Presence and Evolution of a Catalytic Activity in Patients with Severe, Mild or Moderate Hemophilia A

Abstract Abstract 1211 The development of neutralizing anti-Factor VIII (FVIII) antibodies is the major complication of the treatment of patients with hemophilia A (HA). Several mechanisms of inhibition have been described: steric hindrance, immune complex formation and catalytic antibodies. Anti-FVIII catalytic antibodies act like enzymes and lead to the hydrolysis of FVIII. Lacroix-Desmazes et al. had shown the presence of catalytic antibodies in the plasma of patients with severe HA who had developed inhibitors (13/24) (Lacroix-Desmazes et al, NEJM, 2002). Previous studies on catalytic antibodies reported results for patients with severe HA at one time point, exclusively. Thus, we proposed to extend the analysis of catalytic antibodies to patients with a mild or moderate HA and to follow over their lifetime patients who develop inhibitor. We studied plasma samples from 33 patients with HA. Sixteen were patients with severe HA, including 8 patients with inhibitor (Inh+) and 8 patients without inhibitor (Inh-), and 17 were mild or moderate HA patients (7 Inh+ and 10 Inh-). Among Inh+ patients, 6 were treated on-demand (3 severe and 3 moderate HA patients) and 9 were submitted to an immune tolerance induction (ITI) protocol (4 severe and 5 mild or moderate patients with HA). A therapeutic preparation of pooled normal IgG (IVIg) from healthy donors was used as a source of normal IgG. We also used plasma from 13 male healthy donors. As described previously [2], IgG were purified from plasma by affinity-chromatography on protein G followed by a size-exclusion chromatography in presence of urea. Then, catalytic activity was evaluated by the hydrolysis of FVIII after incubation with purified IgG (Lacroix-Desmazes et al, Nature, 1999). Inhibitor titer is measured by modified Bethesda test. Mean FVIII-hydrolyzing rates were determined for healthy donors, HA patients and IVIg, used as control. Catalytic activity of HA patients IgG was significantly higher than those of healthy donors or IVIg (p<0.01 and p<0.001, respectively). Sixty four per cent of patients with HA had catalytic antibodies, regardless the phenotype nor inhibitor presence. In addition, prevalence of FVIII-hydrolyzing antibodies for Inh+ and Inh- HA patients was 94% and 47%, respectively. However, the mean FVIII-hydrolysis rate was comparable for both groups (0.23 ± 0.06 mmol/min/mol). Surprisingly, we showed that the mean catalytic activity of mild or moderate HA patients were significantly lower than those of severe HA patients (0.17 ± 0.05 versus 0.31 ± 0.07). Interestingly, we were able to study the evolution of both catalytic and inhibitory activities for patients who developed inhibitor. We observed 2 profiles: in the first case, FVIII-hydrolysis rate and inhibitor titer followed the same trend, but in the second case, these two parameters showed a dissociated evolution. These results were independent of the type of treatment (on-demand or ITI). For the first time, we studied catalytic activity for patients with mild or moderate HA. In comparison with patients with severe HA, catalytic activity is much lower for patients with mild or moderate HA. In addition, most of patients with severe HA had FVIII-hydrolytic antibodies (88 %), in contrast with previous studies (50%) (Lacroix-Desmazes et al, NEJM, 2002). In the same way, we showed the presence of catalytic antibodies in patients without inhibitor. Moreover, we studied the evolution of catalytic activity and inhibitor titer over the time for patients with inhibitor and showed that catalytic activity did not necessarily follow the same trend as inhibitory activity. The results suggested that catalytic antibodies could not act like neutralizing antibodies. Disclosures: No relevant conflicts of interest to declare.

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Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection

Pharmaceutics ◽

10.3390/pharmaceutics13101656 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1656

Author(s):

Jiapeng Li ◽

Shuhan Liu ◽

Jian Shi ◽

Hao-Jie Zhu

Keyword(s):

Catalytic Activity ◽

Human Lung ◽

Hydrolytic Activity ◽

Airway Epithelial Cells ◽

Protein Profiling ◽

Cell Permeability ◽

Hydrolysis Rate ◽

Proteomics Data ◽

Tenofovir Alafenamide ◽

Hydrolysis Of

ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection.

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Impact of Treatment Regimen with Moroctocog Alfa (AF-CC) on Bleeding Frequency in Pediatric Aged Subjects with Moderately Severe to Severe Hemophilia A

Blood ◽

10.1182/blood-2018-99-111891 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1205-1205

Author(s):

Mark P. Smith ◽

Jeremy Rupon ◽

Yasser Wali ◽

Hala Remawi ◽

Joan Korth-Bradley ◽

...

Keyword(s):

Standard Deviation ◽

Hemophilia A ◽

Pediatric Population ◽

Low Frequency ◽

Primary Objective ◽

Severe Hemophilia ◽

On Demand ◽

Routine Prophylaxis ◽

The Mean ◽

Safety Signals

Abstract Moroctocog alfa (AF-CC) is a B-domain deleted recombinant factor VIII product for the treatment of patients with hemophilia A. It has previously been studied in pediatric aged hemophilia patients, however direct comparisons between treatment regimens have not been evaluated. Importantly, as prophylaxis has been shown to be superior to on-demand treatment, comparisons between different prophylaxis regimens are needed. Reducing the frequency of infusions without sacrificing efficacy may reduce the burden of hemophilia management. This study was an open label, multicenter, randomized crossover study of two routine prophylaxis (RP) regimens of moroctocog alfa (AF-CC) compared to on-demand (OD) therapy in 2 cohorts of children with hemophilia A. The primary objective was to demonstrate that prophylaxis reduces ABR relative to OD therapy. A secondary objective was to compare the efficacy of two different RP regimens. Additional objectives focused on FVIII recovery and safety. Study Design: The study was open to previously treated patients (≥20 exposure days (ED) to any FVIII replacement product) with moderately severe to severe hemophilia A (<2% FVIII) aged 6 months to <16 years. The study enrolled 2 cohorts of subjects who were treated in two segments. The first cohort started with OD treatment for 6 months (segment 1) followed by 1 year of RP of 25 international units (IU)/kg every other day (segment 2). Subjects in the second cohort were randomized to one of 2 RP regimens for 1 year (segment 1) and then crossed over to a second RP regimen for an additional year (segment 2). The RP regimens were high frequency (HF) (25 IU/kg every other day) and low frequency (LF) (45 IU/kg twice weekly). Recovery assessment after administration of 50 IU/kg of moroctocog alfa (AF-CC) was optional. Results: A total of 51 subjects were enrolled in the study: 9 subjects (age 2.4-5.9 yr; median 4.9 yr) in the OD group, 18 subjects (age 1.1-12.7 yr; median 4.7 yr) received LF followed by HF, and 24 subjects (age 1.2 to 9.6 yr; median 4.6 yr) received HF followed by LF. The mean (standard deviation [SD]) ABR for the first cohort (n=9) during the OD segment was 47.0(32.2) and during the RP segment (HF) was 1.5(2.2). In those subjects from cohort 2 that completed both segments (n=38), the mean (standard deviation) ABR for HF was 2.2(4.1) and for LF was 3.3(5.3). Based on a prospectively defined equivalence limit of (-3, 3) bleeds per year, equivalence between these two regimens was demonstrated as the 90% CI for the difference, (0.03, 2.22) fell within this range. The mean (SD) recovery in 6 children aged 3.7 to 5.8 yrs was 1.44(0.61) IU/dL/IU/kg. Three subjects tested positive for FVIII inhibitors, however 2 were considered to be false positives for an overall rate of 2%. There were no new safety signals that emerged during this study. Conclusion: This study met its primary objective of demonstrating that RP reduces the ABR compared to OD. Notably, LF prophylaxis with moroctocog alfa (AF-CC) is as efficacious as a HF prophylaxis regimen. This has important implications as less frequent infusions can improve adherence and the quality of life for patients with hemophilia. Additionally, recovery was as expected in this pediatric population and no new safety signals emerged. Moroctocog alfa (AF-CC) is safe and efficacious for routine prophylaxis in pediatric patients with hemophilia A and a low frequency (twice weekly) prophylaxis regimen is as efficacious as every other day prophylaxis. Disclosures Rupon: Pfizer: Employment. Korth-Bradley:Pfizer Inc.: Employment. Smith:Pfizer: Employment. Rendo:Pfizer: Employment.

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Immune Tolerance Induction Treatment Is Cost-Effective in Adult Patients with Long-Standing High-Responding Inhibitors

Blood ◽

10.1182/blood.v126.23.3530.3530 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3530-3530

Author(s):

Alessandra Nunes Loureiro Prezotti ◽

Silmara Aparecida De Lima Montalvão ◽

Cláudia Lorenzato ◽

Andrea Maia Oliveira ◽

Maria do Perpétuo Socorro Vendramini Orletti ◽

...

Keyword(s):

Hemophilia A ◽

Tolerance Induction ◽

Cost Effective ◽

Adult Patients ◽

Median Period ◽

Long Time ◽

Bleeding Episodes ◽

The Mean ◽

Bypassing Agents ◽

Inhibitor Titer

Abstract Introduction: Despite treatment of hemophilia having achieved considerable improvement over the last decades, inhibitor development remains a major complication for these patients. Inhibitors are alloantibodies that neutralize factor (F) VIII coagulation activity, and occur in approximately 25% of hemophilia A patients. Immune tolerance induction (ITI) is the choice therapy to eradicate these antibodies. There are several ITI protocols with similar success rates, approximately 70%. The presence of long-standing inhibitors in the adult population has usually been associated with poor ITI outcome. In Brazil, as in other countries with constrained resources, limited access to factor concentrates prevented the use of ITI. Thus, almost all patients with inhibitors remained a long time with this complication. Only after 2013 with increased availability of factor concentrates in the country, has assured long-term prophylaxis and ITI treatment been possible. Objective: This study aimed to describe the Brazilian experience in managing ITI in adult patients from seven Hemophilia Treatment Centers with long-standing inhibitor using bypassing agents to control bleeding episodes, considering bleeding frequency, treatment barriers and economic impact of therapy. Patients and Methods: We retrospectively analyzed clinical and laboratory data of hemophilia A patients with high-responding inhibitors (peak titer > 5 Bethesda Unit (BU), anamnestic response to FVIII) over 18 yo who underwent ITI treatment and were using bypass agents before ITI. The initial ITI protocol used was low-dose FVIII concentrate (25-50 IU/kg 3x/week) Results: In Brazil, since the availability of ITI, 39 adult hemophilia A patients with inhibitors, have been submitted to ITI for the first time. In this study, we accessed data from 26 patients, of these 13 (50%) already achieved complete ITI success criteria (inhibitor titer < 0.6 BU; FVIII recovery ≥ 66%, and half-live ≥ 6 h). Another 4 patients with ongoing ITI achieved inhibitor titer < 5 BU and are no longer using bypassing agents. Thus, 17/26 (65%) patients discontinued the bypassing agents in a median period of 2 mo (range 0 to 18 mo) after starting ITI. After 24 months of irregular treatment 2 patients abandoned ITI protocol. Analyses of the 13 patients with complete success revealed mean age at first reported inhibitor of 16.21y (SD ± 11.6; median 21y, range 0.7 to 37y), and mean age at ITI onset of 31.2y (SD ± 10.6; median 27y, range 19 to 47y). The mean period from the first inhibitor detected until starting ITI was 15y (SD ± 5.1), with a 16y median (range 7 to 22y). The historical peak inhibitor mean was 53.1 BU (SD ± 49.5; median 39.4 BU, range 5.5 to 163 BU). The mean pre ITI inhibitor titer was 6.6 BU (SD ± 5.5; median 5.5 BU, range 0.6 to 20.8 BU). Low-dose ITI protocol was initially used for all patients, however 3 patients had the dose increased during treatment (100 IU/kg from 3 to 7 x/week). During ITI, 10/13 patients received prophylaxis with bypassing agents, and 9 began prophylaxis before ITI started. The mean time to achieve inhibitor titer < 0.6 BU was 11.6 mo (SD ± 12.5; median 7 mo, range 0.5 to 44 mo). All 13 patients achieved other ITI success criteria. However, a delay was observed in the majority of patients in FVIII recovery and half-life. Regarding clinical outcome, we observed a significant reduction in both, annualized bleeding rate (ABR), and annualized joint bleeding rate (AJBR), comparing 12 months before ITI, during ITI, and after achieving complete success (figure 1). Regarding economic evaluation, we observed a significant reduction in the median cost of clotting factor consumption, comparing 12 months before starting ITI, and the following years after achieving ITI success. No significant difference was observed between the median cost of 12 months before, and ITI period (figure 2). Conclusion: This study indicated that this treatment is worthwhile even for inhibitor patients with poor prognostic factors for ITI considering both effectiveness to control and prevent bleeding episodes, and economic impact. In this group, 65% of adult hemophilia A high-responding inhibitors patients stopped using bypassing agents after a median period of 2 months after starting ITI. Despite being a high cost treatment, ITI can be cost-effective, especially for patients using bypassing agents. These results reinforce the positive impact of the ITI even in long time inhibitor adult patients. Disclosures Ozelo: Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Biogen: Research Funding.

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Acquired Hemophilia A. Experience Of a Single Center

Blood ◽

10.1182/blood.v122.21.4781.4781 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4781-4781 ◽

Cited By ~ 1

Author(s):

Mauricio A Alzate ◽

Susana S Meschengieser ◽

Alicia Blanco ◽

Silvia Grosso ◽

María A Lazzari ◽

...

Keyword(s):

Autoimmune Disease ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Acquired Hemophilia A ◽

Acquired Hemophilia ◽

Initial Symptoms ◽

Bleeding Episodes ◽

The Mean ◽

Inhibitor Titer

Introduction Acquired hemophilia A is a rare and serious autoimmune disease. Morbidity and mortality are associated with advanced age, comorbidities, toxicity of treatment and bleeding severity. Treatment goals are control of the bleeding and eradication of the inhibitor, while treating the underlying condition if it is present. Objective To describe the baseline characteristics of acquired hemophilia A patients and to assess the response to treatment. Patients and Methods Between November 1991 and April 2013, 27 patients were diagnosed with acquired hemophilia A (mean age 59, range 21-86; 18 women - 66%) in the Departamento de Hemostasia y Trombosis. Five patients were lost from follow-up. APTT mixing studies with normal plasma (1:1) and time-temperature dependent effect on the APTT were performed for a-FVIII diagnosis. Whenever possible, inhibitor activity was titrated by Bethesda method at diagnosis (BU/mL). Medical records were reviewed to evaluate the initial symptoms, underlying diseases, treatments and outcome. Results The mean follow-up was 86 weeks (range 1-640). Underlying etiologies included: idiopathic 70.4%, postpartum 14.8%, malignancy 11.1%, autoimmune disease 3.7%. All patients had bleeding at diagnosis. The most frequent sites of bleeding were: muscular 32%, soft tissue 18%, urinary tract 9%, gastrointestinal tract 6%; being from multiple sites in 9%. At diagnosis, the mean value for FVIII was 6% (range 1-40), and inhibitor titer 220 BU/mL (range 2.2-1173). Initial therapeutic scheme included glucocorticoids in 97% of the patients, 13 in monotherapy (mean age 53 years ± 19), 13 with cyclophosphamide (63 years ± 18) (p= ns), and human immunoglobulin in 1 patient. This last patient died after 1 week of diagnosis due to uncontrolled gastrointestinal bleeding (previous to the era of rVIIa). As a second-line therapy, rituximab was used in 3 patients. Sixty-three (63%) patients achieved complete response (CR) (inhibitor titer < 0.6 BU/mL without bleeding episodes), and 23% achieved partial response (PR) (reduction in inhibitor titer > 50% without bleeding episodes), without differences between monotherapy or combined. Overall, women responded more frequently than men (93.3% vs. 71.4%, p= ns). All patients that received rituximab achieved CR. Conclusions In this study, the overall response rate was higher than 80%. In most cases, the disease has a prolonged course like other autoimmune diseases, with remissions and relapses. Disclosures: No relevant conflicts of interest to declare.

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The Platelet-Derived Microparticles Related to the Clinical Phenotype Heterogeneity of Hemophlia a

Blood ◽

10.1182/blood.v124.21.2828.2828 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2828-2828

Author(s):

Fangmei Qin ◽

Shunhua Huang ◽

Zhitao Li ◽

Jieyu Ye ◽

Jing Sun

Keyword(s):

Hemophilia A ◽

Conflicts Of Interest ◽

Clinical Phenotype ◽

Venous Blood ◽

On Demand ◽

Normal Individuals ◽

Fluorescent Microbeads ◽

The Mean ◽

Phenotype Heterogeneity ◽

Severe Type

Abstract Keywords: Hemophlia A, Phenotype; Heterogeneity; Flowcytometry ; Mircoparticle Background: The clinical phenotype for hemophlia A are typically recognized sub-clinical, mild, moderate and severe according to the residue FVIII level of the plasma. For recent years, there are evidences indicate that the residue FVIII level is not the only factor relate to the clinical phenotype of patients with severe hemophlia A. Platelets are an important role in hemostasis and platelet-derived microparticles (PMPs) are known to mediate a prothrombotic state in patients, we hypothesized that PMPs contributes to the clinical phenotype heterogeneity of hemophilia A patients. Methods: Thirty-one patients with severe hemophlia A (FVIII:C<1%, age 18-33 yrs, median 23.5 yrs) treated on demand were enrolled in this study, including 10 patients with mild type(FVIII:C<1%, but annualized bleed rates<6) and 21 patients with severe type (FVIII:C<1% and annualized bleed rates≥24). Seventeen healthy male donors were selected as normal controls. We also compared the baseline PMPs counts from eleven patients on demand treatment to that when they had three months prophylactic treatment. Venous blood was anticoagulated with sodium citrate (1:9). Plasma was isolated (1550g for 20 min at RT) and then cell free supernatant was ultracentrifuged (18,000g for 20 min at 20°C). The supernatant plasma samples were measured for baseline PMPs counts, with phycoerythrin (PE)-conjugated monoclonal antibodies CD41, CD42b, CD61, CD62P, and CD63 specific for PMPs derived from platelets respectively. Analyzed by flow cytometry, Forward scatter was set in scale using fluorescent microspheres of 1.0μm and standard fluorescent microbeads (0-1.0μm) in diameter were used to set the microparticle gate. Statistics were performed using independent-samples T test Results: There was a trend toward higher the mean level of PMPs counts in mild type patients compared to severe type (mean ± SD: 2.06±1.33 vs 0.95±0.64, p = 0.02), the PMPs counts of normal individuals was significantly higher than severe type(mean ± SD: 1.06±1.20 v s0.95±0.64, p = 0.02), significantly lower than mild type (mean ± SD: 1.06±1.20 vs 2.06±1.33, p = 0.02). The PMPs counts decreased after 3 months low dose (10 iu/kg, 2-3/w) prophylacticlly treatment (mean ± SD: 6.50±0.51 vs 0.45±0.50, p = 0.00). Conclusion/Discussion: Platelet-derived Microparticles (PMPs) are generated in response to platelet activation, our observations demonstrate that the PMPs counts differs in patients of severe hemophlia A, the mean lever of PMPs in mild clinical phenotype patients was higher than the severe type, prophylacticlly treated patients has a lower PMPs lever, PMPs is likely to play a role in clincal phenotype heterogeneity of sever hemophilia A. Disclosures No relevant conflicts of interest to declare.

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A polyclonal antibody preparation with Michaelian catalytic properties

Biochemical Journal ◽

10.1042/bj2790871 ◽

1991 ◽

Vol 279 (3) ◽

pp. 871-881 ◽

Cited By ~ 29

Author(s):

G Gallacher ◽

C S Jackson ◽

M Searcey ◽

G T Badman ◽

R Goel ◽

...

Keyword(s):

Catalytic Activity ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

Catalytic Antibodies ◽

Initial Assessment ◽

Compound I ◽

Keyhole Limpet Haemocyanin ◽

Antibody Preparation ◽

Lower Limits ◽

Hydrolysis Of

1. 4-Nitrophenyl 4′-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4′-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.

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Quantification of small-scale heterogeneity in aquatic aminopeptidase activity

Aquatic Microbial Ecology ◽

10.3354/ame01930 ◽

2020 ◽

Vol 84 ◽

pp. 127-140

Author(s):

BM Gaas ◽

JW Ammerman

Keyword(s):

Chlorophyll Fluorescence ◽

Leucine Aminopeptidase ◽

Hudson River ◽

Aminopeptidase Activity ◽

Small Scale ◽

Analysis Instrument ◽

Sequential Samples ◽

Degree Of Confidence ◽

The Mean ◽

Hydrolysis Of

Leucine aminopeptidase (LAP) is one of the enzymes involved in the hydrolysis of peptides, and is sometimes used to indicate potential nitrogen limitation in microbes. Small-scale variability has the potential to confound interpretation of underlying patterns in LAP activity in time or space. An automated flow-injection analysis instrument was used to address the small-scale variability of LAP activity within contiguous regions of the Hudson River plume (New Jersey, USA). LAP activity had a coefficient of variation (CV) of ca. 0.5 with occasional values above 1.0. The mean CVs for other biological parameters—chlorophyll fluorescence and nitrate concentration—were similar, and were much lower for salinity. LAP activity changed by an average of 35 nmol l-1 h-1 at different salinities, and variations in LAP activity were higher crossing region boundaries than within a region. Differences in LAP activity were ±100 nmol l-1 h-1 between sequential samples spaced <10 m apart. Variogram analysis indicated an inherent spatial variability of 52 nmol l-1 h-1 throughout the study area. Large changes in LAP activity were often associated with small changes in salinity and chlorophyll fluorescence, and were sensitive to the sampling frequency. This study concludes that LAP measurements in a sample could realistically be expected to range from zero to twice the average, and changes between areas or times should be at least 2-fold to have some degree of confidence that apparent patterns (or lack thereof) in activity are real.

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Immobilized Nanoparticles-Mediated Enzymatic Hydrolysis of Cellulose for Clean Sugar Production: A Novel Approach

Current Nanoscience ◽

10.2174/1573413714666180611081759 ◽

2019 ◽

Vol 15 (3) ◽

pp. 296-303 ◽

Cited By ~ 5

Author(s):

Swapnil Gaikwad ◽

Avinash P. Ingle ◽

Silvio Silverio da Silva ◽

Mahendra Rai

Keyword(s):

Catalytic Activity ◽

Enzymatic Hydrolysis ◽

Immobilized Enzyme ◽

Free Enzyme ◽

Magnetic Nanomaterials ◽

Sugar Production ◽

Cellulase Enzyme ◽

Enzymatic Hydrolysis Of Cellulose ◽

Hydrolysis Of Cellulose ◽

Hydrolysis Of

Background: Enzymatic hydrolysis of cellulose is an expensive approach due to the high cost of an enzyme involved in the process. The goal of the current study was to apply magnetic nanomaterials as a support for immobilization of enzyme, which helps in the repeated use of immobilized enzyme for hydrolysis to make the process cost-effective. In addition, it will also provide stability to enzyme and increase its catalytic activity. Objective: The main aim of the present study is to immobilize cellulase enzyme on Magnetic Nanoparticles (MNPs) in order to enable the enzyme to be re-used for clean sugar production from cellulose. Methods: MNPs were synthesized using chemical precipitation methods and characterized by different techniques. Further, cellulase enzyme was immobilized on MNPs and efficacy of free and immobilized cellulase for hydrolysis of cellulose was evaluated. Results: Enzymatic hydrolysis of cellulose by immobilized enzyme showed enhanced catalytic activity after 48 hours compared to free enzyme. In first cycle of hydrolysis, immobilized enzyme hydrolyzed the cellulose and produced 19.5 ± 0.15 gm/L of glucose after 48 hours. On the contrary, free enzyme produced only 13.7 ± 0.25 gm/L of glucose in 48 hours. Immobilized enzyme maintained its stability and produced 6.15 ± 0.15 and 3.03 ± 0.25 gm/L of glucose in second and third cycle, respectively after 48 hours. Conclusion: This study will be very useful for sugar production because of enzyme binding efficiency and admirable reusability of immobilized enzyme, which leads to the significant increase in production of sugar from cellulosic materials.

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Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

International Journal of Molecular Sciences ◽

10.3390/ijms22094512 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4512

Author(s):

Michał Marcinkowski ◽

Tomaš Pilžys ◽

Damian Garbicz ◽

Jan Piwowarski ◽

Damian Mielecki ◽

...

Keyword(s):

Catalytic Activity ◽

Size Exclusion Chromatography ◽

Posttranslational Modifications ◽

Size Exclusion ◽

Saxs Data ◽

Wide Range ◽

Exclusion Chromatography ◽

Demethylase Activity ◽

Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

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