Low Levels Of Enzyme Research Articles

Optimization of operational conditions for adipate ester synthesis in a stirred tank reactor

Esterification of adipic acid and oleyl alcohol in a solvent-free system featuring a stirred tank reactor containing commercially immobilized Candida antarctica lipase B was performed. The process was carried out using an artificial neural network (ANN) trained by the Levenberg-Marquardt (LM) algorithm. The effects of four operative variables, temperature, time, amount of enzyme, and impeller speed, on the reaction yield were studied. By examining different ANN configurations, the best network was found to consist of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The values of the coefficient of determination (R2) and root mean squared error (RMSE) between the actual and predicted responses were determined to be 1 and 0.0058178 for training and 0.99467 and 0.622540 for the testing datasets, respectively. These results imply that the developed model was capable of predicting the esterification yield. The operative variables affected the yield, and their order of contribution was as follows: time > amount of enzyme > temperature > impeller speed. A high percentage of yield (95.7%) was obtained using a low level of enzyme (2.5% w/w), and the temperature, time, and impeller speed were 66.5°C, 354 min (about 6 h), and 500 rpm, respectively. A simple protocol for efficient substrate conversion in a solvent-free system evidenced by high enzyme stability is indicative of successful ester synthesis.

Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse

Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.

Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheat

Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu(2+) sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5 U/mg of total protein for gluten in a SDS gluten assay (1 U of enzyme activity was defined as the decrease in gel height in millimeters in 1 h) and exhibited a high-affinity Km of 65 microM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20 degrees and 35 degrees C. The enzyme was identified to be a prolyl endoprotease.

Expression of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) and its role in neural tube defects

ITPK1 is the rate limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP6 and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP5 or IP6 in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.

Low levels of steroid-metabolizing hepatic enzyme (cytochrome P450 3A) activity may elevate responsiveness to steroids and may increase risk of steroid-induced osteonecrosis even with low glucocorticoid dose

BackgroundThe main purpose of this study was to examine the relationships among osteonecrosis, steroid-metabolizing hepatic enzyme (cytochrome P450 3A; CYP3A) activity, and steroid dose to determine whether it is possible to prevent osteonecrosis in animals with low hepatic CYP3A activity by reducing exogenous steroid doses. MethodsJapanese white rabbits (n = 103) were divided into three groups: a group with CYP3A activity induction (by intramuscular phenobarbital injection, n = 31), a group with CYP3A activity inhibition (by oral itraconazole administration, n = 30), and a control group (n = 42). Three weeks later, all rabbits received a methylprednisolone injection. Each group was divided into two subgroups by dosage of methyl-prednisolone (5 or 10 mg/kg body weight). Three weeks after methylprednisolone injections, the animals were killed and histological examination was performed to determine the incidences of osteonecrosis in the six subgroups. ResultsIncidence in the inhibition subgroup with 5 mg/kg steroid was higher than that in the induction subgroup receiving 10 mg/kg steroid. Thus, suppression of CYP3A activity significantly increased vulnerability to steroid-induced osteonecrosis, while increased CYP3A activity reduced this vulnerability. ConclusionsThese findings suggest that low CYP3A activity may be vulnerable to the effect of steroids and increase risk of osteonecrosis, even with a low dose of steroid.

Metabolic responses to long-term pharmacological inhibition of CB1-receptor activity in mice in relation to dietary fat composition

The antiobesity effects of suppressed endocannabinoid signaling may rely, at least in part, on changes in lipid fluxes. As fatty acids exert specific effects depending on their level of saturation, we hypothesized that the dietary fatty acid composition would influence the outcome of treatment with a CB(1)-receptor antagonist (rimonabant). Mice were treated with rimonabant (10 mg kg(-1) body weight per day) or vehicle while equicalorically fed either a low-fat diet (LF), a high-fat (HF) diet or an HF diet in which 10% of the saturated fatty acids (SFAs) were replaced by poly-unsaturated fatty acids (PUFA) from fish oil (FO). Food intake and body weight were registered daily. Indirect calorimetry was performed and feces were collected. After 3 weeks, mice were killed for blood and tissue collection. Relative to the LF diet, the HF diet caused anticipated metabolic derangements, which were partly reversed by the HF/FO diet. The HF/FO diet, however, was most obesity-promoting despite inhibiting lipogenesis as indicated by low gene expression levels of lipogenic enzymes. On all three diets, rimonabant treatment improved metabolic derangements and led to significantly lower body weight gain than their respective controls. This latter effect appeared largest in the HF/FO group, but occurred without major changes in nutrient absorption and energy expenditure. The effects of chronic rimonabant treatment on body weight gain occurred irrespective of diet-induced changes in lipogenic activity, food intake and daily energy expenditure, and were, in fact, most pronounced in HF/FO mice. The effects of dietary PUFA replacement in an HF diet on expansion of adipose tissue might allow the favorable effects of dietary PUFA on dyslipidemia and hepatic steatosis. In light of other disadvantageous effects of weight gain, this might be a risky trade-off.

Black Americans are More Likely to have Intermediate TPMT Enzyme Levels than White Americans

Purpose: Thiopurine methyltransferase (TPMT) metabolizes azathioprine (AZA) and mercaptopurine (6MP) into active and inactive metabolites. Optimal therapeutic effect is achieved at 6-thioguanine levels (6TG) between 230-400. Achieving therapeutic levels of 6TG is limited by the concurrent production of the inactive hepatotoxic metabolite 6-methylmercaptopurine (6MMP). A patient's TPMT enzyme activity is related to the ratio of production of these metabolites. Black Americans and Caucasians have differing frequencies of mutant alleles resulting in similar phenotypes of low, intermediate and normal levels. It is unclear whether there are racial differences in TPMT levels. Such differences could affect both medication choices and dosage regimens in the management of patients with inflammatory bowel disease. We sought to investigate ethnic variations in TMPT levels. Methods: We performed a retrospective chart review of patients who underwent TPMT level or genetic testing at our institution from June 2004 to June 2009. TPMT testing was performed at Prometheus Laboratories (San Diego, CA). Results: We identified 272 patients who had TPMT levels drawn (96 males, 176 females). Of the patients with quantitative TPMT levels available for review, 80 were self-described black American, 133 Caucasian, and 18 other. These levels are categorized into low, intermediate and normal (with less than 6.0 EU being low, 6.0- 21.0 EU intermediate and greater than 21.0 EU normal). The mean TPMT level for black Americans was 23.2 EU (SD 8.1), for Caucasians 27.7 EU (SD 9.4) and for other patients 25.7 EU (SD 8.7) (p=0.002). The mean levels are all within the normal range. Categorization of these levels into low, intermediate and normal showed that 0/80 (0.0%) of black Americans, 1/133 (0.8%) of Caucasians and 0/18 (0.0%) of others had low enzyme levels. 31/80 (38.8%) of black Americans, 26/133 (19.6%) of Caucasians and 5/18 (27.3%) of others had intermediate levels. 49/80 (61.3%) of black Americans, 106/133 (79.7%) of Caucasians and 13/18 (72.2%) of others had normal levels. The difference among the three categories is statistically significant (Fisher exact test p-value=0.017). Conclusion: Significant racial differences exist in mean TPMT enzyme levels among different American ethnic groups. While mean levels fall within the normal enzyme range, there was a statistically significant difference in the distribution of patients who fall into the intermediate versus the normal enzyme level between black and white Americans with a higher percentage of black Americans in the intermediate range and higher percentage of Caucasians in the normal range. Such differences may have important clinical implications insofar as ability to achieve therapeutic levels of 6TG.

Effects of Lutein on Oxidative Stress in the Term Newborn: A Pilot Study

Background: Oxidative stress (OS) plays a crucial role in pathological conditions during the early neonatal period. The newborns are susceptible to oxidative damage due to high metabolic rate and low levels of antioxidant enzymes. Lutein has been found to have protective functions in adult humans as antioxidant. Aim: To evaluate the effects of lutein on OS in newborns. We tested the hypothesis that lutein would act both by increasing antioxidant capacity and inhibiting OS. Methods: This was a randomized, double-blind, placebo-controlled, single-center study. 20 healthy term newborns were assigned to receive lutein or placebo (lutein and control group, respectively) at 12 and 36 h after birth. Total hydroperoxides (TH), as marker of OS, and biological antioxidant potential (BAP), as marker of antioxidant power, were detected on cord blood and at 48 h of life in all babies. Results: TH significantly increased from birth to 48 h in the control group (p = 0.02), but not in the lutein group. In the lutein group, BAP significantly increased after 48 h (p = 0.02), showing a strengthening of antioxidant activity due to lutein. At 48 h of life, compared with those in the control group, neonates assigned to receive lutein had significantly lower TH levels (p = 0.04) and higher BAP levels (p = 0.028). Conclusions: Lutein administration in newborns increases the levels of BAP decreasing TH. The enhancement of antioxidant activity in plasma clearly results in protecting newborn from perinatal OS. These preliminary results, adding a new contribution in antioxidant strategies, strongly require to be confirmed by RCT.

Thermal inactivation kinetics of alkaline phosphatase in equine milk

Alkaline phosphatase (ALP) is widely used as an indicator of proper pasteurization in bovine milk. Due to interest in the use of equine ALP as a time/temperature integrator (TTI) for evaluation of the efficacy of thermal processing of equine milk, its inactivation kinetics were evaluated in whole and skimmed equine milk. Experimentally determined decimal reduction times showed that equine ALP is more readily inactivated in equine milk than its bovine counterpart. Thus, considering the required 6 D reduction of pathogens and the rather low enzyme level present in equine milk, equine ALP will not be suitable as indicator for correct pasteurization of equine milk under the conditions currently used in the reference method for the determination of ALP in milk-based products.

Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats

Neurons have a high demand for cholesterol to develop and maintain membrane-rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum-free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell-specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post-squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol-converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up-regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.

Computer-assisted myocardial blush quantification after percutaneous coronary angioplasty for acute myocardial infarction: a substudy from the TAPAS trial

Myocardial reperfusion after acute myocardial infarction can be angiographically assessed by the myocardial blush grade (MBG) or TIMI Perfusion Grade. These scores are based on subjective human judgement and lead to a score of four categories. A more operator-independent way of scoring myocardial perfusion may facilitate research in this area. We designed the 'Quantitative Blush Evaluator' (QuBE), a computer program which calculates a score for myocardial perfusion. This program will be freely available as open source software. The inter-observer concordance was 97.7%. We calculated values on prospectively collected angiograms in patients with acute ST-elevation myocardial infarction from the TAPAS trial. Quantitative blush evaluator values could be assessed on 790 out of 980 collected angiograms (81%). The QuBE score correlated significantly with MBG as determined by a core lab. The QuBE score predicted complete ST-elevation resolution, low enzyme levels, and 1 year survival (all P < 0.001). Quantitative blush evaluator value was an independent predictor of mortality at 1 year [OR 0.40 (0.17-0.90), P = 0.02]. The QuBE program provides a practical, freely available computer-assisted assessment of myocardial perfusion. The QuBE score provides a useful surrogate endpoint in trials of therapies aimed at improving myocardial reperfusion.

Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects in 2008

This review highlights some of the research advances in anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects and in allergic skin disease that were reported in the Journal in 2008. Key epidemiologic observations include a rise in anaphylaxis in a population-based study and lower rates of peanut allergy in Israel, where infants consume peanut early compared with the United Kingdom, where dietary introduction is generally delayed. Advances in food allergy diagnosis include IgE epitope mapping that discloses the likelihood and severity of allergy; studies correlating likelihood of clinical reactivity on the basis of food-specific IgE to sesame, peanut, milk, and tree nuts; and an observation that a low baseline angiotensin-converting enzyme level may be associated with having pharyngeal edema during a reaction. Molecular, immunologic, and genetic studies are discerning pathways that are key in development of food allergy, identifying new modalities to interrupt mast cell degranulation, and elucidating risks associated with penicillin allergy. Regarding treatment, clinical studies show a majority of children with milk and egg allergy tolerate these proteins in modest amounts when they are extensively heated in baked goods, and studies show promise for oral immunotherapy to treat milk allergy and sublingual immunotherapy for honey bee venom hypersensitivity. The importance of skin barrier dysfunction has continued to be highlighted in the pathophysiology of atopic dermatitis (AD). Research has also continued to identify immunologic defects that contribute to the propensity of patients with AD to develop viral and bacterial infection. New therapeutic approaches to AD, urticaria, and angioedema have been reported including use of probiotics, biologics, vitamin D, and skin barrier creams.

Roles of polymorphic enzymes CYP2D6 and CYP2C19 for in vitro metabolism of amitriptyline at therapeutic and toxic levels

The metabolism of the tricyclic antidepressant amitriptyline was studied in vitro in the presence of the main metabolite nortriptyline to simulate the steady state with amitriptyline and nortriptyline present in the ratio 1:1. The metabolism of the active metabolite nortriptyline in the presence of amitriptyline was also studied. The contribution of cytochrome P450 (CYP) 2D6, CYP2C19, and CYP3A4 was assessed by in vitro experiments at therapeutic (5 μM) and toxic (50 μM) concentrations for amitriptyline or nortriptyline with human liver microsomes (HLM). The results indicated that nortriptyline acted as a competitive inhibitor at the steady-state concentrations, lowering the amitriptyline metabolism, and thus changed the importance of different metabolic pathways. When the concentration of substrate amitriptyline was increased from therapeutic level to toxic level (in the steady state), the contribution of CYP2D6 significantly decreased for its demethylation and hydroxylation pathways. However, the contribution of CYP3A4 increased according to the increase in substrate concentration. Similar results could be obtained when nortriptyline was used as substrate in the presence and absence of the precursor amitriptyline. For CYP2C19 in the average HLM, its contribution to metabolism of both amitriptyline and nortriptyline was found to be small. However, it was also found that CYP2C19, in a “high activity” CYP2C19 HLM, was active for N-demethylation of both amitriptyline and nortriptyline and likewise the importance of CYP2C19 decreased when the concentration of substrate was increased. The above results indicate that CYP2D6 or CYP2C19 are generally not likely to be critical enzymes related to accidental intoxication under treatment with amitriptyline, but other contributing factors would be important; for example, low levels of various CYP enzymes and/or drug-drug interactions resulting in inhibition of several CYP enzymes may cause amitriptyline poisoning.

Decreased Serum Paraoxonase 1 Activity and Increased Serum Homocysteine and Malondialdehyde Levels in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is one of the most common causes of vision loss. AMD has been classified into two forms: atrophic and exudative forms. The exudative form is associated with choroidal neovascularization of the subretinal macular region, resulting in a sudden loss of central vision. However, the exact cause of AMD remains unknown. Several risk factors have been postulated, including smoking, atherosclerosis, and low levels of antioxidant enzymes. Malondialdehyde (MDA), a lipid peroxidation product, is used as a marker of oxidative stress. Paraoxonase 1 (PON1) metabolizes lipid peroxides and prevents oxidation of low-density lipoprotein. Increased levels of homocysteine may cause vascular endothelial injury by releasing free radicals. The purpose of this study is to investigate the relationships between serum PON1 activity and the serum levels of homocysteine and MDA in AMD. Forty patients with exudative-type AMD (63.3 +/- 5 years) and 40 controls (61+/- 4 years) were assessed in a cross-sectional study. The serum PON1 activity was significantly lower in the patients with AMD than that in the controls (p < 0.001). In contrast, the serum levels of MDA and homocysteine were significantly higher in the patients than those in the controls (p < 0.001, for both). In AMD patients, significant negative correlation was found between PON1 activity and MDA level (r = -0.493, p < 0.05) and between PON1 activity and homocysteine level (r = -0.557, p < 0.05). Increased serum homocysteine and MDA levels may be responsible for the decreased PON1 activity in patients with AMD.

O 6-Methylguanine DNA Methyltransferase Deficiency and Response to Temozolomide-Based Therapy in Patients with Neuroendocrine Tumors

Recent studies suggest that temozolomide has activity in neuroendocrine tumors. Low levels of the DNA repair enzyme, O(6)-methylguanine DNA methyltransferase (MGMT), are associated with sensitivity to temozolomide in other tumor types. We evaluated the prevalence of MGMT deficiency in neuroendocrine tumors and correlated MGMT deficiency with treatment response to temozolomide-based regimens. The prevalence of MGMT deficiency, measured by immunohistochemistry, was assessed in 97 archival neuroendocrine tumor specimens. Rates of treatment response and survival were next evaluated in a cohort of 101 consecutive neuroendocrine tumor patients who had received treatment with a temozolomide-based regimen at one of three institutions. MGMT expression was directly correlated with treatment response in 21 patients who had available tumor tissue and response data. In archival specimens, MGMT deficiency was observed in 19 of 37 (51%) pancreatic neuroendocrine tumors and 0 of 60 (0%) carcinoid tumors (P < 0.0001). In the clinical cohort, 18 of 53 (34%) patients with pancreatic neuroendocrine tumors but only 1 of 44 (2%) patients with carcinoid tumors (P < 0.001) experienced a partial or complete response to temozolomide-based therapy. Among 21 patients with evaluable tumor tissue who had also received treatment with temozolomide, 4 of 5 patients with MGMT-deficient tumors (all pancreatic neuroendocrine tumors) and 0 of 16 patients with tumors showing intact MGMT expression responded to treatment (P = 0.001). MGMT deficiency, measured by immunohistochemistry, is more common in pancreatic neuroendocrine tumors than in carcinoid tumors as is treatment response to temozolomide-based therapy. Absence of MGMT may explain the sensitivity of some pancreatic neuroendocrine tumors to treatment.

Dependence of Bacterial Chemotaxis on Gradient Shape and Adaptation Rate

Simulation of cellular behavior on multiple scales requires models that are sufficiently detailed to capture central intracellular processes but at the same time enable the simulation of entire cell populations in a computationally cheap way. In this paper we present RapidCell, a hybrid model of chemotactic Escherichia coli that combines the Monod-Wyman-Changeux signal processing by mixed chemoreceptor clusters, the adaptation dynamics described by ordinary differential equations, and a detailed model of cell tumbling. Our model dramatically reduces computational costs and allows the highly efficient simulation of E. coli chemotaxis. We use the model to investigate chemotaxis in different gradients, and suggest a new, constant-activity type of gradient to systematically study chemotactic behavior of virtual bacteria. Using the unique properties of this gradient, we show that optimal chemotaxis is observed in a narrow range of CheA kinase activity, where concentration of the response regulator CheY-P falls into the operating range of flagellar motors. Our simulations also confirm that the CheB phosphorylation feedback improves chemotactic efficiency by shifting the average CheY-P concentration to fit the motor operating range. Our results suggest that in liquid media the variability in adaptation times among cells may be evolutionary favorable to ensure coexistence of subpopulations that will be optimally tactic in different gradients. However, in a porous medium (agar) such variability appears to be less important, because agar structure poses mainly negative selection against subpopulations with low levels of adaptation enzymes. RapidCell is available from the authors upon request.

Cysteine, Sulfite, and Glutamate Toxicity: A Cause of ALS?

Amyotrophic lateral sclerosis (ALS) of nonmutant superoxide dismutase (SOD) type may be caused by toxicity of the reduced glutathione (GSH) precursors glutamate and cysteine, and sulfite (a metabolite of cysteine), which accumulate when one or more of the enzymes needed for GSH synthesis are defective. A case is examined where the patient exhibited elevated sulfur on a hair mineral analysis, elevated blood cysteine, positive urine sulfite, elevated urine glutamate, and low whole blood GSH. During the time when strict dietary and supplement measures normalized the patient's whole blood GSH, blood cysteine, and urine sulfite, the patient did not experience additional physical decline. The possible causes of abnormalities of the patient's laboratory test results, as well as the nutrition measures used to normalize them, are discussed in relationship to the functions and importance of cysteine, sulfite, and glutamate in glutathione metabolism in ALS. Since elevated plasma cysteine has been reported in other ALS patients, sulfite and cysteine toxicity may be involved in other cases of ALS. Patients with ALS with nonmutant-SOD should be tested for sulfite toxicity, cysteine, glutamate and GSH levels, and whether they have low levels of GSH metabolism enzymes. Since glutamate metabolism appears to be inhibited by sulfite, research on the effect of sulfite on glutamate levels in patients with ALS should be pursued. Life might be prolonged in those patients with ALS with sulfite toxicity by closely monitoring the blood cysteine and urine sulfite levels and minimizing their dietary intake, as well as increasing GSH by using sublingual GSH. A long-term solution might be found through research to determine methods to increase GSH synthesis without using sulfur-containing supplements that may add to the cysteine and sulfite toxicity.

Analysis of mutant DNA polymerase γ in patients with mitochondrial DNA depletion

We studied six unrelated children with depletion of mitochondrial DNA (mtDNA). They presented with Leigh syndrome, infantile hepatocerebral mtDNA depletion syndrome, or Alpers-Huttenlocher syndrome. Several genes have been implicated in mtDNA depletion. Screening of candidate genes indicated that all six patients were compound heterozygous for missense mutations in the gene for the catalytic subunit of DNA polymerase gamma (POLG). Three of the identified mutations, c.3328C>T (p.H1110Y), c.3401A>G (p.H1134R), and c.3406G>A (p.E1136K), have not been reported earlier. To investigate the functional consequences of the mutations, we carried out a series of biochemical assays in cultured fibroblasts. These studies revealed that fibroblast cultures from the patients with infantile hepatocerebral mtDNA depletion syndrome progressively lost their mtDNA during culturing, whereas fibroblast cultures from patients presenting with Leigh syndrome or Alpers-Huttenlocher syndrome had reduced but stable levels of mtDNA. DNA polymerase gamma activity was below the normal range in all patient cultures, except for one; however, this culture showed low levels of the heterodimeric enzyme and poor DNA polymerase gamma processivity. Parental fibroblast cultures had normal catalytic efficiency of DNA polymerase gamma, consistent with the observation that all carriers are asymptomatic. Thus, we report the first patient with Leigh syndrome caused by POLG mutations. The cell culture experiments established the pathogenicity of the identified POLG mutations and helped to define the molecular mechanisms responsible for mtDNA depletion in the patients' tissues. The assays may facilitate the identification of those patients in whom screening for POLG mutations would be most appropriate.

G.P.11.05 The pharmacological chaperone AT2220 increases trafficking, processing, and cellular activity of acid α-glucosidase and is a potential new treatment for Pompe disease

Pompe disease is an inherited disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity, which reduces lysosomal glycogen turnover and results in progressive skeletal muscle weakness, reduced cardiac function, and respiratory insufficiency. As a potential treatment for Pompe disease, we are developing the pharmacological chaperone AT2220 (1-deoxynojirimycin-HCl), a molecule that directly binds to and stabilizes GAA. To determine the mechanism of action for AT2220, we examined its effect on mutant GAA trafficking, processing, and activity. GAA is synthesized as a precursor protein that trafficks from the endoplasmic reticulum (ER) to lysosomes. In lysosomes, GAA is proteolytically processed into mature forms that have higher affinity for the natural substrate, glycogen. Patient-derived GAA-P545L fibroblasts contain mainly the precursor form and have low levels of enzyme activity, indicative of reduced trafficking and processing of mutant GAA. Incubation of cells with AT2220 increases GAA-P545L trafficking out of the ER, as shown by pulse-chase, glycosidase sensitivity, and lectin affinity experiments. Moreover, AT2220 incubation increases GAA-P545L levels in lysosomes, as evidenced by increased amounts of mature protein, elevated enzyme activity in lysosomal fractions, and increased affinity for glycogen. These results indicate that AT2220 improves mutant GAA trafficking and processing that ultimately leads to increased lysosomal enzyme activity. AT2220 treatment of wild type rodents and monkeys increases mature GAA protein and enzyme activity levels in disease-relevant tissues, including gastrocnemius, heart, and diaphragm. The finding that wild type GAA levels can be increased suggests that AT2220 may increase GAA activity in patients with the common IVS1 (−13 t → g) splice variant that produces low levels of wild type enzyme. Collectively, these data indicate that AT2220 merits further evaluation as a treatment for Pompe disease.

Widespread distribution of DNA glycosylases removing oxidative DNA lesions in human and rodent brains

High metabolic activity and low levels of antioxidant enzymes make neurons particularly prone to damage by reactive oxygen species. Thus, repair of oxidative DNA damage is essential for normal brain function. Base excision repair is the major pathway for repair of oxidative DNA damage, and is initiated by DNA glycosylases recognizing and removing the damaged base. In mammalian cells at least five different DNA glycosylases with overlapping substrate specificity, NEIL1, NEIL2, NEIL3, OGG1 and NTH1, remove oxidative DNA base lesions. Here we report mRNA expression and distribution of these five DNA glycosylases in human and rodent brains using in situ hybridization and Northern blotting supported by glycosylase activity assays. NEIL1, NEIL2, OGG1 and NTH1 showed widespread expression at all ages. In situ hybridization studies in mouse brain showed that expression of mNeil1 increased with age. In newborn mouse brain, mNeil3 revealed a discrete expression pattern in brain regions known to harbour stem cell populations, i.e., the subventricular zone, the rostral migratory stream, and the hilar region of the hippocampal formation. Expression of mNeil3 decreased with age, and in old mice brains could be detected only in layer V of neocortex. MNth1 was constitutively expressed during lifespan. In Northern blots, mOgg1 expression showed a transient decrease followed by an increase after 8 weeks of age. Assays for faPy DNA glycosylase activity revealed increased activity level with age in all brain regions analyzed. The widespread but differential expression of the DNA glycosylases recognizing oxidative base lesions suggests distinct and age dependent roles of these enzymes in genome maintenance in brain. The distribution of mNeil3 is particularly intriguing and points to a specific role of this enzyme in stem cell differentiation.