Hospital infection control in Australia.

Australia is a large country divided into six states and two territories, each of which has infection control programmes. This paper looks at the organization of infection control in Australia, as well as describing the national bodies involved and recent state initiatives in infection control.

Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.

Identification of amino acid residues within GABA(A) receptor beta subunits that mediate both homomeric and heteromeric receptor expression.

GABA(A) receptors are believed to be heteropentamers that can be constructed from six subunit classes: alpha(1-6), beta(1-4), gamma(1-3), delta, epsilon, and pi. Given that individual neurons often express multiple receptor subunits, it is important to understand how these receptors assemble. To determine which domains of receptor subunits control assembly, we have exploited the differing capabilities of the beta2 and beta3 subunits to form functional cell surface homomeric receptors. Using a chimeric approach, we have identified four amino acids in the N-terminal domain of the beta3 subunit that mediate functional cell surface expression of this subunit compared with beta2, which is retained within the endoplasmic reticulum. Substitution of these four amino acids-glycine 171, lysine 173, glutamate 179, and arginine 180-into the beta2 subunit was sufficient to enable the beta2 subunit to homo-oligomerize. The effect of this putative "assembly signal" on the production of heteromeric receptors composed of alphabeta and betagamma subunits was also analyzed. This signal was not critical for the formation of receptors composed of either alpha1beta2 or alpha1beta3 subunits, suggesting that mutation of these residues did not disrupt subunit folding. However, this signal was important in the formation of betagamma2 receptors. These residues did not seem to affect the initial association of beta2 and gamma2 subunits but appeared to be important for the subsequent production of functional receptors. Our studies identify, for the first time, key residues within the N-terminal domains of receptor beta subunits that mediate the selective assembly of GABA(A) receptors.

Subcellular localization and endocytosis of homomeric gamma2 subunit splice variants of gamma-aminobutyric acid type A receptors.

The expression of alpha and beta gamma-aminobutyric acid type A receptor subunits produces GABA-gated channels which require the incorporation of either the gamma2 or gamma3 subunit for benzodiazepine modulation. Here we examine the role of the gamma2 subunit splice variants, gamma2S and gamma2L which differ by eight amino acids in the major intracellular domain, in mediating cell surface expression. Using immunocytochemistry we have demonstrated that when expressed alone, the gamma2S subunit can access the cell surface and internalize constitutively. In contrast, alpha1, beta2 and gamma2L are retained predominantly in the endoplasmic reticulum (ER) when expressed alone. Replacing the insert which differentiates gamma2L from gamma2S (LLRMFSFK) with eight alanines produces a phenotype identical to gamma2S. Both gamma2 subunits fail to produce high molecular weight oligomers observed for alpha1beta2 and alpha1beta2gamma2 heterooligomers and do not form functional ion channels. Surface expression of gamma2S is repressed upon the coexpression of alpha1 or beta2 subunits, resulting in ER-retained heterooligomers, suggesting that homomeric gamma2S is unlikely to occur in vivo. However, its independent maturation to surface competence and preferential assembly with alpha and beta subunits may ensure the production of functional benzodiazepine-sensitive receptors. Furthermore, the presence of the gamma2 subunit appears to confer an endocytotic capacity to these heterooligomeric receptors.

Assembly of GABAA receptors composed of alpha1 and beta2 subunits in both cultured neurons and fibroblasts.

GABAA receptors are believed to be pentameric hetero-oligomers, which can be constructed from six subunits (alpha, beta, gamma, delta, epsilon, and rho) with multiple members, generating a large potential for receptor heterogeneity. The mechanisms used by neurons to control the assembly of these receptors, however, remain unresolved. Using Semliki Forest virus expression we have analyzed the assembly of 9E10 epitope-tagged receptors comprising alpha1 and beta2 subunits in baby hamster kidney cells and cultured superior cervical ganglia neurons. Homomeric subunits were retained within the endoplasmic reticulum, whereas heteromeric receptors were able to access the cell surface in both cell types. Sucrose density gradient fractionation demonstrated that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse-chase analysis revealed that homomers were degraded, with half-lives of approximately 2 hr for both the alpha1((9E10)) and beta2((9E10)) subunits. Oligomerization of the alpha1((9E10)) and beta2((9E10)) subunits was evident, as demonstrated by the formation of a stable 9 S complex, but this process seemed inefficient. Interestingly the appearance of cell surface receptors was slow, lagging up to 6 hr after the formation of the 9 S receptor complex. Using metabolic labeling a ratio of alpha1((9E10)):beta2((9E10)) of 1:1 was found in this 9 S fraction. Together the results suggest that GABAA receptor assembly occurs by similar mechanisms in both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.

Modulation of GABAA receptors by tyrosine phosphorylation.

gamma-Aminobutyric acid type-A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are presumed to be pentameric heteroligomers assembled from four classes of subunits with multiple members: alpha (1-6), beta (1-3), gamma (1-3) and delta (1). Here, GABAA receptors consisting of alpha 1, beta 1 and gamma 2L subunits, coexpressed in mammalian cells with the tyrosine kinase vSRC (the transforming gene product of the Rous sarcoma virus), were phosphorylated on tyrosine residues within the gamma 2L and beta 1 subunits. Tyrosine phosphorylation enhanced the whole-cell current induced by GABA. Site-specific mutagenesis of two tyrosine residues within the predicted intracellular domain of the gamma 2L subunit abolished tyrosine phosphorylation of this subunit and eliminated receptor modulation. A similar modulation of GABAA receptor function was observed in primary neuronal cultures. As GABAA receptors are critical in mediating fast synaptic inhibition, such a regulation by tyrosine kinases may therefore have profound effects on the control of neuronal excitation.

Glutathione in whole blood: a novel determination using double quantum coherence transfer proton NMR spectroscopy.

Double quantum selective coherence transfer proton NMR spectroscopy has been used to observe glutathione in whole blood. The efficient water suppression of this technique avoids the need to resuspend the cells in D2O, hence avoiding equilibrium and kinetic isotope effects. Using this method we estimate the concentration of glutathione in fresh whole rabbit blood at approximately 1.7 mM.