Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

Background

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Methods

Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299?T?>?C), p.V178M (c.532?G?>?A), p.E189G (c.566A?>?G), p.Q286R (c.857A?>?G), p.K315E (c.943A?>?G), p.R379C (c.1135?C?>?T) and p.R385C (c.1153?C?>?T) in comparison to the wildtype protein.

Results

In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K_m values for argininosuccinic acid differed significantly from the wild-type ASL protein.

Conclusion

The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.     

Effects of aging and menopause on serum interleukin-6 levels and peripheral blood mononuclear cell cytokine production in healthy nonobese women

Inappropriate interleukin-6 production is thought to play a role in the development of several age-related conditions including atherosclerosis. This study aimed to determine whether aging affects circulating interleukin-6 (IL-6) levels. Healthy, nonobese women (n = 208, 44.5 ± 0.70 years, 22.4 ± 0.17 kg/m²) were categorized into four age groups (22–31, 32–41, 42–51, and 52–63 years; cross-sectional study). Cytokine levels in serum and those produced from peripheral blood mononuclear cell (PBMC) were measured. The oldest group had the highest circulating levels of IL-6 and oxidized low-density lipoprotein (ox-LDL) and higher PBMC production of IL-6, tumor necrosis factor-α (TNF-α), and interleukin-1 alpha (IL-1β). Additionally, significant interactions between age and menopause were found for serum IL-6 (P = 0.024), and TNF-α (P = 0.011) and IL-1β (P < 0.001) produced from PBMCs. Serum IL-6 levels positively correlated with age, waist–hip ratio (WHR), systolic blood pressure, circulating levels of TNF-α, IL-1β, and ox-LDL, and urinary 8-epi-prostaglandin F₂α. Multiple stepwise regression models identified the following factors for contributing to serum IL-6 levels: serum IL-1β, menopause status, WHR, and serum TNF-α in mode I (R ² = 0.302); serum IL-1β, age, serum TNF-α, and WHR (β = 0.197; P = 0.006) in model II (R ² = 0.283). Sub-analysis was performed according to menopausal status. Serum IL-6 levels were positively associated with levels of IL-6, TNF-α, and IL-1β in PBMC supernatants (unstimulated) from postmenopausal women, whereas these were negatively associated in premenopausal women. In conclusion, circulating IL-6 levels may be interactively influenced by age and menopause. Additionally, estrogen deprivation after menopause may enhance PBMC cytokine production in postmenopausal women, resulting in increased IL-6 levels which are closely related to oxidative stress.     

Comparison of pro-atrial natriuretic peptide and atrial remodeling in marathon versus non-marathon runners

Long-term endurance sports are associated with atrial remodeling and an increased risk for atrial fibrillation (AF) and atrial flutter. Pro-atrial natriuretic peptide (pro-ANP) is a marker of atrial wall tension and elevated in patients with AF. The aim of this study was to test the hypothesis that atrial remodeling would be perpetuated by repetitive episodes of atrial stretching during strenuous competitions, reflected by elevated levels of pro-ANP. A cross-sectional study was performed on nonelite runners scheduled to participate in the 2010 Grand Prix of Bern, a 10-mile race. Four hundred ninety-two marathon and nonmarathon runners applied for participation, 70 were randomly selected, and 56 entered the final analysis. Subjects were stratified according to former marathon participations: a control group (nonmarathon runners, n = 22), group 1 (1 to 4 marathons, n = 16), and group 2 (≥5 marathons, n = 18). Results were adjusted for age, training years, and average weekly endurance training hours. The mean age was 42 ± 7 years. Compared to the control group, marathon runners in groups 1 and 2 had larger left atria (25 ± 6 vs 30 ± 6 vs 34 ± 7 ml/m(2), p = 0.002) and larger right atria (27 ± 7 vs 31 ± 8 vs 35 ± 5 ml/m(2), p = 0.024). Pro-ANP levels at baseline were higher in marathon runners (1.04 ± 0.38 vs 1.42 ± 0.74 vs 1.67 ± 0.69 nmol/L, p = 0.006). Pro-ANP increased significantly in all groups after the race. In multiple linear regression analysis, marathon participation was an independent predictor of left atrial (β = 0.427, p <0.001) and right atrial (β = 0.395, p = 0.006) remodeling. In conclusion, marathon running was associated with progressive left and right atrial remodeling, possibly induced by repetitive episodes of atrial stretching. The altered left and right atrial substrate may facilitate atrial arrhythmias.     

Management of noninfectious mixed cryoglobulinemia vasculitis: data from 242 cases included in the CryoVas survey

Data on the clinical spectrum and therapeutic management of noninfectious mixed cryoglobulinemia vasculitis (CryoVas) in the era of hepatitis C virus screening are lacking. We analyzed data from 242 patients with noninfectious mixed CryoVas included in the French multicenter CryoVas survey. Baseline manifestations were purpura (75%), peripheral neuropathy (52%), arthralgia or arthritis (44%), glomerulonephritis (35%), cutaneous ulcers (16%), and cutaneous necrosis (14%). A connective tissue disease was diagnosed in 30% and B-cell non-Hodgkin lymphoma in 22%, whereas the CryoVas was considered to be essential in 48%. With the use of Cox-marginal structural models, rituximab plus corticosteroids showed the greater therapeutic efficacy compared with corticosteroids alone and alkylating agents plus corticosteroids to achieve complete clinical, renal, and immunologic responses and a prednisone dosage < 10 mg/d at 6 months. However, this regimen was also associated with severe infections, particularly when high doses of corticosteroids were used, whereas death rates did not differ between the therapeutic regimens. The role of each of these strategies remains to be defined in well-designed randomized controlled trials.     

Comparable outcomes between unrelated and related donors after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation in patients with high-risk multiple myeloma

The purpose of this study was to assess the results of allogeneic stem cell transplantation (allo-SCT) after reduced-intensity conditioning (RIC) from matched related donors (MRD) and unrelated donors (URD) in 40 patients with high-risk multiple myeloma (MM) in a single centre. Seventeen (43%) (Group 1) and 23 patients (57%) (Group 2) had URD and MRD, respectively. Thirty-nine patients (98%) received one or more autologous transplantation. The median follow-up was 22 months (1-49). None of our patient experienced a graft rejection. The cumulative incidence of grade II-IV acute GVHD was higher (47%) for the URD vs. (17%) for the MRD (P = 0.092). The cumulative incidence of chronic GVHD was no different between the two groups (24% vs. 30%, respectively). At 2 yr, the TRM probabilities were lower in the unrelated group 12% vs. 22% in the related group (P = 0.4). Also at 2 yrs, for patients receiving unrelated transplantation overall and progression-free survivals, 59% and 42%, respectively compared to patients with related donor transplantation, 66% and 44% (P = 0.241). In conclusion, these results suggest that URD in MM is feasible. The small number of patients with URD emphasizes the need to delineate indications and perform prospective protocols.     

Kinetics of atrial repolarization alternans in a free-behaving ovine model

Kinetics of Atrial Repolarization Alternans. Introduction: Repolarization alternans (Re‐ALT), a beat‐to‐beat alternation in action potential repolarization, promotes dispersion of repolarization, wavebreaks, and reentry. Recently, Re‐ALT has been shown to play an important role in the transition from rapid pacing to atrial fibrillation (AF) in humans. The detailed kinetics of atrial Re‐ALT, however, has not been reported so far. We developed a chronic free‐behaving ovine pacing model to study the kinetics of atrial Re‐ALT as a function of pacing rate. Methods: Thirteen sheep were chronically implanted with 2 pacemakers for the recording of broadband right atrial unipolar electrograms and delivery of rapid pacing protocols. Beat‐to‐beat differences in the atrial T‐wave apex amplitude as a measure of Re‐ALT and activation time were analyzed at incremental pacing rates until the effective refractory period (ERP) defined as stable 2:1 capture. Results: Atrial Re‐ALT appeared intermittently but without periodicity, and increased in amplitude as a function of pacing rate until ERP. Intermittent 2:1 atrial capture was observed at pacing cycle lengths 40 ms above ERP, and increased in duration as a function of pacing rate. Episodes of rapid pacing‐induced AF were rare, and were preceded by Re‐ALT or complex oscillations of atrial repolarization, but without intermittent capture. Conclusion: We show in vivo that atrial Re‐ALT developed and increased in magnitude with rate until stable 2:1 capture. In rare instances where capture failure did not occur, Re‐ALT and complex oscillations of repolarization surged and preceded AF initiation. (J Cardiovasc Electrophysiol, Vol. 23, pp. 1003‐1012, September 2012)