Acceleration of an aldo-keto reductase by minimal loop engineering.

Aldo-keto reductases tighten coenzyme binding by forming a hydrogen bond across the pyrophosphate group of NAD(P)(H). Mutation of the hydrogen bonding anchor Lys24 in Candida tenuis xylose reductase prevents fastening of the "safety belt" around NAD(H). The loosened NAD(H) binding leads to increased turnover numbers (k(cat)) for reductions of bulky-bulky ketones at constant substrate and coenzyme affinities (i.e. K(m Ketone), K(m NADH)).

Transport as a sensitive indicator of quantum criticality.

We consider bosonic transport through one-dimensional spin systems. Transport is induced by coupling the spin systems to bosonic reservoirs kept at different temperatures. In the limit of weak-coupling between spins and bosons we apply the quantum-optical master equation to calculate the energy transmitted from source to drain reservoirs. At large thermal bias, we find that the current for longitudinal transport becomes independent of the chain length and is also not drastically affected by the presence of disorder. In contrast, at small temperatures, the current scales inversely with the chain length and is further suppressed in the presence of disorder. We also find that the critical behaviour of the ground state is mapped to critical behaviour of the current--even in configurations with infinite thermal bias.

Evaluation of the EMIT Mycophenolic Acid Assay from Dade Behring.

The Emit Mycophenolic Acid Assay, a new homogeneous enzyme immunoassay for the quantitative analysis of mycophenolic acid (MPA) in human plasma, was evaluated and compared to a high-performance liquid chromatography (HPLC) method. Coefficients of variation (CV) of the within-run imprecision (n = 10) varied from 2.5% to 4.4% and from 1.3% to 4.9% for the Emit and the HPLC, respectively. The CV's of between-day imprecision (n = 10) ranged from 7.9% to 10.8% for the Emit and from 4.7% to 12.1% for the HPLC. Mean recoveries were 95.6% and 100.1% for Emit and HPLC, respectively. Serial dilution of a patient pool demonstrated a linear relationship between expected (x) and measured (y) concentrations: Emit, y = 0.998x + 0.086; HPLC, y = 1.006x - 0.016. The detection limit and the lower limit of quantification were 0.087 mg/L and 0.20 mg/L for Emit. The detection limit for HPLC was 0.08 mg/L using a signal-to-noise ratio of three. Sample stability under various storage conditions was satisfactory, although storage at -20 degrees C is recommended for storage longer than one day. No cross-reactivity from the major metabolite mycophenolic acid glucuronide (MPAG) was found. A correlation study on 261 patient samples yielded the following regression equation (bivariate Deming procedure): Emit = 1.012HPLC + 0.244, r = 0.970.

Noninvasive monitoring using serum amyloid A and serum neopterin in cardiac transplantation.

The monitoring of allograft function for cardiac transplant patients still relies on endomyocardial routine biopsies. We investigated the diagnostic value of noninvasive monitoring using the parameters serum amyloid A protein and serum neopterin. The circulating levels of the acute phase reactant, amyloid A protein, and the macrophage product, neopterin, were measured serially in 13 patients after cardiac transplantation. The mean period of observation was 240 days. Nine acute cardiac allograft rejections, five cases of viral infection and eight cases of bacterial infection occurred. The levels of serum amyloid A protein and serum neopterin remained low (x = 6.0 mg/dL and 12.6 nmol/L, respectively) during the periods of stable graft function. In contrast, both parameters were significantly elevated (p < 0.01) during the rejection episodes (x = 12.7 mg/dL and 38.0 nmol/L for serum amyloid A protein and serum neopterin, respectively). For a reliable differentiation between rejection and stable graft function, serum amyloid A protein had a diagnostic accuracy of 84% (with a cut-off level of 10 mg/dL) and serum neopterin had one of 75% (with a cut-off level of 23 nmol/L). However, significant increases in the circulating levels of serum amyloid A protein and serum neopterin were also observed during bacterial (x = 14.9 and 88 nmol/L, respectively) and viral (x = 6.2 mg/dL and 44 nmol/L, respectively) infections. The detection of immunological complications after cardiac transplantation using serial measurements of serum amyloid A protein and serum neopterin is possible. These parameters can be used to help in judging both the need and the optimal timing for the otherwise frequent endomyocardial biopsies.

Mycophenolic acid monitoring in HTX patients: a preliminary report.

Mycophenolic acid (MPA) is a new immunosuppressive drug used in clinical trials in combination with cyclosporine A (CsA) and prednisolone. Its immunosuppressive mechanism consists in a non-competitive inhibition of the inosine monophosphate (IMP) dehydrogenase which is the key enzyme in the conversion of inosine monophosphate to guanosine monophosphate. Depletion of the guanine nucleotide pool impairs DNA synthesis and thus inhibits the cell cycle progression of T- and B-lymphocytes. For monitoring of MPA and its metabolite mycophenolic acid glucuronide (MPAG), a high performance liquid chromatography (HPLC) method, and for MPA an EMIT-immunoassay, were established. MPA HPLC and EMIT exhibit a linearity up to 10.00 mumol/l (3.20 micrograms/ml) and 46.95 mumol/l (15.00 micrograms/ml) respectively. Coefficients of variation for intra /inter-assay bias were 5.3/14.1 per cent for HPLC and 4.8/12.3 percent for the EMIT assay. The correlation between both methods was: HPLC = 0.82 EMIT -1.85 (r = 0.955); limits of agreement between the methods were 13.6 and -5.12 mumol/l. In 8 stable heart transplant recipients treated with 2 g mofetil/day, mean and median steady-state MPA concentrations for HPLC and EMIT were 6.86 mumol/l (2.19 micrograms/ml), 4.77 mumol/l (1.52 micrograms/ml) and 10.88 mumol/l (3.48 micrograms/ml), 7.42 mumol/l (2.37 micrograms/ml) respectively, during a period of 3 months. Mean and median MPAG concentrations were 141.21 mumol/l (61.40 micrograms/ml) and 100.70 mumol/l (43.78 micrograms/ml) respectively.

Clinical usefulness of percentage of free serum prostate specific antigen.

Serum concentrations of total and free prostate specific antigen were measured retrospectively in 268 patients, in order to test the usefulness of percentage of free prostate specific antigen in distinguishing between cancer and benign hyperplasia of the prostate and to improve the specificity of cancer screening. Four groups were investigated: 94 urologic patients without prostate disease (controls), 98 patients with a histologically confirmed benign hyperplasia, 76 with histologically established prostatic adenocarcinoma, 18 of them after radical prostatectomy. Total and free prostate specific antigen concentrations were measured in frozen serum, in a retrospective mode, by using an equimolar monoclonal antibody immunoassay. Median percentage of free prostate specific antigen was 20.48% in controls, 17.75% in patients with hyperplasia, 10.52% in patients with cancer and 33.03% in patients after prostatectomy. Median percentage of free prostate specific antigen was significantly lower in men with cancer than in patients with benign hyperplasia (P < 0.0001). The percentage of free prostate specific antigen increased the specificity of cancer screening: a cut-off of 23.6% detected at least 90% of cancers and would have eliminated 34.7% of biopsies in benign hyperplasias. A prospective study is ongoing to confirm these results.

Tissue polypeptide specific antigen for the detection of lymphoproliferative diseases induced by cyclosporin.

AIM: To evaluate the efficacy of tissue polypeptide specific (TPS) antigen for the early detection of cyclosporin A (CyA) induced post-transplant lymphoproliferative diseases. METHODS: Serum concentrations of TPS antigen were analysed using a monoclonal enzyme immunoassay and whole blood CyA concentrations were measured using high pressure liquid chromatography. Infection with Epstein-Barr virus and cytomegalovirus was detected by determining levels of IgM and IgG antibodies directed against viral capsid antigen (VCA). Immunohistochemistry and analysis of clonality were carried out on formalin fixed, paraffin wax embedded tissue. RESULTS: The mean serum concentration of TPS antigen in the eight transplant recipients investigated was 60 U/l during periods without complication (control), 101 U/l during infection, 166 U/l when the diagnosis of a lymphoma was confirmed, and 172 U/l when lymphoma and infection coincided. Increased TPS antigen concentrations were detected in six patients one month before detection of malignancy. After reduction of immunosuppression and the start of tumour regression, TPS antigen concentrations decreased. TPS antigen concentrations increased in the one patient who experienced a recurrence. CONCLUSIONS: Continuous monitoring of TPS antigen concentrations leads to the early discovery of CyA induced lymphoproliferative disease.

Tissue polypeptide specific antigen, neopterin, and CRP for monitoring heart transplant recipients.

OBJECTIVES: The aim of our study was to investigate the usefulness of tissue polypeptide specific antigen, an established tumor marker detecting proliferation of cells, for monitoring heart transplant recipients in order to detect infection and rejection early. METHODS: Tissue polypeptide specific antigen serum levels were compared with neopterin and C-reactive protein serum concentrations. RESULTS: When infections occurred, tissue polypeptide specific antigen serum concentrations were increased approximately five times (mean: 214 U/L, SD 145), while in instances of acute rejection crises, they were increased two times (mean: 76 U/L, SD 16 in comparison with the values during uncomplicated postoperative courses (mean: 45 U/L, SD 26). CONCLUSIONS: Tissue polypeptide specific antigen was the only investigated analyte that showed significant diagnostic validities when infections were compared with rejections. The discrimination between an uncomplicated postoperative course and a rejection or infection episode was only possible with a combination of the three analytes.