as an important substrate for lactose synthesis. Mammary epithelial cells take up glucose from extracellular fluid into the cell through glucose transporter (GLUT). This study was aimed at investigating the expression of GLUT1 glucose transporter in dairy goat mammary gland during puberty, pregnancy, lactation, and involution. Using real-time reverse transcription PCR (qRT-PCR) and Western blotting, we analyzed the expression of GLUT1 mRNA and protein in dairy goat mammary gland. GLUT1 mRNA and protein expression increased during pregnancy and lactation, especially at peak lactation, and decreased strongly after weaning. Furthermore, the location of GLUT1 protein was determined by immunofluorescence laser confocal microscopy. GLUT1 protein localised to the basal and apical plasma membrane of epithelial cells, and also in the cytoplasm. The results from this study showed that GLUT1 is expressed in the dairy goat mammary gland with the greatest expression found in mammary epithelial cells during pregnancy and lactation.Key words: Expression, glucose transporter, goat, mammary gland

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Nelson, Tom, Pankaj Garg, Richard H. Clayton, and Justin Lee. "The Role of Cardiac MRI in the Management of Ventricular Arrhythmias in Ischaemic and Non-ischaemic Dilated Cardiomyopathy." Arrhythmia & Electrophysiology Review 8, no. 3 (August 9, 2019): 191–201. http://dx.doi.org/10.15420/aer.2019.5.1.

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Ventricular tachycardia (VT) and VF account for the majority of sudden cardiac deaths worldwide. Treatments for VT/VF include anti-arrhythmic drugs, ICDs and catheter ablation, but these treatments vary in effectiveness and carry substantial risks and/or expense. Current methods of selecting patients for ICD implantation are imprecise and fail to identify some at-risk patients, while leading to others being overtreated. In this article, the authors discuss the current role and future direction of cardiac MRI (CMRI) in refining diagnosis and personalising ventricular arrhythmia management. The capability of CMRI with gadolinium contrast delayed-enhancement patterns and, more recently, T1 mapping to determine the aetiology of patients presenting with heart failure is well established. Although CMRI imaging in patients with ICDs can be challenging, recent technical developments have started to overcome this. CMRI can contribute to risk stratification, with precise and reproducible assessment of ejection fraction, quantification of scar and ‘border zone’ volumes, and other indices. Detailed tissue characterisation has begun to enable creation of personalised computer models to predict an individual patient’s arrhythmia risk. When patients require VT ablation, a substrate-based approach is frequently employed as haemodynamic instability may limit electrophysiological activation mapping. Beyond accurate localisation of substrate, CMRI could be used to predict the location of re-entrant circuits within the scar to guide ablation.

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Modica, S., A. Morgano, L. Salvatore, M. Petruzzelli, M.-T. Vanier, R. Valanzano, D. L. Esposito, et al. "Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2." Gut 58, no. 9 (February 15, 2009): 1250–59. http://dx.doi.org/10.1136/gut.2008.158386.

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Martin, Sarah L., Anthony K. P. Jones, Christopher A. Brown, Christopher Kobylecki, Grace A. Whitaker, Wael El-Deredy, and Monty A. Silverdale. "Altered Pain Processing Associated with Administration of Dopamine Agonist and Antagonist in Healthy Volunteers." Brain Sciences 12, no. 3 (March 4, 2022): 351. http://dx.doi.org/10.3390/brainsci12030351.

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Striatal dopamine dysfunction is associated with the altered top-down modulation of pain processing. The dopamine D2-like receptor family is a potential substrate for such effects due to its primary expression in the striatum, but evidence for this is currently lacking. Here, we investigated the effect of pharmacologically manipulating striatal dopamine D2 receptor activity on the anticipation and perception of acute pain stimuli in humans. Participants received visual cues that induced either certain or uncertain anticipation of two pain intensity levels delivered via a CO2 laser. Rating of the pain intensity and unpleasantness was recorded. Brain activity was recorded with EEG and analysed via source localisation to investigate neural activity during the anticipation and receipt of pain. Participants completed the experiment under three conditions, control (Sodium Chloride), D2 receptor agonist (Cabergoline), and D2 receptor antagonist (Amisulpride), in a repeated-measures, triple-crossover, double-blind study. The antagonist reduced an individuals’ ability to distinguish between low and high pain following uncertain anticipation. The EEG source localisation showed that the agonist and antagonist reduced neural activations in specific brain regions associated with the sensory integration of salient stimuli during the anticipation and receipt of pain. During anticipation, the agonist reduced activity in the right mid-temporal region and the right angular gyrus, whilst the antagonist reduced activity within the right postcentral, right mid-temporal, and right inferior parietal regions. In comparison to control, the antagonist reduced activity within the insula during the receipt of pain, a key structure involved in the integration of the sensory and affective aspects of pain. Pain sensitivity and unpleasantness were not changed by D2R modulation. Our results support the notion that D2 receptor neurotransmission has a role in the top-down modulation of pain.

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Crosbie, Sarah J., PG Blain, and Faith M. Williams. "An investigation into the role of rat skeletal muscle as a site for xenobiotic metabolism using microsomes and isolated cells." Human & Experimental Toxicology 16, no. 3 (March 1997): 138–45. http://dx.doi.org/10.1177/096032719701600302.

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1 The role of skeletal muscle microsomes as a site of extrahepatic xenobiotic metabolism using n-hexane as a model substrate was investigated. The observed cytochrome P450-dependent metabolism was com pared with that found with liver, and brain micro somal fractions. 2 Rat skeletal muscle microsomes metabolised n-hexane to 1-, 2- and 3-hexanol at rates 40 - 300 times lower than observed with rat liver microsomes. 3 Fast-twitch extensor digitorum longus muscle (EDL) microsomes had twice as much n-hexane hydroxylase activity as the slow-twitch soleus and furthermore the EDL microsomes produced 2-hexanol, a bioactivation product of n-hexane, as a major metabolite. 4 Metabolism of hexane to 1-, 2- and 3- hexanol and 2- hexanone was demonstrated in cultured rat myoblasts. 5 Ethoxyresorufin and pentoxyresorufin O-dealkylation were not detected in either muscle microsomes or myoblasts although immunocytochemical localisation studies were suggestive of the presence of cytochrome P-450. 6 In conclusion, rat skeletal muscle has a low level of xenobiotic metabolism activity. The relevance to neuromuscular toxicity of n-hexane is discussed.

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Skovbjerg, Signe, Lasse Holt-Danborg, Annika W. Nonboe, Zebin Hong, Ásdís K. Frost, Christine R. Schar, Cecilia C. Thomas, Lars Vitved, Jan K. Jensen, and Lotte K. Vogel. "Inhibition of an active zymogen protease: the zymogen form of matriptase is regulated by HAI-1 and HAI-2." Biochemical Journal 477, no. 9 (May 15, 2020): 1779–94. http://dx.doi.org/10.1042/bcj20200182.

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The membrane-bound serine protease matriptase belongs to a rare subset of serine proteases that display significant activity in the zymogen form. Matriptase is critically involved in epithelial differentiation and homeostasis, and insufficient regulation of its proteolytic activity directly causes onset and development of malignant cancer. There is strong evidence that the zymogen activity of matriptase is sufficient for its biological function(s). Activated matriptase is inhibited by the two Kunitz-type inhibitor domain-containing hepatocyte growth factor activator inhibitors 1 (HAI-1) and HAI-2, however, it remains unknown whether the activity of the matriptase zymogen is regulated. Using both purified proteins and a cell-based assay, we show that the catalytic activity of the matriptase zymogen towards a peptide-based substrate as well as the natural protein substrates, pro-HGF and pro-prostasin, can be inhibited by HAI-1 and HAI-2. Inhibition of zymogen matriptase by HAI-1 and HAI-2 appears similar to inhibition of activated matriptase and occurs at comparable inhibitor concentrations. This indicates that HAI-1 and HAI-2 interact with the active sites of zymogen and activated matriptase in a similar manner. Our results suggest that HAI-1 and HAI-2 regulate matriptase zymogen activity and thus may act as regulators of matriptase trans(auto)-activation. Due to the main localisation of HAI-2 in the ER and HAI-1 in the secretory pathway and on the cell surface, this regulation likely occurs both in the secretory pathway and on the plasma membrane. Regulation of an active zymogen form of a protease is a novel finding.

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Orton, Henry W., Iresha D. Herath, Ansis Maleckis, Shereen Jabar, Monika Szabo, Bim Graham, Colum Breen, Lydia Topping, Stephen J. Butler, and Gottfried Otting. "Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites." Magnetic Resonance 3, no. 1 (January 4, 2022): 1–13. http://dx.doi.org/10.5194/mr-3-1-2022.

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Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

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Guettler, Norbert, Edward Nicol, Joern Schmitt, and Kim Rajappan. "Mechanisms of Atrial Fibrillation and Their Impact on Strategies for Catheter Ablation." European Journal of Arrhythmia & Electrophysiology 4, no. 2 (2018): 56. http://dx.doi.org/10.17925/ejae.2018.4.2.56.

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Atrial fibrillation (AF) is the most common sustained arrhythmia, and is ubiquitous in clinical practice. The underlying mechanisms of initiation and maintenance of AF are complex and not completely understood. This knowledge, however, is fundamental for the development of treatment strategies for AF. Within the last 20 years, catheter ablation has played an increasing role as a rhythm control therapy. Based on diverse models for the initiation and maintenance of AF, various ablation strategies have been proposed. The cornerstone of AF ablation has been pulmonary vein isolation (PVI). In persistent AF, however, PVI alone is often not sufficient. This may be because of the structural remodelling of the atria leading to dilation and fibrosis amongst other factors. The optimal strategy for substrate modification, however, is still a matter of investigation. Current studies are concentrating on the ablation of fibrotic areas, especially in the left atrium, either detected by delayed enhancement magnetic resonance imaging or by identification of low-voltage areas as a surrogate marker. The second intensely evaluated strategy is the localisation and ablation of rotational activity. Many further randomised controlled trials will likely be needed to determine the optimal ablation strategy for individual patients.

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36

Soussi-Yanicostas, N., C. Faivre-Sarrailh, J. P. Hardelin, J. Levilliers, G. Rougon, and C. Petit. "Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner." Journal of Cell Science 111, no. 19 (October 1, 1998): 2953–65. http://dx.doi.org/10.1242/jcs.111.19.2953.

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Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.

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Kenny, Stephen, and Bernd Roelfs. "High Aspect Ratio Package Core Production with Electrolytic Deposited Copper." International Symposium on Microelectronics 2010, no. 1 (January 1, 2010): 000855–60. http://dx.doi.org/10.4071/isom-2010-tha3-paper5.

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A typical package core consists of copper clad dielectric which is drilled to produce the necessary through vias serving two functions either as electrical or as thermal conduits. The thermal vias are normally characterised by a low drill pitch and a localisation at the centre of the package core. The vias are plugged after metallisation with a resin material to give the core the required planar surface necessary to provide the basis for the subsequent build up layers. Use of electrolytic deposited copper has been introduced as a new method to fill such through vias, pure copper as a plugging material has obvious advantages due its higher thermal conductivity in comparison to any currently available plugging resins. Significant cost savings are also possible as a fully automated in line processing sequence is available for electrolytic copper deposition in comparison to the more labour intensive resin plugging methods. The package cores utilising this technology have however been restricted to a dielectric thickness in the range 60μm to 100μm and with through via diameter 75μm to 100μm due to limitations in the processing technology. With these dimensions a hole pitch of down to 250μm may be filled reliably and is currently in production, latest results from this technology are shown. Hole filling with higher aspect ratios and particularly with substrates thicker than 200μm has required improvements in processing to ensure uniform copper filling. This paper describes the optimised process for through hole filling and shows the results achieved with dielectric materials 200μm and up to 400μm thick. Current qualification results of substrate 400μm thick with through via 80μm diameter are shown together with the comparison of filling with via pitch variation between 1.0mm and 0.6mm

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Knott, Peter. "Design of a Printed Dipole Antenna Array for a Passive Radar System." International Journal of Antennas and Propagation 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/179296.

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Passive radar (or Passive Coherent Localisation) is an advancing technology for covert operation. The signal transmitted from sources of opportunity such as radio or TV stations is used as illumination for a certain area of interest. Part of the transmitted signal is reflected by radar targets, for example, moving objects such as vehicles or aircraft. Typical radar parameters are derived from the comparison between the direct line-of-sight from the transmitter and the signal scattered from the target object. Such systems are an attractive addition to existing active radar stations because they have the potential to discover low-flying and low-observable targets and no active radar transmitter is required. Printed dipole antennas are very attractive antenna elements for such systems because of their easy fabrication, low-cost, polarisation purity, and low-profile properties. The present paper describes the design of an antenna array using printed dipole elements with flared arms for a passive radar system operating in the GSM900 frequency range. Isolated antenna elements and a small uniform linear antenna array were designed and optimised using computational electromagnetic methods. Several prototypes have been fabricated on conventional microwave PCB substrate material. Preliminary measurement results for antenna matching and far-field radiation patterns are shown.

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Mosienko, Valentina, Seyed Rasooli-Nejad, Kasumi Kishi, Matt De Both, David Jane, Matt Huentelman, Sergey Kasparov, and Anja Teschemacher. "Putative Receptors Underpinning l-Lactate Signalling in Locus Coeruleus." Neuroglia 1, no. 2 (November 16, 2018): 365–80. http://dx.doi.org/10.3390/neuroglia1020025.

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The importance of astrocytic l-lactate (LL) for normal functioning of neural circuits such as those regulating learning/memory, sleep/wake state, autonomic homeostasis, or emotional behaviour is being increasingly recognised. l-Lactate can act on neurones as a metabolic or redox substrate, but transmembrane receptor targets are also emerging. A comparative review of the hydroxy-carboxylic acid receptor (HCA1, formerly known as GPR81), Olfactory Receptor Family 51 Subfamily E Member 2 (OR51E2), and orphan receptor GPR4 highlights differences in their LL sensitivity, pharmacology, intracellular coupling, and localisation in the brain. In addition, a putative Gs-coupled receptor on noradrenergic neurones, LLRx, which we previously postulated, remains to be identified. Next-generation sequencing revealed several orphan receptors expressed in locus coeruleus neurones. Screening of a selection of these suggests additional LL-sensitive receptors: GPR180 which inhibits and GPR137 which activates intracellular cyclic AMP signalling in response to LL in a heterologous expression system. To further characterise binding of LL at LLRx, we carried out a structure–activity relationship study which demonstrates that carboxyl and 2-hydroxyl moieties of LL are essential for triggering d-lactate-sensitive noradrenaline release in locus coeruleus, and that the size of the LL binding pocket is limited towards the methyl group position. The evidence accumulating to date suggests that LL acts via multiple receptor targets to modulate distinct brain functions.

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Bozatzi, Polyxeni, and Gopal P. Sapkota. "The FAM83 family of proteins: from pseudo-PLDs to anchors for CK1 isoforms." Biochemical Society Transactions 46, no. 3 (June 5, 2018): 761–71. http://dx.doi.org/10.1042/bst20160277.

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The eight members of the FAM83 (FAMily with sequence similarity 83) family of poorly characterised proteins are only present in vertebrates and are defined by the presence of the conserved DUF1669 domain of unknown function at their N-termini. The DUF1669 domain consists of a conserved phospholipase D (PLD)-like catalytic motif. However, the FAM83 proteins display no PLD catalytic (PLDc) activity, and the pseudo-PLDc motif present in each FAM83 member lacks the crucial elements of the native PLDc motif. In the absence of catalytic activity, it is likely that the DUF1669 domain has evolved to espouse novel function(s) in biology. Recent studies have indicated that the DUF1669 domain mediates the interaction with different isoforms of the CK1 (casein kinase 1) family of Ser/Thr protein kinases. In turn, different FAM83 proteins, which exhibit unique amino acid sequences outside the DUF1669 domain, deliver CK1 isoforms to unique subcellular compartments. One of the first protein kinases to be discovered, the CK1 isoforms are thought to be constitutively active and are known to control a plethora of biological processes. Yet, their regulation of kinase activity, substrate selectivity and subcellular localisation has remained a mystery. The emerging evidence now supports a central role for the DUF1669 domain, and the FAM83 proteins, in the regulation of CK1 biology.

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Rodenacker, Karsten, Andreas Brühl, Martina Hausner, Martin Kühn, Volkmar Liebscher, Michael Wagner, Gerhard Winkler, and Stefan Wuertz. "QUANTIFICATION OF BIOFILMS IN MULTI-SPECTRAL DIGITAL1 VOLUMES FROM CONFOCAL LASER-SCANNING MICROSCOPES." Image Analysis & Stereology 19, no. 2 (May 3, 2011): 151. http://dx.doi.org/10.5566/ias.v19.p151-156.

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Populations of bacteria in sludge flocs and biofilm marked by fluorescence marked with fluorescent probes are digitised with a confocal laser scanning microscope. These data are used to analyse the microbial community structure, to obtain information on the localisation of specific bacterial groups and to examine gene expression. This information is urgently required for an in-depth understanding of the function and, more generally, the microbial ecology of biofilms. Methods derived from quantitative image analysis are applied to digitised data from confocal laser scanning microscopes to obtain quantitative descriptions of volumetric, topological (and topographical) properties of different compartments of the components under research. In addition to free-moving flocs, also biofilms attached to a substratum in an experimental environment are analysed. Growth form as well as interaction of components are quantitatively described. Classical measurements of volume and intensity (shape, distribution) and distance dependent interaction measurements using methods from mathematical morphology are performed. Mainly image (volume) processing methods are outlined. Segmented volumes are globally and individually (in terms of 3Dconnected components) measured and used for distance mapping transform as well as for estimation of geodesic distances from the substrate. All transformations are applied on the 3D data set. Resulting distance distributions are quantified and related to information on the identity and activity of the probe-identified bacteria.

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Smeets, N., B. van Groen, J. Pertijs, M. Wilmer, B. Smeets, R. Verdijk, and S. de Wildt. "O32 Ontogeny of human kidney OCT2 expression across the paediatric age range." Archives of Disease in Childhood 104, no. 6 (May 17, 2019): e14.2-e14. http://dx.doi.org/10.1136/archdischild-2019-esdppp.32.

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BackgroundIn adults, the organic cation transporter 2 (protein name OCT2, gene name SLC22A2) is localised in the kidney proximal tubules where it mediates organic cation secretion. Hence, the transporter plays a role in the disposition and excretion of several drugs and drug-drug interactions. To better understand the disposition of OCT2 substrate drugs in children, we studied OCT2 localisation and expression in paediatric kidney tissue.MethodsThe expression of OCT2 was visualised in tissue using immunohistochemical staining. Tissues were derived post-mortem from children aged 0 -14 years. Gestational age varied between 24 and 40 weeks. Intensity of the staining at the basolateral membrane was scored by two individual observers using three categories; negative, detectible and high. Agreement between two observers was determined using Cohen’s kappa.Results44 kidney samples (n=17 neonates, n=17 infants, n=7 children, n=3 adolescent) were analysed and scored. There was substantial agreement between two judgements with a kappa of 0.773 (p< 0.005). No age related pattern was observed in the expression of OCT2. Even in the youngest age group, the expression of OCT2 was clearly visible.ConclusionThe kidney expression of OCT2 did not show an age-related pattern. In all age groups, expression levels were similar and OCT2 was properly localised at the basolateral membrane. These findings suggest that, with increasing age, OCT2 will not influence the renal excretion of its substrates.Disclosure(s)Nothing to disclose

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43

Töröcsik, Dániel, Lajos Széles, György Paragh, Zsuzsa Rákosy, Helga Bárdos, László Nagy, Margit Balázs, Aida Inbal, and Róza Ádány. "Factor XIII-A is involved in the regulation of gene expression in alternatively activated human macrophages." Thrombosis and Haemostasis 104, no. 10 (2010): 709–17. http://dx.doi.org/10.1160/th09-11-0805.

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SummaryFactor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.

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Hejgaard, Jørn, William A. Laing, Salla Marttila, Andrew P. Gleave, and Thomas H. Roberts. "Serpins in fruit and vegetative tissues of apple (Malus domestica): expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b." Functional Plant Biology 32, no. 6 (2005): 517. http://dx.doi.org/10.1071/fp04220.

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Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain serpins are well characterised, but serpins have not been identified in eudicot seeds. In apple (Malus domestica Borkh.), the origin of 88 serpin expressed sequence tags (ESTs) identified among 160 000 ESTs from 30 cultivar-, tissue- and time-specific libraries showed that serpin genes are expressed in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with ~85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P2 (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P2–P1′ Glu–Arg–Arg was purified from decorticated seeds and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k a ~4 × 106 m –1 s–1 and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates (k a ~104 m –1 s–1). Chymotrypsin was inhibited at the same site (k a~4 × 103 m –1 s–1), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k d in the order of 10−4 s–1. The bulk of mature seed MdZ1b was localised to the cotyledons. The content of MdZ1b in ripe apples was 5–26 µg per seed, whereas MdZ1b could not be detected in the cortex or skin. Localisation and inhibitory specificity of serpins in monocot and eudicot plants are compared and putative functions are discussed.

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Hulme, Benjamin J., Kathrin K. Geyer, Josephine E. Forde-Thomas, Gilda Padalino, Dylan W. Phillips, Wannaporn Ittiprasert, Shannon E. Karinshak, et al. "Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production." PLOS Pathogens 18, no. 1 (January 13, 2022): e1009828. http://dx.doi.org/10.1371/journal.ppat.1009828.

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α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

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Fang, Zijian, Shiqian Chen, Yusman Manchanda, Stavroula Bitsi, Philip Pickford, Alessia David, Maria M. Shchepinova, et al. "Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells." International Journal of Molecular Sciences 21, no. 21 (November 9, 2020): 8404. http://dx.doi.org/10.3390/ijms21218404.

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The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

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47

Donella-Deana, Arianna, Peter James, Werner Staudenmann, Luca Cesaro, Oriano Marin, Anna Maria Brunati, Maria Ruzzene, and Lorenzo A. Pinna. "Isolation from Spleen of a 57-kDa Protein Substrate of the Tyrosine Kinase Lyn. Identification as a Protein Related to Protein Disulfide-Isomerase and Localisation of the Phosphorylation Sites." European Journal of Biochemistry 235, no. 1-2 (January 1996): 18–25. http://dx.doi.org/10.1111/j.1432-1033.1996.00018.x.

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Jansen, S., M. Pantaleon, and P. Kaye. "236.Differential expression of monocarboxylate cotransporter proteins in preimplantation embryos." Reproduction, Fertility and Development 16, no. 9 (2004): 236. http://dx.doi.org/10.1071/srb04abs236.

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During preimplantation development mouse embryos demonstrate a switch in substrate preference. Pyruvate consumption, high during the first few cleavage stages, declines as the morula develops to a blastocyst, when glucose becomes the preferred substrate. Whilst pyruvate utilisation has been well characterised, changes in the function and expression of pyruvate transporters during this crucial period remain unclear. Pyruvate, lactate and other monocarboxylates are transported across mammalian cell membranes via a specific H+-monocarboxylate cotransporter (MCT). Fourteen members of this family have been identified of which MCT1, MCT2 and MCT4 are well characterised. Although mRNA expression profiles are known during early mouse development (1,2), the specific roles of each protein isoform are unknown. In order to understand these, the expression pattern for each isoform and their cellular localisation during preimplantation development have been determined. Mouse embryos were freshly collected from superovulated Quackenbush mice at 24, 48, 72 and 96 h post-hCG and expression of MCT1, MCT2 and MCT4 analysed by confocal laser scanning immunohistochemistry. Our results confirm that all three MCT proteins are expressed in preimplantation embryos. Immunoreactivity for MCT1 and MCT2 appears diffuse throughout the cytoplasm of cleavage stage embryos. As development proceeds, MCT1 localised to the basolateral membranes of morulae and blastocysts, whilst stronger MCT2 expression was found on the apical trophectoderm as well as the inner cell mass. MCT4 immunoreactivity on the other hand is apparent at cell-cell contact sites in cleavage stage embryos and morulae, but it is not apparent in the blastocyst. The demonstration of different expression patterns for MCT1, MCT2 and MCT4 in mouse embryos implies specific functional roles for each in the critical regulation of H+, pyruvate and lactate transport during preimplantation development. (1) Harding EA, Day ML, Gibb CA, Johnson MH, Cook DI (1999) The activity of the H+-monocarboxylate cotransporter during pre-implantation development in the mouse. Eur. J. Physiol. 438, 397–404. (2) H�rubel F, El Mouatassim S, Gu�rin P, Frydman R, M�n�zo Y (2002) Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development. Zygote 10, 175–181.

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Watanabe, Naoko, Yukio Kume, Yumiko Satoh, Makoto Kaneko, Daiya Takai, Kazuaki Tejima, Masakazu Nagamine, et al. "Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis." Thrombosis and Haemostasis 102, no. 08 (2009): 389–96. http://dx.doi.org/10.1160/th08-11-0732.

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SummaryAlthough hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity.To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle α-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle α-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle α-actin in the livers of rats fed a choline-deficient L-amino aciddefined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

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Jansen, S., M. Pantaleon, and P. L. Kaye. "295. Peroxisome proliferator activated receptor-alpha is involved in H+-monocarboxylate transporter 2 and catalase protein expression in cultured preimplantation mouse embryos." Reproduction, Fertility and Development 17, no. 9 (2005): 125. http://dx.doi.org/10.1071/srb05abs295.

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Cleavage stage embryos consume pyruvate before switching to glucose as the major energy substrate for blastocyst formation. This switch is conditional, because freshly collected two-cell embryos form blastocysts without glucose by increasing pyruvate consumption. Zygotes cultured without glucose cannot adapt in this way and degenerate, but paradoxically demonstrate upregulation of the H+-monocarboxylate transporter protein, MCT2, in morulae. MCT2 is a high affinity transporter implicated in redox shuttling for peroxisomal beta-oxidation of fatty acids.3 Fatty acids may provide energy for embryos2 but peroxisomal beta-oxidation has not been explored in preimplantation development. Rat oocytes possess a primitive peroxisomal system.1 The possibility therefore exists that MCT2 may also be linked to fatty acid metabolism in embryos. Peroxisome proliferator activated receptor (PPAR)-alpha is a transcriptional regulator of fatty acid transport and beta-oxidation, and controls expression of catalase, a major peroxisomal enzyme. This investigation explores the role of PPAR-α in the glucose-driven control of MCT2 expression in mouse embryos. Zygotes (18 h post-hCG) were cultured in KSOM in the presence or absence of glucose, or KSOM with selective agonists of PPAR-α, fenofibrate and WY 14643. Expression of MCT2 and catalase was analysed by confocal laser scanning immunohistochemistry and western blot. Results confirm the presence of catalase throughout preimplantation development. With glucose, cytoplasmic immunoreactivity for catalase was punctate and diffuse, while MCT2 was localised to apical membranes of outer blastomeres in morulae. Without glucose, catalase and MCT2 expression were increased with notable localisation of catalase to nuclei. This response was reflected in morulae cultured in the presence of glucose and PPAR-α agonists. These data suggest that PPAR-α plays a role in controlling catalase and MCT2 expression in embryos, and that conditions in the absence of glucose are more conducive for PPAR-α activation. (1)Figueroa C, Kawada ME, Veliz LP, Hidalgo U, Barros C, Gonzalez S and Santos MJ (2000) Peroxisomal proteins in rat gametes. Cell Biochem Biophys 32, 259–268.(2)Hewitson LC, Martin KL and Leese HJ (1996) Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses. Mol Reprod Dev 43, 323–330.(3)McClelland GB, Khanna S, Gonzalez GF, Butz CE and Brooks GA (2003) Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system? Biochem Biophys Res Commun 304, 130–135.

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Journal articles on the topic 'Substrate localisation'

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