Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microencapsulation of recombinant "universal" cells with immunoprotective membranes is an alternate approach to somatic gene therapy. Therapeutic gene products secreted by these cells can be delivered to different patients without immunosuppression or genetic modification of the host's cells. The encapsulation of different mammalian cell types (epithelial cells, fibroblasts, and myoblasts) is compared among three alginate-based microcapsules: (1) calcium-linked alginate microcapsules with a solubilized core and a poly-L-lysine-alginate-laminated surface; (2) barium-linked alginate beads with a gelled core; and (3) a hybrid formulation of barium-linked alginate beads with a poly-L-lysine-alginate-laminated surface. The mechanical stability of the different microcapsule types, as measured with a cone-and-plate shearing apparatus, was superior in the two barium-linked alginate beads. All cell types maintained high viability (65-90%) in culture after encapsulation. The recombinant gene products secreted by these cells (human growth hormone MW = 22,000, human factor IX MW = 57,000, and murine beta-glucuronidase MW = 300,000) were able to traverse the three microcapsule types at similar rates. Cell numbers within the microcapsules increased twofold to > 20-fold over 4 weeks, depending on the cell type. Epithelial and myoblast cell numbers were not affected by microcapsule formulation; however, fibroblasts proliferated the most in the calcium-linked alginate spheres. These results show that for culturing fibroblasts in a mechanically stable environment the classical calcium-linked microcapsules are adequate. However, where mechanical stability is a more critical requirement, the solid barium-linked gelled beads are more appropriate choices.
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PMID:Encapsulation of various recombinant mammalian cell types in different alginate microcapsules. 982 83

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-beta-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the beta-glucuronidase reporter gene (uidA. Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tuabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed.
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PMID:The Arabidopsis thaliana dhdps gene encoding dihydrodipicolinate synthase, key enzyme of lysine biosynthesis, is expressed in a cell-specific manner. 1035 84

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.
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PMID:Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase. 1040 64

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.
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PMID:Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution. 1072 49

A tetrapeptide derivative PEP1261 [Boc-Lys-(Boc)-Arg-Asp-Ser-(tBu)-OtBu], corresponding to residues 39-42 of human lactoferrin, was tested for its antiinflammatory action in adjuvant induced arthritis in rats. Administration of heat killed Mycobacterium tuberculosis (500 microg/0.1 ml of paraffin oil) intradermally into the foot pad of right hind paw resulted in an increased paw volume and an increase in the levels of reactive oxygen species and beta-glucuronidase as well as a decrease in the antioxidants levels. PEP1261, at an effective dose of 10 mg/kg body wt., exhibited a significant antiarthritic activity as evidenced by lowering of paw volume and inhibited the free radicals toxicity by increasing the antioxidants levels. This peptide derivative was also shown to have a membrane stabilizing action by significantly decreasing the total and free activity of beta-glucuronidase and inhibiting the rate of release of the enzyme from lysosomal rich fraction. Histopathological studies confirmed the above results by showing a decrease in mononuclear cell infiltration, hypertrophy, hyperplasia and pannus formation after PEP1261 treatment in arthritic rats.
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PMID:A novel peptide derivative exhibits anti inflammatory and antioxidant activity in adjuvant induced arthritis in rats. 1193 51

Tetrahydrocurcumin (THC) is an antioxidative substance which is derived from curcumin by hydrogenation. Curcumin is the main component of turmeric and is responsible for the yellow color of curried foods.First, LDL derived from a normal human volunteer was incubated in the presence of an antioxidant with 10 microM CuSO(4) at 37 degrees C for 2 hours.All antioxidants tested (THC, curcumin, probucol, and alpha-tocopherol) dose-dependently (1-10 microM) inhibited the oxidative modification of LDL. Probucol was the strongest, followed by THC, alpha-tocopherol, and curcumin.Next, in order to evaluate the antioxidative activity of THC in vivo, we fed rabbits diets containing 1% cholesterol with or without 0.5% THC and examined their effects on oxidative stress and atherosclerosis. Animals were divided into two groups: the control group rabbits (n = 12) were fed a normal chow diet and the experimental group (n = 12) was fed a diet containing 0.5% THC for one week.Then, 1% cholesterol was added to the diets and the animals were allowed to feed further for either 6 (n = 4 for each group) or 12 weeks (n = 8 for each group). Although serum cholesterol levels rapidly increased after starting the high cholesterol diet, no difference was observed between the control and THC groups.TBARS formation in the absence of added copper ion was inhibited in the LDL separated from THC-treated animals compared with that from control animals.THC treatment tended to inhibit the area covered with atherosclerotic lesions compared with the control, although this was not significant (28.8 +/- 17.5% vs. 40.0 +/- 23.7%, p = 0.2). Formation of N(epsilon)-(hexanoyl) lysine, 4-hydroxynonenal and dityrosine in liver and kidney also had a tendency to be inhibited by THC treatment. Although free THC was not detected in serum and liver, THC was detected in samples treated with beta-glucuronidase and sulfatase, suggesting that THC is present as a conjugate with glucuronic acid or sulfate. In conclusion, the present results suggest that curcuminoids, particularly THC, which are contained in turmeric, may be useful as a functional food factor.
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PMID:The protective effects of tetrahydrocurcumin on oxidative stress in cholesterol-fed rabbits. 1240 34

The Escherichia coli Tat system has unusual capacity of translocating folded proteins across the cytoplasmic membrane. The TatA protein is the most abundant known Tat component and consists of a transmembrane segment followed by an amphipathic helix and a hydrophilic C terminus. To study the operation mechanism of the Tat apparatus, we analyzed the topology of TatA. Intriguingly, alkaline phosphatase (PhoA)-positive fusions were obtained at positions Gly-38, Lys-40, Asp-51, and Thr-53, which are all located at the cytoplasmic C terminus of the TatA protein. Interestingly, replacing phoA with uidA at Thr-53 led to positive beta-glucuronidase fusion, implying cytoplasmic location of the TatA C terminus. To further determine cellular localization of the TatA C terminus, we deleted the phoA gene and left 46 exogenous residues, including the tobacco etch virus (Tev) protease cleavage site (Tcs) after Thr-53, yielding TatA(T53)::Tcs. Unlike the PhoA and UidA fusions, which abolished the TatA function, the TatA(T53)::Tcs construct was able to restore the growth of tatA mutants on the minimal trimethlyamine N-oxide media. In vitro and in vivo proteolysis assay showed that the Tcs site of TatA(T53)::Tcs was accessible from both the periplasm and cytoplasm, indicating a dual topology of the TatA C terminus. Importantly, growth conditions seemed to influence the protein level of TatA and the cytoplasmic accessibility of the Tcs site of TatA(T53)::Tcs. A function-linked change of the TatA topology is suggested, and its implication in protein transport is discussed.
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PMID:Dual topology of the Escherichia coli TatA protein. 1470 31

The Ext 1.2A gene of Nicotiana sylvestris L. encoding an extensin, a cell wall structural protein, was characterized. Ext 1.2A encodes a polypeptide of 311 amino acids having a highly repetitive structure and showing extensin features such as Ser-(Pro)(4) repeats and a high content of Tyr and Lys. The expression profile of the gene was demonstrated using the reporter GUS (beta-glucuronidase) fused to its promoter region (-630/+124, relative to the transcription start site) and by RNA gel blots. The results show that the (-630/+124) Ext 1.2A/GUS gene fusion is expressed in the root transition zone, where cells undergo an isodiametric growth but have not yet reached the rapid elongation phase, in stem inner and outer phloems and in cortical cells at the stem/petiole junction. The Ext 1.2A gene is also induced after wounding of stems, ribs, leaves or roots. The gene fusion is expressed in stem cortical cells, in ribs and at leaf edges upon wounding. These data suggest that the (-630/+124) promoter region contains regulatory elements responsible for expression in roots and stems, as well as for response to wounding in stems and leaves.
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PMID:The Nicotiana sylvestris extensin gene, Ext 1.2A, is expressed in the root transition zone and upon wounding. 1548 88

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.
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PMID:AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis. 1550 Apr 65

Cellular biomarkers of exposure and biological effects were measured in hepatocytes of turbot exposed to either Cd, Cu or Zn at concentrations of 1 and 10 mg/l seawater for 7 days and after depuration for 14 days. Metal content in hepatocyte lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Metallothionein (MT) levels were quantified on liver sections by microdensitometry after immunohistochemical staining with a polyclonal anti cod-MT antibody (MT-OD), and in the cytosolic fraction of hepatocytes by difference pulse polarography (MT-DPP). Lysosomal structural changes (lysosomal volume, surface and numerical densities--Vv(LYS), Sv(LYS) and Nv(LYS-), and surface-to-volume ratio S/V(Lys)) were quantified by image analysis after demonstration of beta-glucuronidase activity on liver cryotome sections. Vacuolisation produced by metal-exposure in hepatocytes was quantified by stereology as volume density of vacuoles (Vv(VAC)). Exposure time and metal concentrations significantly affected Vv(BSD) in lysosomes, MT levels and the degree of vacuolisation after 1 h and 1 day exposure to the three metals. The highest Vv(BSD), MT and Vv(VAC) values were recorded after 7 days exposure in all cases. MT-OD and MT-DPP were significantly correlated with Vv(BSD). Vv(LYS) in hepatocytes increased significantly after exposure to the metals. Exposure biomarkers returned to control values after depuration with the exception of those turbots that had been exposed to 10 mg Cd/l. Alike, Vv(LYS) and Sv(lys) (Cu exposure) and Nv(LYS) (Cd and Zn exposures) returned to control values after depuration. It has been therefore demonstrated that the biomarkers used are reversible and return towards control levels once metal exposure ceases. Overall, it is concluded that Vv(BSD), MT-levels and lysosomal responses are valuable biomarkers to assess metal exposure and its effects in turbot, although in quantitative terms the biomarker response varied between metals.
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PMID:Cellular biomarkers of exposure and biological effect in hepatocytes of turbot (Scophthalmus maximus) exposed to Cd, Cu and Zn and after depuration. 1599 Jan 79


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