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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Covalent DNA-streptavidin conjugates have been utilized for the reversible and site-selective immobilization of various biotinylated enzymes and antibodies by DNA-directed immobilization (DDI). Biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase as well as biotinylated anti-mouse and anti-rabbit immunoglobulins have been coupled to the DNA-streptavidin adapters by simple, two-component incubation and the resulting preconjugates were allowed to hybridize to complementary, surface-bound capture oligonucleotides. Quantitative measurements on microplates indicate that DDI proceeds with a higher immobilization efficiency than conventional immobilization techniques, such as the binding of the biotinylated proteins to streptavidin-coated surfaces or direct physisorption. These findings can be attributed to the reversible formation of the rigid, double-stranded DNA spacer between the surface and the proteins. Moreover, BIAcore measurements demonstrate that DDI allows a reversible functionalization of sensor surfaces with reproducible amounts of proteins. Ultimately, the simultaneous immobilization of different compounds using microstructured oligonucleotide arrays as immobilization matrices demonstrate that DDI proceeds with site selectivity due to the unique specificity of Watson-Crick base pairing.
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PMID:DNA-Directed immobilization: efficient, reversible, and site-selective surface binding of proteins by means of covalent DNA-streptavidin conjugates. 1003 62

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.
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PMID:Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin. 1009 62

Tat proteins (trans-activating proteins) are present in all known lentiviruses and are early RNA binding proteins that regulate transcription. Tat from the human immunodeficiency virus type-1 is a protein comprising 86 amino acids and encoded by 2 exons. The first 72 amino acids are encoded by exon 1 and exhibit full trans-activating activity. The second exon encodes a 14-amino-acid C-terminal sequence that is not required for trans-activation but does contain an RGD motif, which is important in binding to alphavbeta3 and alpha5beta1 integrins. Tat has an unusual property for a transcription factor; it can be released and enter cells freely, yet still retain its activity, enabling it to up-regulate a number of genes. Tat also has an angiogenic effect; it is a potent growth factor for Kaposi sarcoma-derived spindle cells, and, separately, it has been shown to bind to a specific receptor, Flk-1/KDR, on vascular smooth muscle cells, as well as to integrin-like receptors present on rat skeletal muscle cells and the lymphocyte cell line H9. It appears that the basic domain of tat is important, not only for translocation but also for nuclear localisation and trans-activation of cellular genes. As such, targeting of tat protein or, more simply, the basic domain provides great scope for therapeutic intervention in HIV-1 infection. There is also opportunity for tat to be used as a molecular tool; the protein can be manipulated to deliver non-permeable compounds into cells, an approach that already has been employed using ovalbumin, beta-galactosidase, horseradish peroxidase, and caspase-3.
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PMID:HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches. 1053 42

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.
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PMID:Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay. 1056 85

Submandibular glands obtained post-mortem from mature ferrets of both sexes were examined with the use of light microscopical histochemical methods for proteins, mucosubstances and enzymes associated with cell functions or organelles. Demilunar cells showed carboxylated mucosubstances that were mainly non-sulphated, and diffuse activity for peroxidase, E600-sensitive esterase and acid phosphatase. Thiol groups were also detected in these cells. Central acinar cells showed sulphated mucosubstances, disulphides and reticular staining for thiamine pyrophosphatase. Intercalary ducts showed diffuse activity for NADH and NAD(P)H dehydrogenases. Striated ducts contained protein, tryptophan, disulphides, neutral mucosubstances and E600-sensitive esterase periluminally. Basally, the striated ductal cells showed variable activity for peroxidase, cytochrome oxidase, succinate dehydrogenase and acid phosphatase. Basolateral plasma membranes of these cells exhibited ouabain-sensitive Na,K-ATPase activity. The collecting ducts were characterized by variable periluminal staining for acid phosphatase, beta-glucuronidase, acid beta-galactosidase and E600-resistant esterase. The results suggest that the histological appearances of the acini of the submandibular gland of the ferret are dependent on the synthesis of secretory acid glycoproteins, that the striated ducts are involved with the secretion of tryptophan-rich product comprising neutral glycoproteins and showing esterase activity and with marked transport of ions and that the collecting ducts are involved with absorption.
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PMID:Morphological phenotypes and functional capabilities of submandibular parenchymal cells of the ferret investigated by protein, mucosubstance and enzyme histochemistry. 1066 22

Chemiluminescent 1,2-dioxetane enzyme substrates provide a highly sensitive and versatile detection method for immunoblots and other membrane-based detections. 1,2-Dioxetane substrates, coupled with either alkaline phosphatase or beta-galactosidase enzyme labels, generate glow light emission kinetics, with a signal duration that is significantly longer than most enhanced luminol/horseradish peroxidase chemiluminescent detection systems. The long-lived, high-intensity light signal is ideal for imaging using a variety of formats, including X-ray film, photographic film, chemiluminescence phosphor imaging screens, and the rapidly expanding selection of camera imaging systems.
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PMID:Chemiluminescent immunodetection protocols with 1,2-dioxetane substrates. 4. 1081 17

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.
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PMID:Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase. 1098 24

The increasing demand for on-line measurement of milk composition directs science and industry to search for practical solutions, and biosensors may be a possibility. The specific objective of this work was to develop an electrochemical biosensor to determine lactose concentration in fresh raw milk. The sensor is based on serial reactions of three enzymes--beta-galactosidase, glucose oxidase, and horseradish peroxidase--immobilized on a glassy carbon electrode. The sequential enzymatic reactions increase the selectivity and sensitivity of the sensor. The sensor requires dilution of the raw milk and the addition of 5-aminosalicylic acid. Lactose concentrations in raw milk measured by the sensor were in good agreement with those measured by a reference laboratory using infrared technology. The results were obtained in milk samples that varied in fat and protein composition. From the results, we conclude that an electrochemical biosensor for determination of lactose concentration in fresh raw milk can be developed, and that the biosensor presented in this study maintained the qualities required for further development into an online sensor in the milking parlor.
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PMID:A three-cascaded-enzymes biosensor to determine lactose concentration in raw milk. 1100 21

Direct comparisons are made between covalently linked streptavidin and silver ion coated microplates. Both coatings can immobilize biotinylated molecules. Silver ion coated microplate wells can immobilize 1.8 times higher amounts of biotin labeled horseradish peroxidase. The quantitation range and capacity for the capture of horseradish peroxidase using biotin labeled horseradish peroxidase are also greater for silver ion coated microplates. Approximately twice as many anti-horseradish peroxidase antibodies can be immobilized per well using silver ion coated microplates. Higher capacities are presumed to be due to the smaller footprint of silver ions as compared to streptavidin. A direct comparison between the two coatings for a beta-galactosidase ELISA showed that while the silver ion coated microplates gave higher readings, the streptavidin coated microplates exhibited smaller well-to-well variation. However, higher well to well variation for the silver microplates is attributed to the high density of anti-beta-galactosidase antibodies on the microplates and the weak binding of clone GAL-13 to beta-galactosidase, rather than the silver coating. These studies suggest silver ion coated microplates are a desirable alternative to streptavidin plates for quantitative immunoassays.
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PMID:A comparison of silver ion to streptavidin coated microplates. 1116 40

Blood-brain barrier (BBB) permeability during sepsis with Escherichia coli or Streptococcus pneumoniae was examined in a mouse model and measured by a circulating beta-galactosidase tracer. The leakage of brain microvascular vessels during sepsis was confirmed by transmission electron microscopic examination of brain tissues stained with horseradish peroxidase. The increase of BBB permeability induced by E. coli and S. pneumoniae, which was maximal at 3 h and 12 h after injection, respectively, was transient because of rapid clearance of the bacteria from the blood. Tumour necrosis factor-alpha (TNF-alpha) was stained on microvascular vessels of the brain during sepsis and intravenous injection of recombinant TNF-alpha also increased the BBB permeability. The increase in BBB permeability induced by either E. coli or S. pneumoniae could be inhibited by anti-TNF-alpha antibody. It was concluded that circulating TNF-alpha generated during sepsis induced the increase in BBB permeability.
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PMID:Tumour necrosis factor-alpha causes an increase in blood-brain barrier permeability during sepsis. 1154 83


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