Enzyme Activity In Extracts Research Articles

Evaluation of Cytotoxicity, Antimicrobial and Antioxidant Enzyme Activity of Diploid and Tetraploid Platycodon grandiflorum

This experiment was conducted to obtain the have higher contents of pharmaceutical constituents as well as higher yield from colchicine induced diploid and tetraploid extracts of Platycodon grandiflorum. In order to determine the biological activity, this study was focused to evaluate the cytotoxicity, antimicrobial on the bronthus disease bacteria, antioxidant enzyme activity of diploid and tetraploid extracts in P. grandiflorum. The activities of antioxidant enzyme according to different solvent extracts were measured as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX). The cytotoxicity of methanol extracts of P. grandiflorum showed significant differences between tetraploid and diploid. That is, the cytotoxic effect against human cancer cell was higher in tetraploid than in diploid. At all extracts concentration, tetraploid samples showed high toxicity and the <TEX>$IC_{50}$</TEX> (concentration causing 50% cell death) value showed the highest on HCT-116 cell (<TEX>$105.91{\mu}g/mL$</TEX>), and exhibited significant activity against the Hep 3B cell (<TEX>$140.67{\mu}g/mL$</TEX>), SNU-1066 cell (<TEX>$154.01{\mu}g/mL$</TEX>), Hela cell (<TEX>$158.37{\mu}g/mL$</TEX>), SNU-601 cell (<TEX>$182.67{\mu}g/mL$</TEX>), Calu-6 cell (<TEX>$190.42{\mu}g/mL$</TEX>), MCF-7 cell (<TEX>$510.19{\mu}g/mL$</TEX>). Antimicrobial activities of diploid P. grandiflorum were relatively low compared to tetraploid P. grandiflorum on most of the bacterial strains. In tetraploid P. grandiflorum, K. pneumoniae showed the clear zone formation (18~19 mm) of growth inhibition, followed by the clear zone formation of 13~15 mm on C. diphtheria and S. pyogenes. The antimicrobial activities in diploid P. grandiflorum were the highest on K. pneumonia (14~15 mm), and showed the clear zone formation of 11~12 mm on C. diphtheria and 12~13 mm on S. pyogenes. The antimicrobial activity is thought to look different depending on the bacterial strains and the polyploidy of P. grandiflorum. The root extract of P. grandiflorum had the highest (97.2%) SOD enzyme activity in ethyl acetate partition layer of tetraploid while water partition layer of diploid showed the lowest (48.6%) SOD enzyme activity. The activity of CAT showed higher values in the root of tetraploid than in the diploid of P. grandiflorum in all partition layers except butyl alcohol. The activities of APX and POD showed higher values in the root of tetraploid than in the diploid of P. grandiflorum in all fraction solvents except water layer. These results indicate that the tetraploid P. grandiflorum can be used as a source for developing cytotoxic agent and antimicrobials which can act against bronchus diseases bacterial strains.

Specific activity of thymidine nucleotide biosynthetic enzymes in hairy roots extracts of Sesamum indicum L. transformed by two strains of Agrobacteruim rhizogenes

The study concluded induction of transformed hairy roots from leaves and decapitated seedlings of Sesamum indicum L. using two strains of Agrobacterium rhizogenes considered as natural vector of transformation. The strain R1601 stimulated roots on leaves and seedlings during 20 days of inoculation placed on solidified Arnon and Hoagland medium. Whereas they involved 12 days when inoculated with the strain R15834. Generally strain R15834 was efficient in inducing these roots and their numbers than strain R1601 which approached 54.4% and 41.6% respectively. The results indicated an increase in the specific activity of enzymes Thymidlate synthase (TS), Dihydrofolate reductase (DHFR), Serine hydroxy methyl transfrase (SHMT) in extract of transformed hairy roots producing agropine by stain R15834 and approach 4.610, 1.057, 0.480 µmolmin mg of protein respectively compared with the activity of 1.256, 0.097, 0.125µ molmin mg in the control samples. This was coupled with an increase in amount of DNA and RNA that approached 105, 1020 µg per gram respectively compared to 44, 462 µg per gram in control samples. The transformation of these hairy roots was pointed out through the separation of agropine spots from their extracts when electrophoreted in the presence of standared agropine.

Measuring the activity of 1-deoxy-D-xylulose 5-phosphate synthase, the first enzyme in the MEP pathway, in plant extracts.

The first enzyme in the methylerythritol phosphate (MEP) pathway is 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS). As such this enzyme is considered to be important in the control of plastidial isoprenoid production. Measuring the activity of DXS in plant extracts is therefore crucial to understanding the regulation of the MEP pathway. Due to the relatively low amounts of DXS, the activity of this enzyme can only be measured using highly sensitive analytical equipment. Here, a method is described to determine the DXS enzyme activity in a crude plant extract, by measuring DXP production directly using high performance liquid chromatography linked to a tandem triple quadrupole mass spectrometry detector (LC-MS/MS).

In vivo hypotensive effect and in vitro inhibitory activity of some Cyperaceae species

In 1820, French naturalist August Saint Hillaire, during a visit in Espírito Santo (ES), a state in southeastern Brazil, reported a popular use of Cyperaceae species as antidote to snake bites. The plant may even have a hypotensive effect, though it was never properly researched. The in vitro inhibitory of the angiotensin converting enzyme (ACE) activity of eigth ethanolic extracts of Cyperaceae was evaluated by colorimetric assay. Total phenolic and flavonoids were determined using colorimetric assay. The hypotensive effect of the active specie (Rhychonospora exaltata, ERE) and the in vivo ACE assay was measured in vivo using male Wistar Kyoto (ERE, 0.01-100mg/kg), with acetylcholine (ACh) as positive control (5 µg/kg, i.v.). The evaluation of ACE in vivo inhibitory effect was performed comparing the mean arterial pressure before and after ERE (10 mg/kg) in animals which received injection of angiotensin I (ANG I; 0,03, 03 and 300 µg/kg, i.v.). Captopril (30 mg/kg) was used as positive control. Bulbostylis capillaris (86.89 ± 15.20%) and ERE (74.89 ± 11.95%, ERE) were considered active in the in vitro ACE inhibition assay, at 100 µg/mL concentration. ACh lead to a hypotensive effect before and after ERE's curve (-40±5% and -41±3%). ERE showed a dose-dependent hypotensive effect and a in vivo ACE inhibitory effect. Cyperaceae species showed an inhibitory activity of ACE, in vitro, as well as high content of total phenolic and flavonoids. ERE exhibited an inhibitory effect on both in vitro and in vivo ACE. The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.

Evaluating the plant growth promoting ability of thermotolerant bacteria and cyanobacteria and their interactions with seed spice crops

The potential of eight thermotolerant bacteria (seven Bacillus spp. and one actinobacterium Kocuria sp.) and two cyanobacteria (Anabaena laxa and Calothrix elenkinii) as plant growth promoting (PGP) agents was evaluated with seed spices – coriander, cumin and fennel, under controlled conditions in potting mix fortified with microbial cultures. Amendment with Anabaena brought about 25% enhanced germination in cumin over control, while Calothrix enhanced root/shoot length significantly in all the three crops, especially fennel. Fortification with microbes led to 30–50% increase in shoot/root length, which was reflected as two–three fold enhancement in the vigour index of the plants. Among the bacterial strains, T4 (Bacillus pumilus) was most promising in terms of PGP traits in fennel and cumin crop. Plant dry weight and peroxidase activity of shoots and roots were enhanced by 5–10-folds in all the microbe inoculated treatments, with highest values in Calothrix treated coriander seedlings. β-1,3 endoglucanase activity showed twofold enhancement in shoots from Anabaena inoculated coriander seedlings. The fungicidal activity of the root extracts from the bacteria treated treatments of coriander seedlings against Macrophomina phaseolina was highest, while root extracts of fennel seedlings were able to show largest zones of inhibition against Fusarium moniliforme. This study highlighted the promise of fortification with both heterotrophic and photosynthetic microbes in plant growth promotion, and their significant role in enhancing and eliciting peroxidase/endoglucanase enzyme and fungicidal activity of plant extracts of seed spices.

CONSTRUCTION OF METHYLOTROPHIC YEAST HANSENULA POLYMORPHA STRAINS OVERPRODUCING FORMALDEHYDE REDUCTASE

The aim of the presented work was to obtain recombinant strains of methylotrophic yeast Hansenula polymorpha overproducing formaldehyde reductase (FR) which could be used bioanalyticaly. Methods. The yeast cells were grown in a synthetic Burkholder medium containing the following (g/l): КН2РО4 – 1; (NH4)2SO4 – 3.5; MgSO4½7H2O – 0.5; CaCl2 – 0.1; with the standard content of trace elements and 0.05% (wt/vol) yeast extract. 1% (wt/vol) glucose or 1% methanol (vol/vol) served as the sources of carbon. Enzyme activities in the cell-free extracts were determined by spectrophotometry (Shimadzu-UV-1650) at 340 nm by the rates of NADH formation for alcohol dehydrogenase (ADH), or NADH uptake for formaldehyde reductase (FR) at room temperature (20–25 °C). The activity (A) for each enzyme (in μmol∙min–1∙mg–1 protein) was calculated by the formula: A = A+substrate – A–substrate, taking into account the nonspecific background reactions. Results. We constructed the expression plasmid р21ADH1Sc contained ADH1 Saccharomyces cerevisiae gene under the strong constitutive promotеr of the H. polymorpha glyceraldehyde 3-phosphate dehydrogenase gene (GAP1). H. polymorpha NCYC 495 (leu1-1) yeast cells were transformed by electroporation. Selection of the integrants was carried out in accordance with their resistance to zeocine. Presence of the recombinant plasmid, containing the ADH1 S. cerevisiae gene, in genome of transformants was checked using polymerase chain reaction (PCR). Conclusions. Recombinant strains were characterized by 4–6 times higher activity of alcohol dehydrogenase (ADH) and 2–3 times higher activity of FR in cellfree extracts, compared to the parental strain. Stable transformants have elevated resistance to formaldehyde (up to 10 mM) in methanol containing medium.

Thermal and High-Pressure Stability of Pectin-Converting Enzymes in Broccoli and Carrot Purée: Towards the Creation of Specific Endogenous Enzyme Populations Through Processing

The thermal and pressure stability of broccoli and carrot pectin-converting enzymes, in particular pectinmethylesterase (PME), β-galactosidase (β-Gal), and α-arabinofuranosidase (α-Af), were investigated in vegetable puree matrices. In situ enzyme inactivation by thermal and high-pressure processing (respectively 5 min at 25–80 °C at 0.1 MPa and 10 min at 0.1–800 MPa at 20 °C) was evaluated by measuring the residual enzyme activity in crude extracts of treated carrot, broccoli floret, and broccoli stem puree samples. PME was completely inactivated in all vegetable puree matrices after a 5-min treatment at 80 °C. After a treatment at 800 MPa (20 °C, 10 min) only 77–90 % of pressure stable PME was inactivated, depending on the matrix. β-Gal and α-Af enzymes were inactivated in the vegetable puree matrices by thermal treatments respectively at 67.5–72.5 and 80 °C. These enzymes showed some pressure resistance: treatments respectively at 600–700 and 600–750 MPa were necessary for one log-reduction of β-Gal and α-Af activity in the different purees at 20 °C. Under the assumption of a first-order inactivation model, inactivation rate constants and their temperature or pressure dependency were determined for the different enzymes. Based on differences in process stability of the enzymes in the individual puree matrices, the feasibility for the creation of specific endogenous enzyme populations by selective processing was evaluated.

브로콜리잎 분말 첨가 재래식 고추장의 이화학적 및 기능적 특성

기능성 고추장을 제조할 목적으로 브로콜리잎 분말을 첨가하여 고추장을 제조한 후 80% 메탄올, 80% 에탄올, 물로 추출하여 총페놀 함량과 항산화 및 항암활성을 측정하여 재래식 고추장 및 개량식 고추장과 비교하였다. 총페놀 함량은 80% 메탄올과 80% 에탄올 추출물에서 브로콜리 고추장이 각각 524.2와 541.9 mg GAE/100 g으로 재래식 고추장의 431.0과 499.9 mg GAE/100 g보다 각각 22%와 8% 높았다. DPPH radical 소거능은 80% 메탄올 추출물에서는 브로콜리 고추장이 재래식 고추장보다 1.6배 높았으나, 80% 에탄올 추출물에서는 브로콜리 고추장과 재래식 고추장이 비슷하였다. 활성산소 흡수능력은 80% 메탄올과 80% 에탄올 추출물에서 브로콜리 고추장이 재래식 고추장보다 높았으나 유의적인 차이를 나타내지 않았으며 개량식 고추장보다는 각각 55%와 23% 높았다. ACE 저해 활성은 80% 메탄올 추출물에서 브로콜리 고추장은 재래식 고추장과는 유사하였으나 개량식 고추장보다는 2.6배 높았다. 항암활성은 브로콜리 고추장의 80% 에탄올 추출물인 경우 대장암 세포주(HT-29)와 폐암 세포주(NCI-H1229)에서 각각 10.7과 18.3%의 생장 저해효과를 나타내었다. 따라서 브로콜리잎의 건조분말을 첨가하여 재래식 방법으로 제조한 고추장은 총페놀 함량이 높으며, 재래식 고추장이나 공장식 고추장보다 다소 높은 항산화 활성과 암세포 성장 저해효과를 나타내어 브로콜리 분말의 첨가에 의해 고추장의 기능성이 향상되었다.

Deleterious effects of Myracrodruon urundeuva leaf extract and lectin on the maize weevil, Sitophilus zeamais (Coleoptera, Curculionidae)

Deterioration and degradation of grains by storage insect pests lead to economic losses of several billion dollars and affect food security. Sitophilus zeamais is responsible for pre- and post-harvest damages to maize. The high toxicity of synthetic insecticides and the development of resistance by insects to the chemicals currently used stimulate the investigation of plant-derived insecticides as new alternatives for pest control. In this study, we report the effects of diets containing Myracrodruon urundeuva leaf extract (10–150 mg/g) and lectin (MuLL; 3–150 mg/g) on the survival, feeding, and nutrition of the storage pest S. zeamais. The digestive enzyme activity in gut extracts from the insects reared on the leaf extract (25 mg/g) or MuLL (15 mg/g) diets was also evaluated. The leaf extract induced mortality (LC50: 72.4 mg/g), while MuLL (30–150 mg/g) exerted strong feeding deterrence. The leaf extract and MuLL promoted the loss of biomass, as reflected in the negative values for relative biomass gain rates and efficiencies in converting ingested food. Protease, trypsin-like, acid phosphatase, and amylase activities in the insects reared on leaf extract or MuLL diets were significantly (P < 0.05) lower than those in the control insects. MuLL ingestion also significantly reduced (P < 0.05) endoglucanase and alkaline phosphatase activities. In conclusion, the leaf extract and MuLL have the potential for S. zeamais control by killing adults and preventing the use of a food source, respectively. The deleterious effects of the extract and lectin on S. zeamais may be linked to enzyme inhibition and consequent suppression of digestive processes.

Two Roles for Aconitase in the Regulation of Tricarboxylic Acid Branch Gene Expression in Bacillus subtilis

Previously, it was shown that an aconitase (citB) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing Bacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of the citB promoter. B. subtilis aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [citB2]) and the other designed to be defective in RNA binding (R741E [citB7]). The citB2 mutant was a glutamate auxotroph and accumulated citrate, while the citB7 mutant was a glutamate prototroph. Unexpectedly, the citB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of the citB promoter and accumulated high levels of aconitase protein. These strains and the citB null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (citZ) transcript was present at higher-than-normal levels in the citB null mutant, due at least in part to a >3-fold increase in the stability of the citZ transcript compared to the wild type. Purified B. subtilis aconitase bound to the citZ 5' leader RNA in vitro, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the citZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.

Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O) by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L−1 of anthracene (PAH). The optimum conditions of pH (4.0–9.0), temperature (5–70 °C), reaction time (10–90 min) and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 °C, 20 °C higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract.

Proteins linked to drought tolerance revealed by DIGE analysis of drought resistant and susceptible barley varieties

Drought is a major threat to world agriculture. In order to identify proteins associated with plant drought tolerance, barley varieties bred in the UK (Golden Promise) and Iraq (Basrah) were compared. The variety Basrah showed physiological adaptations to drought when compared to Golden Promise, for example relative water content of roots and shoots after 1 week of drought was much higher for Basrah than for Golden Promise. DIGE analysis was carried out on proteins from roots and leaves under control and drought conditions. Twenty-four leaf and 45 root proteins were identified by MALDI-TOF MS. The relative expression patterns of the identified proteins fell into a number of distinct classes. The variety Basrah is characterised by constitutive expression or higher drought-induced expression levels of proteins regulating ROS production and protein folding. Photosynthetic enzymes, by contrast, were downregulated in Basrah. Enzyme assays showed a good correlation between DIGE-derived protein abundance estimates and enzyme activity in extracts. Overall, this study shows that the enhanced drought tolerance of variety Basrah is driven by an enhanced regulation of ROS under drought.

Identification of naphthalene metabolism by white rot fungus Armillaria sp. F022

AbstractArmillaria sp. F022, a white rot fungus isolated from tropical rain forest (Samarinda, Indonesia) was used to biodegrade naphthalene in cultured medium. Transformation of naphthalene by Armillaria sp. F022 which is able to use naphthalene, a two ring-polycyclic aromatic hydrocarbon (PAH) as a source of carbon and energy was investigated. The metabolic pathway was elucidated by identifying metabolites, biotransformation studies and monitoring enzyme activities in cell-free extracts. The identification of metabolites suggests that Armillaria sp. F022 initiates its attack on naphthalene by dioxygenation at its C-1 and C-4 positions to give 1,4-naphthoquinone. The intermediate 2-hydroxybenzaldehyde and salicylic acid, and the characteristic of the meta-cleavage of the resulting diol were identified in the long-term incubation. A part from typical metabolites of naphthalene degradation known from mesophiles, benzoic acid was identified as the next intermediate for the naphthalene pathway of this Armillaria sp. F022. Neither phthalic acid, catechol and cis, cis-muconic acid metabolites were detected in culture extracts. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase) produced by Armillaria sp. F022 were detected during the incubation.

Fluorescence-based active site probes for profiling deubiquitinating enzymes

Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel fluorescence imaging.

Fate and cometabolic degradation of benzo[a]pyrene by white-rot fungus Armillaria sp. F022

Armillaria sp. F022, a white-rot fungus isolated from a tropical rain forest in Samarinda, Indonesia, was used to biodegrade benzo[a]pyrene (BaP). Transformation of BaP, a 5-ring polycyclic aromatic hydrocarbon (PAH), by Armillaria sp. F022, which uses BaP as a source of carbon and energy, was investigated. However, biodegradation of BaP has been limited because of its bioavailability and toxicity. Five cosubstrates were selected as cometabolic carbon and energy sources. The results showed that Armillaria sp. F022 used BaP with and without cosubstrates. A 2.5-fold increase in degradation efficiency was achieved after addition of glucose. Meanwhile, the use of glucose as a cosubstrate could significantly stimulate laccase production compared with other cosubstrates and not using any cosubstrate. The metabolic pathway was elucidated by identifying metabolites, conducting biotransformation studies, and monitoring enzyme activities in cell-free extracts. The degradation mechanism was determined through the identification of several metabolites: benzo[a]pyrene-1,6-quinone, 1-hydroxy-2-benzoic acid, and benzoic acid.

Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

[NiFe]-hydrogenases are multimeric proteins. Thelarge subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands iscatalyzed by the combined actions of HypF andHypE using carbamoylphosphate as a substrate.We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi

HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

Phenotypic Correction of a Mouse Model for Primary Hyperoxaluria With Adeno-associated Virus Gene Transfer

Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors.

Glutathione-dependent formaldehyde dehydrogenase from golden pothos (Epipremnum aureum) and the production of formaldehyde detoxifying plants

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is an enzyme involved in formaldehyde metabolism in eukaryotes. FALDH cDNA was cloned from golden pothos, which is reported to effectively purify gaseous formaldehyde from enclosed room atmosphere. FALDH cDNAs from Arabidopsis, rice and golden pothos were overexpressed in transgenic Arabidopsis, and the enzyme activity was compared to determine the one most suitable for the molecular breeding of formaldehyde-detoxifying plants. The transgenic lines exhibited modified levels of the FALDH transcript, i.e. 10–800% compared to the endogenous transcript, due to either an overexpression or a cosuppression phenotype. The enzyme activity in the crude leaf extract was not proportionate, but did correlate with the transcription levels with certain exceptions. The FALDHs from the three plant species indicated similar enzymatic activity on average. The capacity to detoxify exogenous formaldehyde in the transformants with the FALDHs was determined at the whole plant level. Plants overexpressing FALDH from the three plant species displayed up to a 40% increase in their efficiency to take up exogenous formaldehyde as compared with the wild-type plants. On the other hand, no difference in the survival rate was observed among the transformants and wild type plants on formaldehyde-containing agar medium. These results show the FALDH from golden pothos to be similarly or more effective for detoxifying formaldehyde in transgenic plants compared with Arabidopsis and rice, and that this cDNA is applicable to the molecular breeding of formaldehyde detoxifying plants.

The Effect of Freezing and Pressure of 50 MPA and 100 MPA on the Proteolytic Activity of Enzymes in Edam Cheese

This study examined the effects of pressure treatment at 50 MPa / 0.5 h and 100 MPa / 0.5 h, at 18 ± 2oC, on the proteolytic activity of edam cheese subject to freezing after week 1, 4, 6 and 8 of ripening. The aim of the study was to test the utility of high pressures in cheese ripening under model conditions. Sensory and chemical analysis was performed on Edam cheeses after varied periods of ripening. The chemical analysis involved determination of active acidity of cheese, water and salt content and the content of different fractions of nitrogen compounds. Subsequently, the cheeses were frozen in a freezer (freezing rate 1 cm h –1 ) or in an alcohol bath (freezing rate 0.1 cm h –1 ), in order to speed up the lysis of starter cells. Extracts of frozen cheeses were subjected to high pressure treatment. Proteolytic activity of enzymes was determined by the Westhoff method in frozen, frozen and pressurised and control cheese samples after 1 and 2 weeks of incubation at 30 o C. The results of the sensory and chemical analysis displayed the normal course of the ripening process. The proteolytic activity of enzymes increased as the cheese ripened. No significant difference was found between the proteolytic activity in extracts from control cheese and in those from frozen and pressurised cheese. Freezing the cheese after 8 weeks of ripening lowered the activity of proteolytic enzymes. The proteolytic activity of enzymes in extracts from frozen cheeses after 8 weeks of ripening, pressurised at 50 MPa / 0.5 h was higher than in those subjected to the pressure of 100 MPa / 0.5 h. Pressure treatment of extracts from frozen cheeses at 50 MPa or 100 MPa did not result in expected acceleration of proteolysis.