Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.

MONEY AND EDUCATION: HOW ECONOMIC DISTRIBUTION CORRESPONDS TO ACADEMIC SUCCESS

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Route maintenance using mobility prediction for mobile ad hoc networks

Next generation networks (3G & beyond) will support real-time multimedia applications through traditional wide-area networking concepts as well as hot-spot (WLAN) and ad hoc networking concepts. In order to fulfill the vision of next generation networks a method of maintaining a real-time flow despite frequent topology changes and irregularity in user movement is required. Mobility prediction has been identified as having applications in the areas of link availability estimation and pro-active routing in ad hoc networks. In this work we present the mobility prediction based algorithm for route maintenance in mobile ad hoc networks. Simulation study of the algorithm proves it to offer significant benefits to dynamic source routing (DSR)

The pragmatics of text facilitation : the study of academic English of native and non-native writers

In this paper I describe the discursive strategies related to the writer–reader textual reciprocity. I focus on one way of achieving such reciprocity -- the employment by the writer of facilitative schematic structures and metalanguage where one text segment signposts information conveyed in the segment that follows. I refer to these facilitative schematic structures as "organising relational schemata". I see organising relations as the most explicit components of the rhetorical structure of texts: they illuminate the main message and aid the reader's cognitive processes in the orientation of how information is conveyed by text.
This paper discusses the way the choices of organising relations and associated metalanguage by the writers in different cultures and different discourse communities contribute to the communicative homeostasis in the world of text. It shows how the influence of a native culture and intellectual style together with the forces operating within the writer's international disciplinary community interact in the authorial guidance in the scholarly prose.
I introduce and exemplify three types of organising relational structures: Advance Organisers, Introducers and Enumerators. I trace the utilisation of these three types of relations in sociology research papers written in English and produced in "Anglo" and Polish academic discourse comunities by native English speaking and native Polish speaking scholars. The relational typology adopted is based on a study by Golebiowski (2002), which proposed a theoretical framework for the examination of discoursal structure of research papers, referred to as FARS – Framework for the Analysis of the Rhetorical Structure of Texts. FARS entails a relational taxonomy which displays a pattern of rhetorical relations utilised by the writer to achieve textual coherence.
I describe intertextual differences in the frequency of occurrence of organising relations, their degree of explicitness and their positioning in the hierarchical structure of texts. Differences in the mode of employment of textual organisers suggest that the rhetorical structure of English research prose produced by non-native speakers cannot escape being shaped by the characteristics and conventions of the authors’ first language. They are also attributed to cultural norms and conventions as well as educational systems prevailing within the discourse communities which constitute the social contexts of texts.