Journal articles on the topic 'D251N'

ABSTRACTThedegQgene ofBacillus subtilis(natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out thedegQgene inBacillus subtilis(natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found indegS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His6mutant proteins were expressed inEscherichia colicells and subjected to anin vitrophosphorylation assay. Compared with the wild type, mutant DegS-His6proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSUin vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

DLEC1 has been suggested as a tumor suppressor gene in several cancers. DLEC1 D215N somatic mutation (COSM36702) was identified in a melanoma cell line through whole genome sequencing. However, little is known about the implication and prevalence of this mutation in primary melanomas or in melanocytic nevi. The aim of this study was to genotype DLEC1 D215N mutation in melanoma tissue and melanocytic nevi samples to confirm its occurrence and to estimate its prevalence. Primary melanomas (n=81) paired with synchronous or asynchronous metastases (n=21) from 81 melanoma patients and melanocytic nevi (n=28) were screened for DLEC1 D215N mutation. We found the mutation in 3 primary melanomas and in 2 melanocytic nevi, corresponding to a relatively low prevalence (3.7% and 7.1%, resp.). The pathogenic role of DLEC1 215N mutation is unclear. However, since the mutation has not been previously described in general population, its involvement in nevogenesis and melanoma progression remains a possibility to be clarified in future studies.

Abstract TMEM106B encodes a lysosomal membrane protein and was initially identified as a risk factor for frontotemporal lobar degeneration. Recently, a dominant D252N mutation in TMEM106B was shown to cause hypomyelinating leukodystrophy. However, how TMEM106B regulates myelination is still unclear. Here we show that TMEM106B is expressed and localized to the lysosome compartment in oligodendrocytes. TMEM106B deficiency in mice results in myelination defects with a significant reduction of protein levels of proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), the membrane proteins found in the myelin sheath. The levels of many lysosome proteins are significantly decreased in the TMEM106B-deficient Oli-neu oligodendroglial precursor cell line. TMEM106B physically interacts with the lysosomal protease cathepsin D and is required to maintain proper cathepsin D levels in oligodendrocytes. Furthermore, we found that TMEM106B deficiency results in lysosome clustering in the perinuclear region and a decrease in lysosome exocytosis and cell surface PLP levels. Moreover, we found that the D252N mutation abolished lysosome enlargement and lysosome acidification induced by wild-type TMEM106B overexpression. Instead, it stimulates lysosome clustering near the nucleus as seen in TMEM106B-deficient cells. Our results support that TMEM106B regulates myelination through modulation of lysosome function in oligodendrocytes.

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.

ABSTRACT The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.

The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Brugada syndrome is an inherited cardiac arrhythmia that follows autosomal dominant transmission and can cause sudden death. This paper reported a case of Brugada syndrome in a 43-year-old male patient with no clinical symptoms. Brugada-type 2-ECG changes were accidentally detected. Flecainide test was done and proved positive. Gene analysis revealed a novel missense mutation in the SCN5A gene with a genetic variation of D252N. This novel mutation has not been reported on any genetic databases related to Brugada syndrome. Functional protein analysis software suggested that the mutation occurs in the highly conserved gene and probably has a damaging effect. This is the first Brugada syndrome case reported with a mutation in the SCN5A gene in Vietnam.

Crystal structures of inactive variants of HIV-1 protease bound to peptides have revealed how the enzyme recognizes its endogenous substrates. The best of the known substrates is, however, a nonnatural substrate that was identified by directed evolution. The crystal structure of the complex between this substrate and the D25N variant of the protease is reported at a resolution of 1.1 Å. The structure has several unprecedented features, especially the formation of additional hydrogen bonds between the enzyme and the substrate. This work expands the understanding of molecular recognition by HIV-1 protease and informs the design of new substrates and inhibitors.

A GH (glycoside hydrolase) family 54 α-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii α-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl–enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl–enzyme intermediate was proposed. Based on the kcat values of a series of aryl-α-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, βlg=−0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pKa values (>6) of leaving phenols, with βlg=−1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

Рассматривается экстремальная задача отыскания точных констант между наилучшимисовместными полиномиальными приближениями аналитических функций и его промежуточныхпроизводных в пространстве Бергмана. Пусть $U:=\{z:|z|<1\}$ -- единичный круг вкомплексной плоскости, $B_{2}:=B_{2}(U)$ -- пространство Бергмана функций $f$,аналитических в круге $U$, c конечной $L_2$ нормой;$B_{2}^{(r)}:=B_{2}^{(r)}(U)$ $(r\in\mathbb{Z}_{+},$ $B_{2}^{(0)}:=B_{2})$ -- классфункций $f\in B_{2}$, у которых $f^{(r)}\in B_{2}$. В работе найдены точные константы внеравенствах типа Джексона -- Стечкина для характеристики гладкости $\Lambda_{m}(f)$,$m\in\mathbb{N},$ определенных при помощи усреднения норм конечных разностей $m$-го порядкастаршей производной функции $f$, принадлежащей пространству Бергмана $B_{2}$. Также решенаэкстремальная задача наилучшего совместного полиномиальная приближения класса$W_{2,m}^{(r)}(\Phi):=W_{2}^{(r)}(\Lambda_{m},\Phi)$ $(m\in\mathbb{N}$,$r\in\mathbb{Z}_{+})$ функций из $B_{2}^{(r)},$ $r\in\mathbb{Z}_{+}$, у которой значениехарактеристики гладкости $\Lambda_{m}(f)$ ограничено сверху мажорантой $\Phi,$ и класса$W_{p,m}^{(r)}(\varphi,h):=W_{p}^{(r)}(\Lambda_{m},\varphi,h)$$(m\in\mathbb{N}$, $r\in\mathbb{Z}_{+}$, $h\in[0,2\pi],$ $1\le p<\infty,$$\varphi$ - весовая на $[0,h]$ функция) из $B_{2}^{(r)}$, укоторого усредненное с заданным весом значение характеристикигладкости $\Lambda_{m}(f)$ ограничено сверху единицей. Следует отметить,что изложенные в статье результаты являются обобщениями недавно опубликованныерезультаты второго автора [10] для совместного приближения периодическихфункций тригонометрическими полиномами на случай совместного приближенияаналитических в единичном круге функций комплексными алгебраическимиполиномами в пространстве Бергмана.

ABSTRACT Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme.

ABSTRACT Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.

ABSTRACTWe developed a novel process for efficient synthesis ofl-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation ofl-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesizel-THA. By using these recombinant enzymes,l-THA was obtained byl-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube.l-THA was obtained in a small amount with a molar yield of 0.076% by using intactEscherichia coliexpressing theasnOgene, and thus, two asparaginase-deficient mutants ofE. coliwere investigated. A remarkably increasedl-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of theasnOgene was enhanced by using the T7 promoter inE. coliinstead of thelacpromoter, thel-THA yield was significantly increased to 92%. By using a combination of theE. coliasparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently,l-THA was successfully obtained froml-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production ofl-THA.

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

Abstract Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. Results: Five multiplex assays were developed, comprising the following 15 SNPs: −82T→C (*22); 86G→C (R29T, *17); 136A→G (M46V, *11); 296G→A (G99E, *12); 415A→G (K139E, *8, *13); 419G→A (R140Q, *14); 516G→T (Q172H, *6, *7, *9, *13, *19, *20), 547G→A (V183I); 769G→A (D257N); 785A→G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T→C (I328T, *16, *18); 1006C→T (R336C, *19); 1172T→A (I391N, *15); 1282C→A (P428T, *21); 1459C→T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. Conclusions: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development

ABSTRACT Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1′ makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1′ and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.

ABSTRACTWe previously established that cells infected with herpes simplex virus 2 (HSV-2) are disrupted in their ability to form stress granules (SGs) in response to oxidative stress and that this disruption is mediated by virion host shutoff protein (vhs), a virion-associated endoribonuclease. Here, we test the requirement for vhs endoribonuclease activity in disruption of SG formation. We analyzed the ability of HSV-2 vhs carrying the point mutation D215N, which ablates its endoribonuclease activity, to disrupt SG formation in both transfected and infected cells. We present evidence that ablation of vhs endoribonuclease activity results in defects in vhs-mediated disruption of SG formation. Furthermore, we demonstrate that preformed SGs can be disassembled by HSV-2 infection in a manner that requires vhs endoribonuclease activity and that, befitting this ability to promote SG disassembly, vhs is able to localize to SGs. Together these data indicate that endoribonuclease activity must be maintained in order for vhs to disrupt SG formation. We propose a model whereby vhs-mediated destruction of SG mRNA promotes SG disassembly and may also prevent SG assembly.IMPORTANCEStress granules (SGs) are transient cytoplasmic structures that form when a cell is exposed to stress. SGs are emerging as potential barriers to viral infection, necessitating a more thorough understanding of their basic biology. We identified virion host shutoff protein (vhs) as a herpes simplex virus 2 (HSV-2) protein capable of disrupting SG formation. As mRNA is a central component of SGs and the best-characterized activity of vhs is as an endoribonuclease specific for mRNAin vivo, we investigated the requirement for vhs endoribonuclease activity in disruption of SG formation. Our studies demonstrate that endoribonuclease activity is required for vhs to disrupt SG formation and, more specifically, that SG disassembly can be driven by vhs endoribonuclease activity. Notably, during the course of these studies we discovered that there is an ordered departure of SG components during their disassembly and, furthermore, that vhs itself has the capacity to localize to SGs.

The interactions between Na+ (and K+) and Asp-201 of β-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type β-galactosidase (0.36 ± 0.09 mM) was about 10× lower than for K+ (3.9 ± 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-β-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-β-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.Key words: β-galactosidase, sodium, potassium, binding, aspartic acid.

Abstract Background Vancomycin-resistant Enterococcus faecium (VREfm) are important causes of bloodstream infections (BSI) in patients (pts) with cancer, liver disease, and foreign bodies. Daptomycin (DAP) is commonly used to treat VRE BSI, but DAP resistance (DAP-R) is increasing. Current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility. DAP triggers the LiaFSR cell membrane stress response pathway, resulting in the extracellular release of LiaX, a protein that functions as a sentinel molecule for DAP, and controls the cell membrane response. Methods We used 6 reference Efm isolates to optimize a whole-cell enzyme-linked immunosorbent assay (ELISA) method for LiaX detection. We then assessed 86 clinical Efm BSI isolates recovered from 3 hospitals in Houston and Detroit for DAP MICs and used whole genome sequencing to assess for substitutions in LiaFSR/LiaXYZ proteins. We collected patient and microbiological data by chart review. Results All DAP-R reference strains had increased detection of LiaX compared to DAP-S strains (p&lt; 0.0001). Of the 86 pts with Efm BSI, 73.2% had malignancy, 9.3% had liver disease, and 76.7% had foreign bodies. The source of 52.3% of BSIs was determined to be gastrointestinal. Two of the 86 isolates were DAP-R by CLSI breakpoints. The LiaX test and DAP MICs had a categorical agreement in 62.8% of isolates. All isolates with discordant ELISA/MIC results had DAP-S MIC (median 2, IQR 1-3)but increased LiaX. Using the genomic information, we identified 41 sites of amino acid (AA) changes in the LiaFSR/XYZ proteins of the ELISA/MIC discordant isolates. The substitutions LiaR S19F and LiaS E153K/D251E were associated with discordancy (p=0.0036 and p=0.0018, respectively). Conclusion Detection of LiaX is likely to indicate activation of the DAP-mediated cell membrane response in Efm and may be an indicator of DAP-R. Important discrepancies between LiaX and standard DAP MIC determination were found, highlighting the limitation of MIC determination. Further characterization of the discrepant isolates by time-kill assays and evaluation of patient clinical outcomes are warranted to fully validate the performance and clinical utility of the LiaX ELISA. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

ABSTRACT Both induction of transcription of the ferric citrate transport genes and transport of ferric citrate by the Escherichia coli outer membrane receptor FecA require energy derived from the proton motive force (PMF) of the inner membrane. The energy is transduced to FecA by the inner membrane complex, TonB, ExbB, and ExbD. Region 160 of TonB and the conserved TonB box of other TonB-dependent receptors are implicated as sites of interaction. In the present study, the postulated TonB box (D80A81L82T83V84) of FecA was deleted in frame, with a subsequent loss of both FecA functions. DALTV of FecA could be functionally replaced with the core TonB boxes of FhuA (DTITV) and FepA (DTIVV). Each residue of the TonB box of FecA was sequentially replaced with cysteine residues, and only the D80C replacement showed a loss (reduction) of both FecA functions. A physical interaction between TonB and FecA was demonstrated using both in vivo site-specific disulfide bond cross-linking and nonspecific formaldehyde (FA) cross-linking. Pairwise combinations of FecA (DALTV)/Cys substitutions were cross-linked via disulfide bond formation with TonBQ160C, TonBQ162C, and TonBY163C. Unexpectedly, this cross-linking was not enhanced by substrate (ferric citrate). In contrast, the TonB-FecA interaction was enhanced by ferric citrate in the FA-cross-linking assay. Energy derived from the PMF was not required for the TonB-FecA interaction in either the disulfide- or FA-cross-linking assay. TonB/CysExbB/ExbD(D25N) was still able to cross-link with the FecA (DALTV)/Cys derivatives in a tonB tolQ background, even though ExbD25N renders the TonB/ExbBD complex nonfunctional (V. Braun, S. Gaisser, C. Herrmann, K. Kampfenkel, H. Killmann, and I. Traub, J. Bacteriol. 178:2836-2845, 1996). TonB cross-linked to FecA via FA was not inhibited by either carbonylcyanide-m-chlorophenylhydrazone or 1 mM 2,4-dinitrophenol, which dissipate the electrochemical potential of the cytoplasmic membrane and disrupt both FecA functions. The studies shown here demonstrate the significance of the TonB box for FecA functions and are consistent with the view that it is the structure and not the sequence of the TonB box that is important for activity. Demonstrated here for the first time is the physical interaction of TonB and FecA, which is enhanced by ferric citrate.

Abstract The role of the β3 MIDAS in αIIbβ3 ligand binding is well established, but the role of the nearby ADMIDAS is less well defined. Thus, we studied HEK293 cells expressing normal αIIbβ3 (normal cells) or the ADMIDAS mutants β3 D126A and D127A (mutant cells). Both mutant cells adhered as well or better than normal cells to immobilized fibrinogen under static conditions in the presence of either Ca2+/Mg2+ or Mn2+. Under low shear flow conditions (0.15 dyne/cm2), adhesion of normal cells and D126A mutant cells to fibrinogen was similar in the presence of either Ca2+/Mg2+ or Mn2+. Adherent D126A mutant cells, however, demonstrated greater resistance to detachment at increasing shear rates in the presence of Ca2+/Mg2+ (e.g., at 20.4 dynes/cm2, only 40 ± 10% of normal cells remained vs 85 ± 8% of D126A mutant cells; mean ± SD; p<0.001). Substituting Mn2+ for Ca2+/Mg2+ increased the resistance to detachment of the normal cells (60 ± 20% remaining at 20.4 dynes/cm2; p=0.01), but the value was still less than the mutant cells in the presence of either Ca2+/Mg2+ (see above; p<0.01) or Mn2+ (84 ± 4%; p<0.01). The increased strength of adhesion we observed in the αIIbβ3 ADMIDAS mutant cells is similar to that found in α4β7 ADMIDAS mutant cells (Chen et al, JBC 2004) and is consistent with the findings in isolated β3 βA (I-like) domains (Pesho et al. JBC 2006). The binding of 7E3, whose epitope is near the ADMIDAS, to the D126 mutant cells was similar to its binding to the normal cells, but 7E3 binding to the D127A mutant cells was reduced by 89 ± 7% (n = 4; p<0.001). 7E3 decreased adhesion of normal cells to fibrinogen by 88 ± 4%, but it only decreased adhesion of D126A mutant cells by 3 ± 9%, and it did not inhibit adhesion of D127A cells at all. To provide a structural context for the role of the ADMIDAS in ligand binding to αIIbβ3, we compared results from nanosecond time-scale molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 or the D126A mutant in the presence of Ca2+/Mg2+. Calculations were carried out using the OPLS all-atom force-field of the GROMACS simulation package. With respect to normal αIIbβ3, the mutant receptor demonstrated reduced fluctuations in the β3 207–210 and 335 regions and increased fluctuations in the β3 282–284 region. In addition, the ADMIDAS metal ion moved ~3 Å away from the MIDAS and became more solvent exposed. Rearrangements of the coordination of the ADMIDAS involving S123, D251, and D127 were also observed in the D126A mutant compared to normal αIIbβ3. Steered MD simulations were used to investigate the unbinding of eptifibatide from its binding site. The unbinding force for the D126A mutant was similar to that for the normal αIIbβ3. Quantitative estimations of the binding energies of eptifibatide to normal and D126A mutant αIIbβ3 from Molecular Mechanics/Poisson Boltzman Surface Area analysis of the MD trajectories also yielded similar results. Thus, the much greater resistance of D126A mutant cells to detachment from fibrinogen at increasing shear rates does not appear to be explained by differences in fibrinogen-αIIbβ3 interactions at the sites involved in the binding of eptifibatide. Potential alternative mechanisms involve differences in fibrinogen’s access to the binding site, interactions with other sites, or changes in fibrinogen avidity due to receptor clustering.

Abstract The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at − 120 mV (73.2 ± 3.7%), − 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Abstract We wished to determine if Mycoplasma bovis infection can negatively impact milk quality and production in Holstein dairy cows. For this Research Communication, milk samples (271) from Holstein cows from 3 herds were screened for M. bovis by real-time PCR. Positive (n = 21) and negative animals (n = 21) were matched by herd, age, lactations and days in milk (DIM). Pairs were evaluated in 7 stages of lactation: D1–50, D51–100, D101–150, D151–200, D201–250, D251–300, and D ≥ 301. A mixed model was used to assess the effect of groups (M. bovis+ × M.bovis−), time (lactation) and groups × time interaction. Cows positive for M. bovis had lower average milk production per day and high somatic cells count (SCC).

AbstractOncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein–protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C &gt; T (rs56318881) (R33stop), 190C &gt; T (rs56379106) (R64W), 559C &gt; T (rs5030839) (R187stop) and 752A &gt; T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T &gt; G (rs4986992) (synonymous), 402T &gt; C (rs146727732) (synonymous), 445G &gt; A (rs4987076) (V149I), 613A &gt; G (rs72554609) (M205V) and 640T &gt; G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G &gt; A (rs72554610) (E261K), and 787A &gt; G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G &gt; A (rs4986782) (R187Q) substantially and significantly reduced them. 560G &gt; A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G &gt; A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.

It seems clear that people who are deaf ... struggle continually against the meanings that others impose on their experience, and the way that this separates them from others. They struggle for acknowledgement of the way they see their lives and wish to live them, and aspire to connection?with other people, to share and belong. (David Moorhead. Knowing Who I Am. 1995. 85.) Nga Tapuwae and Before I am deaf but, before that part of my life started, I was hearing and worked for many years as a librarian in New Zealand. My first job was in a public library located within a secondary school Nga Tapuwae Secondary College in South Auckland. Its placement was a 1970’s social experiment to see if a public library could work within the grounds of a community college (and the answer was no, it could not). The experience was a great introduction for me to the Maori and Polynesian cultures that I had not previously encountered. Until then, I was wary of both groups, and so it was a revelation to realise that although there were many social problems in the area including low literacy, many of the children and teenagers were bright, talented individuals. They simply did not connect to the Anglo-Saxon reading materials we offered. Years later, my interest in the social dynamics of literacy led to my enrolment in a post-graduate literacy degree in Melbourne. This action may have saved my life because at the end of this course, a minor ailment resulted in a visit to the university doctor who diagnosed me with the life-threatening medical condition, Neurofibromatosis Type 2 (NF 2). NF2 is a late onset genetic condition in which one’s body grows tumours, always on both hearing nerves, sometimes elsewhere as well. The tumours usually cause deafness and can cause death. I was told I needed to have my tumours removed and would probably become fully deaf as a result. This is how my life as I knew it changed direction and I started the long journey towards becoming deaf. Diagnosis and Change Predictably, once diagnosed, friends and colleagues rallied to comfort me. I was told things probably weren’t as bad as they sounded. Helen Keller was mentioned several times as an example of someone who had succeeded despite being deaf and blind. ‘Really,’ my friends asked, ‘how bad can it be? ‘Inside myself however, it couldn’t have been worse. A day later the enormity of it all hit me and I became inconsolable. A friend drove me back to the doctor and she did two things that were to change my life. She referred me to the University’s counselling services where, happily, I was counselled by Elizabeth Hastings who later went on to become Australia’s first Disability Services Commissioner. Secondly, the doctor organised for me to visit the HEAR Service at the Victorian Deaf Society (VDS). Again by happy accident, my friend and I stumbled into the ‘wrong building’ where I ended up meeting John Lovett, who was Deaf and the CEO there, via an interpreter. When I met John Lovett I was distraught but, unlike other people, he made no attempt to stop me crying. He simply listened carefully until I realised he understood what I was saying and stopped crying myself. He said my fears that I could end up alone and lonely were valid and he suggested the best thing I could do for myself was to join the ‘Deaf community’; a community. I had never heard of. He explained it was made up of people like him who used Australian sign language (Auslan) to communicate. He was so engaging and supportive that this plan sounded fine to me. By the time we finished talking and he walked me over to the HEAR Service, I was so in his thrall that I had enrolled for a Deaf awareness workshop, an Auslan class, and had plans to join the Deaf community. Had I stayed on and learned Auslan, my life may well have followed a different path, but this was not to be at that time. Becoming Hearing Impaired (HI) Across at the HEAR service, an alternate view of my potential future was put to me. Instead of moving away from everything familiar and joining the Deaf community, I could learn to lip-read and hopefully use it to stay in the workforce and amongst my hearing friends. I had a cousin and aunt who were late deafened; my cousin in particular was doing well communicating with lip-reading. I discussed this with friends and the idea of staying with the people I already knew sounded far less confronting than joining the Deaf community and so I chose this path. My surgeon was also optimistic. He was confident he could save some of my hearing. Suddenly learning Auslan seemed superfluous. I phoned John Lovett to explain, and his response was that I should do what suited me, but he asked me to remember one thing: that it was me who decided to leave the Deaf Community, not that the Deaf community had not wanted me. He told me that, if I changed my mind, I could always go back because the door to the Deaf community would always be open and he would be still be there. It would be a decade before I decided that I wanted to go back through that door, and around that time this great man passed away, but I never forgot my promise to remember our conversation. It, and a few other exchanges I had with him in the following years, stayed at the back of my mind, especially as my residual hearing sank over the years, and the prospect of total deafness hung over me. When I had the surgery, my surgeon’s optimism proved unfounded. He could not save any hearing on my left side and my facial and balance nerves were damaged as well. The hospital then decided not to operate again, and would only attempt to remove the second tumour if it grew and threatened my health again. Consequently, for close to a decade, my life was on hold in many ways. I feared deafness—for me it signalled that my life as I knew it would end and I would be isolated. Every hearing test was a tense time for me as I watched my remaining hearing decline in a slow, relentless downward path on the graph. It was like watching the tide go out knowing it was never going to come in as fully again. My thinking started to change too. Within a week of my diagnosis I experienced discrimination for the first time. A library school that had offered me a place in its post graduate librarianship course the following year made it clear that they no longer wanted me. In the end it did not matter as I was accepted at another institution but it was my first experience of being treated less favourably in the community and it was a shock. After the surgery my life settled down again. I found work in public libraries again, rekindled an old relationship and in 1994 had a baby boy. However, living with a hearing loss is hard work. Everything seemed tiring, especially lip-reading. My ears rejected my hearing aid and became itchy and inflamed. I became aware that my continual hearing problems were sometimes seen as a nuisance in work situations. Socialising lost a lot of its appeal so my social world also contracted. Around this time something else started happening. Outside work, people started expressing admiration for me—words like ‘role model’ and ‘inspiring’ started entering the conversation. Any other time I might have enjoyed it but for me, struggling to adapt to my new situation, it felt odd. The whole thing reminded me of being encouraged to be like Helen Keller; as if there is a right way to behave when one is deaf in which you are an inspiration, and a wrong way in which one is seen as being in need of a role model. I discussed this with Elizabeth Hastings who had helped me prepare mentally for the surgery and afterwards. I explained I felt vulnerable and needy in my new situation and she gave me some useful advice. She thought feeling needy was a good thing as realising one needs people keeps one humble. She observed that, after years of intellectualising, educated people sometimes started believing they could use intellectualisation as a way to avoid painful emotions such as sadness. This behaviour then cut them off from support and from understanding that none of us can do it alone. She believed that, in always having to ask for help, people with disabilities are kept aware of the simple truth that all people depend on others to survive. She said I could regard becoming deaf as a disability, or I could choose to regard it as a privilege. Over the years the truth of her words became increasingly more evident to me as I waded through all the jargon and intellectualisation that surrounds discussion of both deafness and the disability arena, compared to the often raw emotion expressed by those on the receiving end of it. At a personal level I have found that talking about emotions helps especially in the face of the ubiquitous ‘positive thinking’ brigade who would have us all believe that successful people do not feel negative emotions regardless of what is happening. The Lie Elizabeth had initially sympathised with my sadness about my impending deafness. One day however she asked why, having expressed positive sentiments both about deaf people and people with disabilities, I was saying I would probably be better off dead than deaf? Up until that conversation I was unaware of the contradictions between what I felt and what I was saying. I came to realise I was living a lie because I did not believe what I was telling myself; namely, that deaf people and people with disabilities are as good as other people. Far from believing this, what I really thought was that being deaf, or having a disability, did lessen one’s worth. It was an uncomfortable admission, particularly sharing it with someone sitting in a wheelchair, and especially as up until then I had always seen myself as a liberal thinker. Now, faced with the reality of becoming deaf, I had been hoist by my own petard, as I could not come to terms with the idea of myself as a deaf person. The Christian idea of looking after the ‘less fortunate’ was one I had been exposed to, but I had not realised the flip side of it, which is that the ‘less fortunate’ are also perceived as a ‘burden’ for those looking after them. It reminded me of my initial experiences years earlier at Nga Tapuwae when I came face to face with cultures I thought I had understood but did not. In both cases it was only when I got to know people that I began to question my own attitudes and assumptions and broadened my thinking. Unfortunately for deaf people, and people with disabilities, I have not been the only person lying to myself. These days it is not common for people to express their fears about deaf people or people with disabilities. People just press on without fully communicating or understanding the other person’s attitude or perspectives. When things then do not work out, these failures reinforce the misconceptions and these attitudes persist. I believe it is one of the main reasons why true community inclusion for deaf and people with disabilities is moving so slowly. Paying for access is another manifestation of this. Everyone is supportive of access in principle but there is continuous complaint about paying for things such as interpreting. The never-ending discussions between deaf people and the wealthy movie industry about providing more than token access to captioned cinema demonstrate that the inclusion lie is alive and well. Until it can be effectively addressed through genuine dialogue, deaf people, hard of hearing people and people with disabilities will always be largely relegated to life outside the mainstream. Collectively we will also continue to have to endure this double message that we are of equal value to the community while simultaneously being considered a financial burden if we try to access it in ways that are meaningful to us. Becoming Deaf In 2002 however all this thinking still lay ahead of me. I still had some hearing and was back living in New Zealand to be close to my family. My relationship had ended and I was a solo mother. My workplace had approved leave of absence, and so I still had my job to go back to in Melbourne if I wanted it. However, I suspected that I would soon need the second tumour removed because I was getting shooting pains down my face. When my fears were confirmed I could not decide whether to move back to Melbourne or let the job go, and risk having trouble finding one if I went back later. I initially chose to stay longer as my father was sick but eventually I decided Melbourne was where I wanted to be especially if I was deaf. I returned, found temporary employment, and right up to the second surgery I was able to work as I could make good use of the small amount of hearing I still had. I thought that I would still be able to cope when I was made fully deaf as a result of the surgery. It was, after all, only one notch down on the audiogram and I was already ‘profoundly deaf’ and still working. When I woke up after the surgery completely deaf, it felt anti-climactic. The world seemed exactly the same, just silent. At home where I was surrounded by my close family and friends everything initially seemed possible. However, when my family left, it was just my seven-year-old son and myself again, and on venturing back into the community, it quickly became clear to me that at some level my status had changed. Without any cues, I struggled to follow speech and few people wanted to write things down. Although my son was only seven, people communicated with him in preference to me. I felt as if we had changed roles: I was now the child and he was the adult. Worse was soon to follow when I tried to re-enter the workforce. When I had the surgery, the hospital had installed a gadget called an auditory brainstem implant, (ABI) which they said would help me hear. An ABI is similar to a cochlear implant but it is attached to the brainstem instead of the cochlear nerve. My cochlear nerve was removed. I hoped my ABI would enable me to hear enough to find work but, aside from clinical conditions in which there was no background noise and the staff knew how to assist, it did not work. My most humiliating moment with it came when it broke down mid job interview and I spent half the time left trying to get it going again in full view of the embarrassed interview panel, and the other half trying to maintain my composure whilst trying to lip-read the questions. The most crushing blow came from the library where I had happily worked for seven years at middle management level. This library was collaborating with another institution to set up a new library and they needed new staff. I hopefully applied for a job at the same level I had worked at prior to becoming deaf but was unsuccessful. When I asked for feedback, I was told that I was not seen as having the skills to work at that level. My lowest point came when I was refused a job unpacking boxes of books. I was told I did not have experience in this area even though, as any librarian will attest, unpacking boxes is part of any librarian’s work. When I could not find unskilled work, it occurred to me that possibly I would never work again. While this was unfolding, my young son and I went from being comfortable financially to impoverished. My ex-partner also decided he would now make childcare arrangements directly with my son as he was annoyed at being expected to write things down for me. My relationship with him, some family members, and my friends were all under strain at that time. I was lost. It also became clear that my son was not coping. Although he knew the rudiments of Auslan, it was not enough for us to communicate sufficiently. His behaviour at school deteriorated and one night he became so frustrated trying to talk to me that he started to pull out his own hair. I calmed him and asked him to write down for me what he was feeling and he wrote down ‘It is like you died. It is like I don’t have a Mum now’. It was now clear to me that although I still had my friends, nobody including myself knew what to do. I realised I had to find someone who could understand my situation and I knew now it had to be a Deaf person. Fortunately, by this stage I was back learning Auslan again at La Trobe University. The week after the conversation with my son, I told my Auslan teacher what had happened. To my relief she understood my situation immediately. She told me to bring my son to class, at no cost, and she would teach him herself. I did and my life started to turn around. My son took to Auslan with such speed and application that he was able to not only converse with her in one month but immediately started using Auslan with me at home to get the things he wanted. We were able to re-establish the mother/son relationship that we both needed. I was also able to help my son talk through and deal with all the changes that me becoming deaf had foisted upon him. He still uses Auslan to talk to me and supplements it using speech, copious finger spelling, notes and diagrams. More than anything else, this relationship has kept me anchored to my long-term goal of becoming a clear signer. Encouraged by my son’s success, I put all my energy into learning Auslan and enrolled in a full time TAFE Auslan course. I also joined a chat group called ‘Here to Hear’ (H2H). The perspectives in the group ranged from strongly oral to strongly Deaf but for me, trying to find a place to fit in any of it, it was invaluable. Almost daily I chatted with the group, asking questions and invariably someone responded. The group acted as a safety net and sounding board for me as I worked out the practicalities of living life deaf. The day of my fateful interview and the ABI humiliation, I came home so shaken that I used the Irish remedy of a couple of swigs of whisky, and then went online and posted an account of it all. I can still remember the collective indignation of the group and, as I read the responses, beginning to see the funny side of it . . . something I could not have done alone. I also made use of easy access to Deaf teachers at TAFE and used that to listen to them and ask advice on situations. I found out for example, that if I instructed my son to stand behind me when people in shops insisted on addressing him, they had no alternative but to talk to me; it was a good clear message to all concerned that my son was the child in this relationship. About this time, I discovered the Disability Discrimination Act (DDA) that Elizabeth Hastings had worked so hard on, filed my first DDA complaint, and received my first apology at the mediation session that followed. My personal life also improved, relationship by relationship as everyone adjusted. Slowly the ice melted in most of my relationships; some relationships faded and were replaced with new ones with signing people, and eventually hearing people again. My life moved forward. Through a member of ‘Here to Hear’, I was invited to apply for my first post deaf job—covering holiday leave at a Deaf sports organisation. I practically finger-spelt my way through the interview but not only did they offer me the job, they were delighted to have me. I was able to buy a few things with the money I earned, and suddenly it felt as if everything was possible again. This acceptance of me by Deaf people had a profound impact on me. I mixed with people more, and it was not too long before I was able to use my basic signing skills to use Auslan interpreters and re-enter the workplace. I have discovered over time that living in silence also has advantages—no more noisy parties or rubbish trucks clanging at dawn and in its place a vastly heightened visual awareness that I enjoy. Before I was deaf I thought it would be lonely in the silence but in fact many of life’s best moments—watching rain hit and then run down a window, swimming in the sea, cooking and being with good friends—do not rely upon sound at all; they feel the same way they always did. Sometimes I have felt somewhat of an outsider in the Deaf community. I have sometimes been taken aback by people’s abruptness but I have learned over time that being succinct is valued in Auslan, and some people like to come straight to the point. At crisis points, such as when I asked for help at the Victorian Deaf Society and my Auslan class, it has been a huge relief to talk to Deaf people and know immediately that they understand just from reading their eyes. Having access to an additional world of deaf people has made my life more enjoyable. I feel privileged to be associated with the Deaf community. I can recall a couple of Christmases ago making dinner for some signing friends and suddenly realising that, without noticing, everything had become alright in my world again. Everyone was signing really fast – something I still struggle with; but every now and then someone would stop and summarise so I felt included. It was really relaxed and simply felt like old times, just old times without the sound thrown in. Le Page and Tabouret-Keller, two ethnographers, have this to say about why people communicate the ways they do: The individual ... creates for himself the patterns of his linguistic behaviour so as to resemble those of the group or groups with which from time to time he wishes to be identified, or so as to be unlike those from whom he wishes to be distinguished ... . We see speech acts as acts of projection; the speaker is projecting his inner universe, implicitly with the invitation to others to share it ... he is seeking to reinforce his models of the world, and hopes for solidarity from those with whom he wishes to identify. (181) This quote neatly sums up why I choose to communicate the ways I do. I use Auslan and speech in different situations because I am connected to people in both groups and I want them in my life. I do not feel hugely different from anyone these days. If it is accepted that I have as much to contribute to the community as anyone else, becoming deaf has also meant for me that I expect to see other people treated well and accepted. For me that means contributing my time and thoughts, and advocating. It also means expecting a good level of access to interpreters, to some thought provoking captioned movies in English, and affordable assistive technologies so I can participate. I see this right to participate and engage in genuine dialogue with the rest of the community as central to the aspirations and identity of us all, regardless of who we are or where others think we belong. References Le Page, R.B., and Andree Tabouret-Keller. Acts of Identity: Creole-Based Approaches to Language and Ethnicity. London: Cambridge University Press, 1985. Moorhead, D. “Knowing Who I Am.” In S. Gregory, ed., Deaf Futures Revisited. Block 3, Unit 10, D251 Issues in Deafness. Open University, 1995.