The blood of 104 medical inpatients was examined at various intervals during storage for 72 hours using a Coulter S counter. Over this period remarkable stability of the white cell count, red cell count, haemoglobin, and mean cell haemoglobin was demonstrated. This permits a useful interpretation of indices obtained in routine postal samples sent to the laboratory by general practitioners for screening. Furthermore, the implications of high correlations between red cell indices in freshly examined blood are considered and the regression line MCV = 2.5 MCH + 16 is derived. Using this relationship the normal range of the Coulter S MCHC is defined as 32.6-33.7 g/dl. The significance of the Coulter MCHC in present-day practice is reassessed and the importance of recognizing values for MCV and MCH not coinciding with the regression line is briefly discussed.
Phosphatidylinositol (4,5)‐bisphosphate (PIP2) is known to regulate the function of ion channels and transporters. The human dopamine (DA) transporter (hDAT) is a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). AMPH’s addictive properties are mediated, at least in part, through elevation of extracellular DA by inducing DA efflux through the DAT. Thus, the objective of our study is to understand how to precisely manipulate the DAT to prevent DA efflux without altering its physiological function of DA uptake. This understanding is paramount to the development of pharmacological therapies for AMPH abuse. Here, we demonstrate that PIP2 directly binds to the hDAT. This binding occurs through electrostatic interactions with positively charged hDAT N‐terminal residues and is shown to facilitate AMPH‐induced, DAT‐mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT‐PIP2 interactions and AMPH‐induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH‐induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate organismal behaviors, such as locomotion. Grant Funding Source : Supported by DGE0909667 & F31 DA 035535‐01(PJH), P22893(HHS), DA13975(AG), P01 DA12408(AG, HW, JAJ)
Mechanisms by which epigenetic modifications regulate alternative splicing are largely unexplored. Differential alternative splicing and histone modification enrichment are key mechanisms for neuronal gene regulation. Both are grossly altered in mouse brain following investigator-administered cocaine. Our group and others have identified the histone modification, H3K36me3, as a putative splicing regulator. In the current study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with enrichment of H3K36me3 at differentially spliced junctions. The splice factor motif for Srsf11 was enriched in splice junctions of cocaine induced alternative exons, yet Srsf11 expression was unchanged by cocaine treatment. Rather, Srsf11 was both differentially spliced and enriched in H3K36me3. Epigenetic editing showed that H3K36me3 functions directly in alternative splicing of Srsf11. Finally, both Srsf11-targeted and global H3K36me3 enrichment enhanced cocaine self-administration. These findings established the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.
Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP 2 ) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP 2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP 2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP 2 -synthesis. We tested the effects of the verified scarcity of PIP 2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP 2 in pull-down assays. On decreased PIP 2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP 2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP 2 . Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP 2 -dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP 2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
