In Type 1 diabetic patients with good glycaemic control, blood glucose variability is lower during continuous subcutaneous insulin infusion than during multiple daily injections with insulin glargine.

AIMS: The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) with glargine is uncertain. In this randomized cross-over study, we compared CSII and MDI with glargine in patients with Type 1 diabetes well controlled with CSII. The primary end-point was glucose variability. METHODS: Thirty-nine patients [38.1 +/- 9.3 years old (mean +/- sd), diabetes duration 16.6 +/- 8.2 years, glycated haemoglobin (HbA(1c)) 7.6 +/- 0.8%], already on CSII for at least 6 months, were randomly assigned to CSII with lispro or MDI with lispro and glargine. After 4 months they were switched to the alternative treatment. During the last month of each treatment blood glucose variability was analysed using glucose standard deviation, mean amplitude of glycaemic excursions (MAGE), lability index and average daily risk range (ADRR). As secondary end-points we analysed blood glucose profile, HbA(1c), number of episodes of hypo- and hyperglycaemia, lipid profile, free fatty acids (FFA), growth hormone and treatment satisfaction. RESULTS: During CSII, glucose variability was 5-12% lower than during MDI with glargine. The difference was significant only before breakfast considering glucose standard deviation (P = 0.011), significant overall using MAGE (P = 0.016) and lability index (P = 0.005) and not significant using ADRR. Although HbA(1c) was similar during both treatments, during CSII blood glucose levels were significantly lower, hyperglycaemic episodes were fewer, daily insulin dose was less, FFA were lower and treatment satisfaction was greater than during MDI with glargine. The frequency of hypoglycaemic episodes was similar during both treatments. CONCLUSIONS: During CSII, glucose variability is lower, glycaemic control better and treatment satisfaction higher than during MDI with glargine.

The benzodiazepine binding site of GABAA receptors.

The GABAA receptor belongs, along with the nicotinic acetylcholine receptor, the glycine receptor and the 5-HT3 receptor, to a family of homologous transmitter-gated ion channels mediating fast synaptic transmission. Many classes of drug interact with the GABAA receptor, which is the major inhibitory ion channel in the mammalian brain. Among these drugs are the allosteric modulators acting at the benzodiazepine binding site. In this article, Erwin Sigel and Andreas Buhr discuss recent studies that have identified amino acid residues that are thought to form the binding pocket for these compounds. These residues are probably located at subunit interfaces of the protein pentamer and at least some of them are homologous to residues implicated in channel agonist binding. This implies pseudosymmetry of channel agonist and channel modulatory sites, which may be, as recent data indicate, a general principle realized in other pseudosymmetric protein complexes.

The glucose transporter of Escherichia coli. Purification and characterization by Ni+ chelate affinity chromatography of the IIBCGlc subunit.

The IIBCGlc transmembrane subunit of the glucose transporter of E. coli containing a carboxy-terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography. The protein was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% of the phosphotransferase activity present in cell membranes was recovered. Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange of subunits in the IIBCGlc dimer and it holds promise for the investigation of other protein-protein interactions.

Subunit stoichiometry of oligomeric membrane proteins: GABAA receptors isolated by selective immunoprecipitation from the cell surface.

GABAA receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study alpha 1 beta 3 GABAA receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABAA receptors. The subunit ratio was determined by quantitation of the amount of [35S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the alpha 1 beta 3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of alpha 1/beta 3 subunits was found to be 1.1 +/- 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2 alpha 2 beta. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3 alpha 2 beta. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins.

Amino acid residue 200 on the alpha1 subunit of GABA(A) receptors affects the interaction with selected benzodiazepine binding site ligands.

Mutant alph1 subunits of the GABA(A) receptor were coexpressed in combination with the wild-type beta2 and gamma2 subunits in human embryonic kidney (HEK) 293 cells. The binding properties of various benzodiazepine site ligands were determined by displacement of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4]benzodia zepine-3-carboxylate ([3H]Ro 15-1788). The mutation G200E led to a decrease in zolpidem and 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine (CL 218872) affinity amounting to 16- and 8-fold. Receptors containing a conservative T206V substitution showed a 41- and 38-fold increase in methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and CL 218872 affinity combined with a decrease in diazepam and zolpidem affinity, amounting to 7- and 10-fold. Two mutations, Q203A and Q203S showed almost no effects on the binding of benzodiazepine site ligands, indicating that this residue is not involved in the binding of benzodiazepines and related compounds.

Intercostal nerve transfer to lumbar nerve roots. Part I: development of an animal model and cadaver studies.

This is a description of the development of an animal model for intercostal nerve transfer and saphenous nerve grafting to lumbar nerve roots in the dog. Further, the technical feasibility of intercostal nerve transfer and autogenous sural nerve grafting to the lumbar and sacral nerve roots in the adult and infant human cadaaver is demonstrated graphically.

Intercostal nerve transfer to lumbar nerve roots. Part II: Neuropathologic findings in the animal model.

These are the neuropathologic findings in dogs 8-11 months after a unilateral intercostal nerve transfer and anastomosis to a lumbar nerve root were performed. This is a follow-up study of a procedure that was initially carried out in the adult and infant human cadaver and subsequently in an animal model by Malik and Buhr, as reported in this issue of Spine. At the time of sacrifice, the animals in this study had lost the limp that they had postoperatively. The results demonstrate the viability of the intercostal nerve transfer and its anastomosis to the lumbar motor root. There was supplied by a lumbar nerve root that had been operated upon, but the basis of this was not definitely established.

A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity.

Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat gamma2 or gamma3, in combination with alpha1 and beta2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a gamma3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma2-containing receptors. Eight chimeras between gamma2 and gamma3 were constructed followed by nine different point mutations in gamma2, each to the homologous amino acid residue found in gamma3. Chimeric or mutant gamma subunits were coexpressed with alpha1 and beta2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of gamma2 (gamma2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (gamma2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose-response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the gamma2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Membrane topology of the glucose transporter of Escherichia coli.

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It consists of a transmembrane subunit (IIBCGlc) and a hydrophilic subunit (IIAGlc). Both subunits are transiently phosphorylated. The IIBCGlc subunit is 477 residues long and consists of two domains. The amino-terminal hydrophobic domain is involved in glucose binding and translocation, the carboxyl-terminal domain contains the phosphorylation site (Cys421). Protein fusions between IIBCGlc and beta-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the IIBCGlc subunit. The protein fusions were generated by progressively deleting ptsG from its 3' end and ligating the truncated gene to lacZ and 'phoA lacking promoter and leader sequences. LacZ fusions of high activity (32 out of 54) occur at the amino and carboxyl termini and three internal clusters, and 41 active PhoA fusions occur in four internal clusters. Accordingly the hydrophobic domain of IICGlc (residues 19-336) is suggested to contains eight membrane-spanning segments, with the amino terminus and the COOH-terminal hydrophilic domain (IIBGlc) located on the cytoplasmic face of the membrane. A sequence comparison of IIBCGlc with three related proteins indicates that the periplasmic loops differ in size and sequence while the cytoplasmic loops are better conserved.

The glucose transporter of Escherichia coli. Overexpression, purification, and characterization of functional domains.

The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc). The IICBGlc subunit consists of two domains. The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc. The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Biochem. 219, 945-952).

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.

Trace analysis of pentachlorophenol (PCP) in wood and wood-based products--comparison of sample preparation procedures.

The main problem with routine analyses of pentachlorophenol (PCP) and sodium pentachlorophenolate (Na-PCP) in wood and wood-based products is to determine critical PCP-contents. This task requires a reliable analytical method and statistical testing. An analytical procedure is described, which permits the determination of PCP and Na-PCP with sufficient sensitivity and accuracy. A medium size sieve (4 x 4 mm quadratic mesh) was found suitable for the grinding step. Different extraction techniques and solvents were tested systematically. Extraction by a combination of ultrasonication and shaking in the solvent mixture toluene/sulfuric acid showed best recoveries. The eluted PCP and Na-PCP were derivatized with acetic anhydride and determined by GC/ECD. The limits of detection and determination were 0.14 mg/kg and 0.40 mg/kg, respectively.

Subtle changes in residue 77 of the gamma subunit of alpha1beta2gamma2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site.

Recombinant alpha1beta2gamma2 gamma-aminobutyric acid type A (GABAA) receptors were functionally expressed in Xenopus oocytes. Upon the mutation F77L, diazepam and Ro 15-1788 retained the ability to interact with the benzodiazepine binding site, but zolpidem lost this ability. To quantify these data, radioligand binding experiments were performed using membrane preparations of transiently transfected human embryonic kidney 293 cells. The amino acid gamma77, phenylalanine, was also mutated to tyrosine, tryptophan, and isoleucine. Although there was little effect on Ro 15-1788 binding upon mutation to tyrosine, the loss in affinity for diazepam was from 12 to 2,720 nM. The change to leucine, in contrast, resulted in little change in the diazepam affinity, whereas there was a strongly reduced affinity for zolpidem from 17 to 4,870 nM and for methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) from 1.9 to 1,780 nM, respectively. The change to tryptophan resulted in two-phasic displacement curves, and only about 50% of the [3H]flunitrazepam binding could be displaced by zolpidem, DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam still resulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in Xenopus oocytes showed that all mutant channels displayed a comparatively small change (<4.3-fold) in their apparent agonist affinity and that these channels could still be functionally modulated by ligands of the benzodiazepine binding site. We conclude that subtle changes in gammaF77 drastically affect benzodiazepine pharmacology and that this residue probably interacts directly with most ligands of the benzodiazepine binding site and therefore defines part of the benzodiazepine binding pocket.

The glucose transporter of Escherichia coli. Mutants with impaired translocation activity that retain phosphorylation activity.

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It is a complex consisting of a transmembrane subunit (IIGlc) and a hydrophilic subunit (IIIGlc). Both subunits are transiently phosphorylated. IIIGlc is phosphorylated at a histidyl residue by the cytoplasmic phosphoryl carrier protein phospho-heat-stable phosphoryl carrier protein; IIGlc is phosphorylated at a cysteinyl residue by phospho-IIIGlc. The IIGlc subunit consists of two domains. The N-terminal hydrophobic domain is presumed to span the membrane several times; the C-terminal cytoplasmic domain includes the phosphorylation site. IIGlc phosphorylates glucose and methyl-alpha-D-glucopyranoside in transit across the inner membrane but can also phosphorylate intracellular glucose. Ten mutants resistant against extracellular toxic methyl-alpha-D-glucopyranoside yet capable of phosphorylating intracellular glucose were isolated. Strong impairment of transport activity in these mutants was accompanied by only a slight decrease of phosphorylation activity. Amino acid substitutions occurred at six sites that are clustered in three presumably hydrophilic loops in the transmembrane domain of IIGlc: M17T, M17I, G149S, K150E, S157F, H339Y, and D343G. We presume that the three polypeptide segments are directly involved in sugar translocation and/or binding but are of little importance for phosphorylation activity, folding, and membrane localization of IIGlc.

The benzodiazepine binding pocket of recombinant alpha1beta2gamma2 gamma-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains.

Wild-type alpha1beta2gamma2 gamma-aminobutyric acid (GABA)A receptors and receptors containing a point-mutated subunit gamma2F77Y were expressed by transient transfection in human embryonic kidney 293 cells. Mutant receptors bound the benzodiazepine binding site ligand [3H]flumazenil with similar, subnanomolar affinity as wild-type receptor. Displacement studies with diazepam showed that the affinity for this compound was reduced 250-fold on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chemical entity presumably interacting with the phenyalanine residue in position 77 of the gamma2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substituent of diazepam is located close to gammaF77. Similarly, we investigated the possible location of alpha1T206 and gamma2M130. Electrophysiological data obtained with the wild-type receptor furthermore suggest a simple overlap between positive allosteric modulators acting at the benzodiazepine binding site with its antagonists.

Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces.

Posterior sternoclavicular dislocations.

Twelve cases of posterior sternoclavicular dislocation were seen over 15 years at the Victoria General Hospital and the Izaak Walton Kiliam Hospital for Children in Halifax, NS. Two patients required open reduction of the dislocation but the rest were treated by closed reduction. One of the former group required threaded K wires for stability, but all other dislocations were deemed stable after reduction and application of a figure-of-eight bandage and an arm sling. There were no failures of reduction and no recurrent dislocations; the authors have been successful even in late closed reduction (up to 5 days) and therefore recommend it highly as the primary treatment. Open reduction is much more difficult and hazardous.

Residues at positions 206 and 209 of the alpha1 subunit of gamma-aminobutyric AcidA receptors influence affinities for benzodiazepine binding site ligands.

Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.

The glucose transporter of Escherichia coli. Assignment of the 1H, 13C and 15N resonances and identification of the secondary structure of the soluble IIB domain.

The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane-spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421). A functional form of the IIB domain was over-expressed separately and isotopically labelled with 13C and 15N. A variety of 15N-edited and 13C, 15N triple-resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented. The protein is comprised of four beta-strands forming an antiparallel beta-sheet, two larger alpha-helices at the N- and C-termini and a smaller helical structure of residues 52-58.