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gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).

Convective Concrete is about a research-driven design process of an innovative thermal mass concept. The goal is to improve building energy efficiency and comfort levels by addressing some of the shortcomings of conventional building slabs with high thermal storage capacity. Such heavyweight constructions tend to have a slow response time and do not make use of the available thermal mass effectively. Convective Concrete explores new ways of using thermal mass in buildings more intelligently. To accomplish this ondemand charging of thermal mass, a network of ducts and fans is embedded in the concrete wall element. This is done by developing customized formwork elements in combination with advanced concrete mixtures. To achieve an efficient airflow rate, the embedded lost formwork and the concrete itself function like a lung. SPOOL, Vol. 4 No. 2: Energy Innovation #4

Convective Concrete is about a research-driven design process of an innovative thermal mass concept. The goal is to improve building energy efficiency and comfort levels by addressing some of the shortcomings of conventional building slabs with high thermal storage capacity. Such heavyweight constructions tend to have a slow response time and do not make use of the available thermal mass effectively. Convective Concrete explores new ways of using thermal mass in buildings more intelligently. To accomplish this ondemand charging of thermal mass, a network of ducts and fans is embedded in the concrete wall element. This is done by developing customized formwork elements in combination with advanced concrete mixtures. To achieve an efficient airflow rate, the embedded lost formwork and the concrete itself function like a lung. SPOOL, Vol. 4 No. 2: Energy Innovation #4

Plasma catecholamine concentrations in 40 patients shortly after accidental injury rose progressively with increasing severity of injury. Norepinephrine and epinephrine concentrations were unrelated other than by a common rise with severity; dopamine was closely related norepinephrine and not independently related to injury severity. Plasma glucose concentrations rose after injury; however, this was related only to the plasma epinephrine concentration and not independently to injury severity. Plasma lactate concentrations, in contrast, showed components related both to severity of injury and independently to norepinephrine and epinephrine concentrations. Plasma insulin concentrations were uniformly low, especially with respect to the hyperglycemia, in patients with high plasma epinephrine concentrations. Plasma concentrations of free fatty acids and of cortisol were unrelated to plasma catecholamine concentrations, as were pulse rate and blood pressure. These relationships confirm the expected role of the sympathoadrenal system in the metabolic changes following injury in man.

Plasma catecholamine concentrations in 40 patients shortly after accidental injury rose progressively with increasing severity of injury. Norepinephrine and epinephrine concentrations were unrelated other than by a common rise with severity; dopamine was closely related norepinephrine and not independently related to injury severity. Plasma glucose concentrations rose after injury; however, this was related only to the plasma epinephrine concentration and not independently to injury severity. Plasma lactate concentrations, in contrast, showed components related both to severity of injury and independently to norepinephrine and epinephrine concentrations. Plasma insulin concentrations were uniformly low, especially with respect to the hyperglycemia, in patients with high plasma epinephrine concentrations. Plasma concentrations of free fatty acids and of cortisol were unrelated to plasma catecholamine concentrations, as were pulse rate and blood pressure. These relationships confirm the expected role of the sympathoadrenal system in the metabolic changes following injury in man.

Recently, research has demonstrated potentiation of the dopamine transporter’s function by ethanol. This, coupled with data showing that the modulation of transporter uptake is determined by changes in trafficking and not function, implied that ethanol increases the number of transporters on the cell surface. To identify which amino acid sites may be targets for ethanol, hybrid transporters were constructed that share different ratios of amino acid sequences with the dopamine transporter and the norepinephrine transporter, a similar protein that lacks ethanol-mediated potentiation. Dopamine transporter mutants were created at the four most promising amino acid residues using site directed mutagenesis. Two mutants expressed in Xenopus oocytes showed some sensitivity to ethanol and two did not. The quadruple transporter mutant, which contains all four amino acid mutations, demonstrated the most interesting phenotype of inability to take up dopamine. The present study confirms this lack of function in human embryonic kidney (HEK) cells, a mammalian expression system, and addresses whether or not the IGLF dopamine transporter is on the surface, yet not functional, or is simply not trafficked to the cell surface. To accomplish this, HEK cells stably expressing the wild-type or quadruple mutant transporter were created. Radioactive uptake assays were used to determine the extent to which each cell type was able to take up dopamine. Cell surface biotinylation and Western blots were then used to identify surface transporters Both preliminary results and the current study showed lack of uptake in HEK cells expressing the mutant, while the latter determined that the mutant protein was present on the cell surface, albeit to a lesser extent than wild-type protein. By demonstrating that the transporter is indeed being trafficked to the surface, the implication that the mutant is simply not functional as a dopamine transporter becomes the more likely possibility. ; Biological Sciences, School of

Recently, research has demonstrated potentiation of the dopamine transporter’s function by ethanol. This, coupled with data showing that the modulation of transporter uptake is determined by changes in trafficking and not function, implied that ethanol increases the number of transporters on the cell surface. To identify which amino acid sites may be targets for ethanol, hybrid transporters were constructed that share different ratios of amino acid sequences with the dopamine transporter and the norepinephrine transporter, a similar protein that lacks ethanol-mediated potentiation. Dopamine transporter mutants were created at the four most promising amino acid residues using site directed mutagenesis. Two mutants expressed in Xenopus oocytes showed some sensitivity to ethanol and two did not. The quadruple transporter mutant, which contains all four amino acid mutations, demonstrated the most interesting phenotype of inability to take up dopamine. The present study confirms this lack of function in human embryonic kidney (HEK) cells, a mammalian expression system, and addresses whether or not the IGLF dopamine transporter is on the surface, yet not functional, or is simply not trafficked to the cell surface. To accomplish this, HEK cells stably expressing the wild-type or quadruple mutant transporter were created. Radioactive uptake assays were used to determine the extent to which each cell type was able to take up dopamine. Cell surface biotinylation and Western blots were then used to identify surface transporters Both preliminary results and the current study showed lack of uptake in HEK cells expressing the mutant, while the latter determined that the mutant protein was present on the cell surface, albeit to a lesser extent than wild-type protein. By demonstrating that the transporter is indeed being trafficked to the surface, the implication that the mutant is simply not functional as a dopamine transporter becomes the more likely possibility. ; Biological Sciences, School of

Two common characteristics of the active site structures of intermediate complexes formed in kinase reactions have been observed by magnetic resonance techniques. First, in creatine, arginine, adenylate and pyruvate kinases (EC2.73.2, 2.7.3.3, 2.7.4.3 and 2.7.1.40, respectively) water is progressively excluded and the structure at the active site is progressively immobilized as each reactant is successively added to the enzyme, as monitored by electron spin resonance (e.s.r) and the enhancement of the proton relaxation rate of water (PRR) due to paramagnetic manganese(II) probe. Significant, and often wide-spread, changes in the protein conformation accompanying successive additions of reaction components are shown with 1H n.m.r. studies of pyruvate kinase. The second characteristic is that, for the ternary enzyme-Mn-nucleotide complexes, two parameters, the e.s.r. spectrum and PRR enhancement values, fall within a range of 10% for all enzymes investigated, with the exception of bovine brain creatine kinase. These similarities suggest a homology in teriary structure at the active sites of these enzymes. An unsuspected aspect of substrate and cofactor specificity has been revealed by e.s.r. spectroscopy of the manganese(II) complexes of the transition-state analogue of creatine kinase (E-MnADP-formate-creatine) and of the ternary phosphoenolpyruvate complex. In the former case, replacement of ADP, the normal substrate, by its substrate analogues IDP or 2acute-deoxyadenosine diphosphate produced two interconvertible species of the transition-state analogue complexes, observed in the e.s.r. spectra as an isotropic species and a highly anisotropic species. With the normal substrate, only the anisotropic species is observed. Similarly, in the case of the complex pyruvate kinase-Mn-phosphoenolpyruvate, when the normal monovalent activator K+ is replaced by the inert tetramethylammonium ion, again two interconvertible species rather than the normal one species are observed by e.s.r. spectroscopy. The implications of these phenomena for the relation of specificity to catalytic efficiency are discussed.

Two common characteristics of the active site structures of intermediate complexes formed in kinase reactions have been observed by magnetic resonance techniques. First, in creatine, arginine, adenylate and pyruvate kinases (EC2.73.2, 2.7.3.3, 2.7.4.3 and 2.7.1.40, respectively) water is progressively excluded and the structure at the active site is progressively immobilized as each reactant is successively added to the enzyme, as monitored by electron spin resonance (e.s.r) and the enhancement of the proton relaxation rate of water (PRR) due to paramagnetic manganese(II) probe. Significant, and often wide-spread, changes in the protein conformation accompanying successive additions of reaction components are shown with 1H n.m.r. studies of pyruvate kinase. The second characteristic is that, for the ternary enzyme-Mn-nucleotide complexes, two parameters, the e.s.r. spectrum and PRR enhancement values, fall within a range of 10% for all enzymes investigated, with the exception of bovine brain creatine kinase. These similarities suggest a homology in teriary structure at the active sites of these enzymes. An unsuspected aspect of substrate and cofactor specificity has been revealed by e.s.r. spectroscopy of the manganese(II) complexes of the transition-state analogue of creatine kinase (E-MnADP-formate-creatine) and of the ternary phosphoenolpyruvate complex. In the former case, replacement of ADP, the normal substrate, by its substrate analogues IDP or 2acute-deoxyadenosine diphosphate produced two interconvertible species of the transition-state analogue complexes, observed in the e.s.r. spectra as an isotropic species and a highly anisotropic species. With the normal substrate, only the anisotropic species is observed. Similarly, in the case of the complex pyruvate kinase-Mn-phosphoenolpyruvate, when the normal monovalent activator K+ is replaced by the inert tetramethylammonium ion, again two interconvertible species rather than the normal one species are observed by e.s.r. spectroscopy. The implications of these phenomena for the relation of specificity to catalytic efficiency are discussed.