A service model for delivering care closer to home.

Upton Surgery (Worcestershire) has developed a flexible and responsive service model that facilitates multi-agency support for adult patients with complex care needs experiencing an acute health crisis. The purpose of this service is to provide appropriate interventions that avoid unnecessary hospital admissions or, alternatively, provide support to facilitate early discharge from secondary care. Key aspects of this service are the collaborative and proactive identification of patients at risk, rapid creation and deployment of a reactive multi-agency team and follow-up of patients with an appropriate long-term care plan. A small team of dedicated staff (the Complex Care Team) are pivotal to coordinating and delivering this service. Key skills are sophisticated leadership and project management skills, and these have been used sensitively to challenge some traditional roles and boundaries in the interests of providing effective, holistic care for the patient.This is a practical example of early implementation of the principles underlying the Department of Health's (DH) recent Best Practice Guidance, 'Delivering Care Closer to Home' (DH, July 2008) and may provide useful learning points for other general practice surgeries considering implementing similar models. This integrated case management approach has had enthusiastic endorsement from patients and carers. In addition to the enhanced quality of care and experience for the patient, this approach has delivered value for money. Secondary care costs have been reduced by preventing admissions and also by reducing excess bed-days. The savings achieved have justified the ongoing commitment to the service and the staff employed in the Complex Care Team. The success of this service model has been endorsed recently by the 'Customer Care' award by 'Management in Practice'. The Surgery was also awarded the 'Practice of the Year' award for this and a number of other customer-focussed projects.

Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neurons.

Creatine uptake by neurons requires a specific creatine transporter (CRT). The purpose of the present work was to investigate the activity and localization of the CRT in primary cultures of hippocampal neurons obtained from 18-day rat embryos. Creatine uptake increased as the neurons differentiated in culture. Immunofluorescence microscopy showed most of the CRT was associated with dendrites, although some CRT was present in axons and axon terminals. Neurons contained high levels of Na(+)-dependent creatine transport activity (K(m) = 45.5 µM; V(max), 1719 pmol creatine/min/mg protein) which was inhibited by competitive inhibitors of the CRT. The IC(50) for guanidinoacetate, a precursor of creatine, was 712 µM, ∼ 15-fold higher than the K(m) for creatine. Incubation of neurons with 1 mM creatine resulted in the accumulation of high levels of creatine which affected the V(max) but not the K(m) for creatine transport. The rate of creatine release from neurons increased in the absence of Na(+) showing the importance of the electrochemical gradient for creatine retention. This is the first detailed study of the CRT in neurons and identifies primary cultures of rat hippocampal neurons as a good model for future studies of the CRT in relation to the effects of creatine on neuronal function and viability.

Interactions of nitrosylhemoglobin and carboxyhemoglobin with erythrocyte.

Nitrosylhemoglobin (HbFe(II)NO) has been detected in vivo, and its role in NO transport and preservation has been discussed. To gain insight into the potential role of HbFe(II)NO, we performed in vitro experiments to determine the effect of oxygenated red blood cells (RBCs) on the dissociation of cell-free HbFe(II)NO, using carboxyhemoglobin (HbFe(II)CO) as a comparison. Results show that the apparent half-life of the cell-free HbFe(II)CO was reduced significantly in the presence of RBCs at 1% hematocrit. In contrast, RBC did not change the apparent half-life of extracellular HbFe(II)NO, but caused a shift in the HbFe(II)NO dissociation product from methemoglobin (metHbFe(III)) to oxyhemoglobin (HbFe(II)O(2)). Extracellular hemoglobin was able to extract CO from HbFe(II)CO-containing RBC, but not NO from HbFe(II)NO-containing RBC. Although these results appear to suggest some unusual interactions between HbFe(II)NO and RBC, the data are explainable by simple HbFe(II)NO dissociation and hemoglobin oxidation with known rate constants. A kinetic model consisting of these reactions shows that (i) deoxyhemoglobin is an intermediate in the reaction of HbFe(II)NO oxidation to metHbFe(III), (ii) the rate-limiting step of HbFe(II)NO decay is the dissociation of NO from HbFe(II)NO, (iii) the magnitude of NO diffusion rate constant into RBC is estimated to be approximately 10(4)M(-1)s(-1), consistent with previous results determined from a competition assay, and (iv) no additional chemical reactions are required to explain these data.

Selective amino acid substitutions convert the creatine transporter to a gamma-aminobutyric acid transporter.

The creatine transporter (CRT) is a member of a large family of sodium-dependent neurotransmitter and amino acid transporters. The CRT is closely related to the gamma-aminobutyric acid (GABA) transporter, GAT-1, yet GABA is not an effective substrate for the CRT. The high resolution structure of a prokaryotic homologue, LeuT has revealed precise details of the substrate binding site for leucine (Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Nature 437, 215-223). We have now designed mutations based on sequence comparisons of the CRT with GABA transporters and the LeuT structural template in an attempt to alter the substrate specificity of the CRT. Combinations of two or three amino acid substitutions at four selected positions resulted in the loss of creatine transport activity and gain of a specific GABA transport function. GABA transport by the "gain of function" mutants was sensitive to nipecotic acid, a competitive inhibitor of GABA transporters. Our results show LeuT to be a good structural model to identify amino acid residues involved in the substrate and inhibitor selectivity of eukaryotic sodium-dependent neurotransmitter and amino acid transporters. However, modification of the binding site alone appears to be insufficient for efficient substrate translocation. Additional residues must mediate the conformational changes required for the diffusion of substrate from the binding site to the cytoplasm.

Substituted cysteine accessibility of the third transmembrane domain of the creatine transporter: defining a transport pathway.

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.

Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells.

The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (95% purity) of the approximately 70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises approximately 3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly alpha-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts ( approximately 100 microg) that make it feasible to consider structural studies of a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family.