Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency

Abstract Background: Assay of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism, is important for the study of severe and mild deficiency states. Because the conventional assay measures in the reverse direction, lacks sensitivity, and uses nonphysiologic substrates, the exact measurement and characterization of residual activity in easily accessible tissues have been difficult. Methods: To measure MTHFR in the physiologic direction, we determined the NADPH-dependent conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by use of HPLC with fluorescence detection. Results: MTHFR activity in control fibroblast in the presence of FAD was maximal between pH 6.3 and 6.9, increased linearly up to 40 min and 80 μg protein/assay, and showed Kms of 30 μmol/L for NADPH and 26 μmol/L for 5,10-methylenetetrahydrofolate. Intraassay variation (CV) was 10%, interassay variation was 7.2%, and variation among 10 subcultures of the same cell line was 18%. Mean (SD) control activity was 431 (150) μU/mg protein (range, 242–910; n = 75), which is 2.5-fold higher than that with the reverse assay. After heat treatment (46 °C for 5 min), the activity showed a trimodal distribution corresponding to the 677TT (thermolabile; 15%), 677CT (35%), and 677CC (51%) genotypes. We found clearly measurable activity ranging from 2.6% to 25.6% of the mean control value in 15 patients with MTHFR deficiency, including 11 cell lines with zero activity in the reverse assay. Ten patients had complete enzyme deficiency. Conclusion: This assay allows reliable determination of residual activity in mutant fibroblasts and characterization of kinetic parameters for natural substrates.

Download Full-text

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19951229 ◽

1995 ◽

Vol 60 (7) ◽

pp. 1229-1235 ◽

Cited By ~ 2

Author(s):

Ivana Zoulíková ◽

Ivan Svoboda ◽

Jiří Velek ◽

Václav Kašička ◽

Jiřina Slaninová ◽

...

Keyword(s):

Amino Acid ◽

Biological Activities ◽

Residual Activity ◽

Detailed Examination ◽

Amino Acid Residues ◽

Linear Peptide ◽

Zone Electrophoresis ◽

Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

Download Full-text

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Polymers ◽

10.3390/polym13152452 ◽

2021 ◽

Vol 13 (15) ◽

pp. 2452

Author(s):

Chia-Jung Hsieh ◽

Ju-Chuan Cheng ◽

Chia-Jung Hu ◽

Chi-Yang Yu

Keyword(s):

Carbonic Anhydrase ◽

High Activity ◽

Co2 Sequestration ◽

Residual Activity ◽

Entrapment Efficiency ◽

Free Form ◽

Uncatalyzed Reaction ◽

The Stability ◽

Time Required

Capturing and storing CO2 is of prime importance. The rate of CO2 sequestration is often limited by the hydration of CO2, which can be greatly accelerated by using carbonic anhydrase (CA, EC 4.2.1.1) as a catalyst. In order to improve the stability and reusability of CA, a silica-condensing peptide (R5) was fused with the fastest known CA from Sulfurihydrogenibium azorense (SazCA) to form R5-SazCA; the fusion protein successfully performed in vitro silicification. The entrapment efficiency reached 100% and the silicified form (R5-SazCA-SP) showed a high activity recovery of 91%. The residual activity of R5-SazCA-SP was two-fold higher than that of the free form when stored at 25 °C for 35 days; R5-SazCA-SP still retained 86% of its activity after 10 cycles of reuse. Comparing with an uncatalyzed reaction, the time required for the onset of CaCO3 formation was shortened by 43% and 33% with the addition of R5-SazCA and R5-SazCA-SP, respectively. R5-SazCA-SP shows great potential as a robust and efficient biocatalyst for CO2 sequestration because of its high activity, high stability, and reusability.

Download Full-text

Abstract 1340: Disruption of a Hepatocyte Growth Factor/c-Met Receptor Autocrine Loop Severely Impairs the Potency of Pluripotent Adipose Stromal Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_274-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Liying Cai ◽

Brian H Johnstone ◽

Zhong Liang ◽

Dmitry Traktuev ◽

Todd G Cook ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Rank Order ◽

Residual Activity ◽

Autocrine Loop ◽

Major Mode ◽

Factor C ◽

Hepatocyte Growth

Background Paracrine stimulation of endogenous repair, rather than direct tissue regeneration, is increasingly accepted as a major mode of therapeutic stem and progenitor cell action; yet, this principle has not been fully established in vivo . Adipose-derived stem cells (ASCs) secrete many factors and promote reperfusion and tissue repair in ischemia models. RNA interference was used to silence the expression of the abundant protein, hepatocyte growth factor (HGF), to determine its contribution to ASC potency in vivo . Methods and Results Dual-cassette lentiviral vectors, expressing GFP and either a small hairpin RNA (shRNA) specific for HGF mRNA (shHGF) or a control sequence (shCtrl), were used to stably transduce ASCs (ASC-shHGF or ASC-shCtrl). ASC-shHGF secreted 5-fold less HGF, which resulted in a reduced ability of these cells to promote survival, proliferation and migration of mature and progenitor endothelial cells in vitro ( p <0.01). HGF knockdown also severely impaired the ability of ASCs to promote reperfusion in a mouse hindlimb ischemia model. Perfusion of the ischemic leg at 15 d in mice treated with ASC-Ctrl was 84±4%, compared to only 69±5% for ASC-shHGF ( p <0.05). Even so, ASC-shHGF retained residual activity as indicated by greater reperfusion ( p <0.05) than with saline treatment (58±6%). Capillary densities in ischemic tissues from each group followed a similar rank order (ASC-Ctrl>ASC-shHGF>saline) ( p <0.05 between each group). While there was no difference in total GFP + cells in ischemic limbs at 5 d after infusion, indicating similar homing potentials, 3-fold fewer ASC-shHGF were present in ischemic tissues at 15 d compared to ASC-shCtrl ( p <0.01). This was accompanied by an increase in TUNEL-positive ASC-shHGF cells (61 ± 0.1%) compared to ASC-Ctrl (41% ± 3.2%) in ischemic tissues at 5 d ( p <0.01); suggesting that attenuated potency of ASC-shHGF was related to reduced survival in ischemic tissues. Conclusions These results indicate that secretion of HGF is critically important for ASC potency. In addition to promoting endogenous repair, the data suggest that an important effect of HGF is autocrine promotion of ASC survival in ischemic tissue. Enhanced donor cell survival is an important goal for increasing the efficacy of cell therapy.

Download Full-text

Antisense Inhibition of Methylenetetrahydrofolate Reductase Reduces Cancer Cell Survival In vitro and Tumor Growth In vivo

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-04-2047 ◽

2005 ◽

Vol 11 (5) ◽

pp. 2047-2052 ◽

Cited By ~ 23

Author(s):

Jitka Stankova ◽

Jijun Shang ◽

Rima Rozen

Keyword(s):

Tumor Growth ◽

Cell Survival ◽

Cancer Cell ◽

Methylenetetrahydrofolate Reductase ◽

Antisense Inhibition ◽

Cancer Cell Survival

Download Full-text

SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

BMC Infectious Diseases ◽

10.1186/s12879-019-4700-1 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Gabrielle Sherella Dijksteel ◽

Peter H. Nibbering ◽

Magda M. W. Ulrich ◽

Esther Middelkoop ◽

Bouke K. H. L. Boekema

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Antimicrobial Agents ◽

Ex Vivo ◽

Residual Activity ◽

Gram Negative Bacteria ◽

Tissue Samples ◽

Gram Negative ◽

Viable Bacteria

Abstract Background Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol]. Methods Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity. To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05–1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30 min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed. Results All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at ≥0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization. Conclusions SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.

Download Full-text

Essential Oils as a Potential Treatment Option for Pediculosis

Planta Medica ◽

10.1055/a-1161-9189 ◽

2020 ◽

Vol 86 (09) ◽

pp. 619-630 ◽

Cited By ~ 1

Author(s):

Kerdalidec Candy ◽

Mohammad Akhoundi ◽

Valérie Andriantsoanirina ◽

Rémy Durand ◽

Christiane Bruel ◽

...

Keyword(s):

Essential Oils ◽

Head Louse ◽

Treatment Options ◽

Residual Activity ◽

Environmental Toxicity ◽

Pediculus Humanus ◽

Synthetic Chemicals ◽

Major Factors ◽

Ectoparasite Infestation

AbstractPediculosis is a prevalent ectoparasite infestation caused by lice. The head louse (Pediculus humanus capitis) and body louse (Pediculus humanus humanus) are obligatory parasites whose only known hosts are humans. Pediculosis is probably the most common ectoparasitic infestation, affecting up to 80% of the population in several countries, and particularly prevalent in the infant population worldwide. Several treatment options, including shampoos and creams containing insecticides, have been introduced for the treatment of pediculosis. Recently, the use of synthetic chemicals to control human lice has raised concerns pertaining to human health and the environment. Therefore, increasing efforts have been undertaken to develop effective pediculicides with low environmental toxicity and minimal environmental residual activity. In this study, we focus on the essential oils derived from 22 plant genera, their constituents, and the major factors that play important roles in the effectiveness of these oils in the treatment of pediculosis. Furthermore, we discuss the advantages and limitations of the mentioned essential oils, and ultimately suggest those demonstrating the most effective in vitro pediculicidal activities. The genera such as Aloysia, Cinnamomum, Eucalyptus, Eugenia, Lavandula, Melaleuca, Mentha, Myrcianthes, Origanum, Pimpinella, and Thymus appear to be more efficient against lice. These genera are rich in anethole, 1,8-cineole, cinnamaldehyde, p-cymene, eugenol, linalool, limonene, pulegone, terpinen-4-ol, and thymol compounds.

Download Full-text

Folate Insufficiency Due to MTHFR Deficiency Is Bypassed by 5-Methyltetrahydrofolate

Journal of Clinical Medicine ◽

10.3390/jcm9092836 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2836

Author(s):

Maša Vidmar Golja ◽

Alenka Šmid ◽

Nataša Karas Kuželički ◽

Jurij Trontelj ◽

Ksenija Geršak ◽

...

Keyword(s):

Methylenetetrahydrofolate Reductase ◽

Fold Increase ◽

Mthfr Gene ◽

Folate Supplementation ◽

Disease States ◽

Metabolic Defects ◽

Key Enzyme ◽

Cell Processes ◽

Activity Groups ◽

Mthfr Deficiency

Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells’ metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.

Download Full-text