Correction of I-cell defect by hybridization with lysosomal enzyme deficient human fibroblasts

I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G_M2 gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mobility of the isoenzymes, and in a decreased activity in the medium. Fusion of I-cells with fibroblasts from G_M1 gangliosidosis, type 1, led to enhancement of β-galactosidase (β-gal) activity. This complementation must be the result of the presence of normal polypeptide chains in I-cells, whereas the other cell types provide a factor that causes the intracellular retention of the enzymes. Restoration of β-gal was also observed in heterokaryons after fusion of I-cells with β-galactosidase/neuraminidase-deficient (β-gal⁻/neur⁻) variants, indicating that the neuraminidase(s) and the posttranslational modification of β-gal are affected in a different way in I-cell disease and in β-gal⁻/neur⁻ variants. Fusion of I-cells with mannosidosis fibroblasts resulted in a restoration of the acidic form of α-mannosidase and in a decrease of the extracellular activity of both this enzyme and the hexosaminidase enzyme, indicating that fusion of I-cells with different types of fibroblasts with a single lysosomal enzyme deficiency not only leads to complementation for one particular enzyme but also to a correction of the basic defect in I-cells.     

Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture

Summary The simultaneous production of endoglucanase and -glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and -glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and -glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C     

Improved gene correction efficiency with a tailed duplex DNA fragment

A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.     

Stabilization of a tetrameric enzyme (α-amino acid ester hydrolase from Acetobacter turbidans) enables a very improved performance of ampicillin synthesis

The stabilized derivative of the enzyme α-amino acid ester hydrolase from Acetobacter turbidans has been found to be very adequate as biocatalyst of the synthesis of the very relevant antibiotic ampicillin. This enzyme resulted much more adequate than the Penicillin G Acylase (PGA) from Escherichia coli (the most used enzyme). The stabilization of the enzyme was required because under optimal conditions (absence of phosphate and 40% of MeOH), no-stabilized derivatives or soluble enzyme from A. turbidans become very rapidly inactivated. Under these conditions, this new stabilized derivative exhibited a very high selectivity for the transferase activity compared to the esterase one, as well as a very low hydrolytic activity towards the antibiotic. Moreover, this new biocatalyst did not recognize -phenylglycine as substrate in the synthetic process. By using the racemic mixture of / phenylglycine methyl ester, 85% of the -ester could be transformed to ampicillin. In contrast, the enzyme from E. coli exhibited a high hydrolytic activity for the ampicillin yielding low synthetic yields. This enzyme also resulted much less enantioselective producing both isomers of the antibiotic.     

Inactive S298R disassembles the dodecameric L-aspartate 4-decarboxylase into dimers

l-Aspartate 4-decarboxylase catalyzes the conversion of aspartate to alanine and CO₂. The wild-type enzyme was observed as dodecamers at pH 5.0. The mutation of Ser298 into Arg resulted in an almost complete loss of the enzyme activity, and caused regional structural distortion and defects in the enzyme assembly, as shown in circular dichroism spectra and gel filtration profiles. Mutating Tyr207 and Pro257 into His also resulted in inactivation of the enzyme, but did not affect the overall structure. Computer modeling suggests that Ser298 is located on the surface, and its mutation may result in enzyme disassembly, whereas Tyr207 and Pro257 are near the active site, and their mutations may cause local structure perturbation.     

Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros^mut248 mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.     

Purification and properties of a glutathione-S-transferase from corn which conjugates s-triazine herbicides

A glutathione-S-transferase involved in atrazine conjugation was purified 43-fold from corn with a total yield of 36%. The purified enzyme has a MW of 45 000 as determined by gel filtration. The estimated activation energy of the enzyme is 6.4 kcal/mol and the optimum pH for activity between 8 and 8.5. Substrate specificity studies with s-triazines indicated that atrazine was the best substrate followed by simazine and propazine. The Cl group at the 2-position was essential for enzyme activity, and replacement by a SCH₃ group resulted in a total loss of activity. The absence of an alkyl group resulted in a reduction of conjugation and 2-chloro-4,6-bis-amino-s-triazine was the poorest substrate. With insecticidal substrates (organophosphates), conjugating activity was observed only with diazinon and little or no activity was observed with ethyl parathion, malathion and etrimfos. No activity was found using methyl iodide as a substrate. The purified enzyme has properties similar to those of an aryl-S-transferase. Quinones were inhibitors of this enzyme.     

Alkali-thermostable and cellulase-free xylanase production by an extreme thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>

The optimization of cultural variables resulted in a marked enhancement in the secretion of cellulase-free and alkali-thermostable xylanase (EC 3.2.1.8) by an extreme thermophile Geobacillus thermoleovorans. The enzyme secretion was enhanced when the medium was supplemented with xylan (0.15%) and Tween-80 (0.1% v/v). In wheat bran-tryptone medium, the peak in enzyme production was attained within 42 h in a fermenter as compared to 72 h in shake flasks. Optimization of the culture conditions resulted in a 7.72-fold enhancement in enzyme production. The cellulase-free xylanase was optimally active at pH 8.5 and 80°C, and it was found to be useful in the pre-bleaching process of paper pulps.     

Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences.

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain ofEscherichia coli

Summary The influence of complex compounds on the growth of a recombinant strain ofEscherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.     

Mechanisms of cerebral radiation syndrome

Postradiation DNA breaks activate the repair enzyme, Adenosine diphosphoribosil transferase, which consumes NAD as a substrate and causes neuronal NAD and then ATP pool depletion; here this is considered to be the crucial links of the cerebral radiation syndrome (CRS) mechanism. Two ways of its metabolic correction were examined: (a) prevention of postradiation NAD depletion by administration of the enzyme inhibitor and (b) shunting NAD-dependent oxidative phosphorilation path of ATP resynthesis by administration of substrate of NAD-independent oxidation.     

DNA Polymerase-associated and autonomous vertebrate 3′→5′ exonuleases

Using methods of gel filtration and ultracentrifugation, cell-free extracts from 12 objects representing the main vertebrate representatives (bony fish, amphibian, reptiles, birds, mammals, including human) were studied. The enzyme activity of autonomous 3′→5′-exonucleases (AE) has been established to be 25–90% of the total 3′→5′ exonuclease activity of the extracts. A part of the AE is revealed in a zone of the DNA polymerases of the α-family and can be separated by changing Chromatographic conditions or by repeated fractionation. The high activity of AE allows suggesting their substantial participation in the replicative correction of the DNA-poly-merase errors as well as in the postreplicative correction of the heteroduplexes in the vertebrate DNA.     

Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max

A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli. Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum DNA. Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene has an apparent molecular weight of 67 000.     

Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice

Background aimsMucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-l-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I.MethodsWe produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice.ResultsAn increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme.ConclusionsThe results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Tyrosyl Residue at or Near the Active Site of Maize NADP-malic Enzyme

Incubation of maize NADP-malic enzyme with tetranitromethane (TNM) resulted in a total loss of enzyme activity. The loss of enzyme activity was not observed at pH 6.3 but at pH 8.0. NADP-malic enzyme was inactivated to almost 90 % by incubation with an 80-fold molar excess of TNM for 5 min at 30 °C. The substrate malate or Mg²⁺ alone gave no protection, while NADP provided considerable protection. NADP in the presence of malate and Mg²⁺ totally protected the enzyme activity, suggesting that tyrosine residue may be located at or near the active site of maize NADP-malic enzyme. The spectral analysis of the modified enzyme indicated that modification of at least one tyrosine residue per subunit resulted in complete loss of the enzyme activity. The fluorescence study of unmodified and modified enzymes postulated that essential tyrosine residue at maize NADP-malic enzyme is possibly involved in malate binding. This revised version was published online in September 2006 with corrections to the Cover Date.     

Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic activities of the Ca2+ and Mg2+-activated adenosine triphosphatase of Escherichia coli

The effect of removal or modification of the polypeptide subunits (α, β γ, δ, and ?) of the Ca²⁺ and Mg²⁺-activated ATPase of Escherichia coli was investigated. Removal of the δ-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of the γ-polypeptide, as found in the ATPase of an energy transfer coupling mutant (etc-15), resulted in diminution of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.