Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

Imbalances in GABA (γ-aminobutyric acid) homoeostasis underlie psychiatric and movement disorders. The ability of the 65 kDa isoform of GAD (glutamic acid decarboxylase), GAD65, to control synaptic GABA levels is influenced through its capacity to auto-inactivate. In contrast, the GAD67 isoform is constitutively active. Previous structural insights suggest that flexibility in the GAD65 catalytic loop drives enzyme inactivation. To test this idea, we constructed a panel of GAD65/67 chimaeras and compared the ability of these molecules to auto-inactivate. Together, our data reveal the important finding that the C-terminal domain of GAD plays a key role in controlling GAD65 auto-inactivation. In support of these findings, we determined the X-ray crystal structure of a GAD65/67 chimaera that reveals that the conformation of the catalytic loop is intimately linked to the C-terminal domain.

Quantification of BK viral load in asymptomatic renal allograft recipients.

INTRODUCTION: Polyoma BK virus (BKV) has recently been identified to cause renal allograft dysfunction, which manifests as polyomavirus-associated nephropathy (PVAN). However, the presence and level of BKV DNA in renal allograft patients with good and stable renal function have remained undetermined. METHODS: In this prospective study, serum samples were collected from a total of 45 renal allograft recipients with serum creatinine <155 µmol/L. In 17 patients, whose duration of transplantation was under 2 years, samples were collected at 3-4-month intervals for up to 2 years after transplantation. BK viral load was quantified using quantitative polymerase chain reaction (Q-PCR). RESULTS: The BK viral load in asymptomatic renal allograft recipients was independent of the duration of transplantation and did not correlate with allograft function. The mean (± SD) level of viremia was 552.80 ± 1931.00 genome copies/mL, with 92.9% of patients having low levels of viremia corresponding to <1 × 10(3) copies/mL. In contrast, patients with proven PVAN had levels in the range of 10(6) copies/mL. CONCLUSIONS: The prevailing BK viral load in asymptomatic renal allograft patients is quantifiably low. Our findings may guide optimal immunosuppressive modulation in PVAN cases, where judicious manipulation of immunosuppression is required without inciting allograft rejection.

Relationship of pretransplantation polyoma BK virus serologic findings and BK viral reactivation after hematopoietic stem cell transplantation.

BACKGROUND: Reactivation of polyoma BK virus (BKV) infection is consistently associated with hemorrhagic cystitis in persons who undergo hematopoietic stem cell transplantation (HSCT). In this study, we examined the relationship of reactivation of BKV infection with pre-HSCT serologic findings of BKV antibody. METHODS: Serial urine samples (n=1118) obtained from 140 HSCT recipients were prospectively obtained, and BKV loads were quantified by quantitative polymerase chain reaction. Pre-HSCT anti-BKV immunoglobulin G (IgG) levels were determined by indirect immunofluorescence. RESULTS: In 68 patients, there was significant peaking (i.e., > or = 3-log increase) in the urine BKV load (median peak, 1.7x10(9) copies/mL; range, 1.1x10(4) to 3.2x10(14) copies/mL) occurring at a median time of 24.5 days (range, 7-49 days). In 72 patients, low-level BKV viruria occurred without peaking (median BKV load, 10 copies/mL; range, 9.9x10(3) to 1.2x10(10) copies/mL) at a median time of 24.5 days (range, 7-49 days). Pre-HSCT anti-BKV IgG was positively related to elevated urine BKV load during HSCT (P<.001). Binary logistic regression revealed that pre-HSCT anti-BKV IgG level was the only statistically significant factor (P=.009) to be associated with a > or = 3-log increase in the peak urine BKV load (positive and negative predictive values, 69% and 68%, respectively). Nine patients developed hemorrhagic cystitis at a median of 56 days (range, 29-160); 7 of these patients were evaluable and were found to have a > or = 3-log increase in the peak BKV load. In binary logistic regression, peaking of the urine BKV load (P=.026) and graft-versus-host disease (P=.033) were found to be statistically significant risks for hemorrhagic cystitis. CONCLUSIONS: The identification of the serologic status of BKV as a significant risk factor for BKV viruria suggests that it should be included as an integral part of the pre-HSCT evaluation.