DNase

Recombinant DNase preparations have been used in the treatment of cystic fibrosis since the end of 1993. This genetic disorder is common, particularly in ethnic groups of northern European extraction, where the frequency of occurrence can be as high as 1 in 2500 live births. A higher than average incidence has also been recorded in southern Europe, as well as in some Jewish and African-American populations. 

A number of clinical symptoms characterize cystic fibrosis. Predominant among these is the presence of excess sodium chloride in cystic fibrosis patient sweat. Indeed, measurement of chloride levels in sweat remains the major diagnostic indicator of this disease. Another characteristic is the production of an extremely viscous, custard-like mucus in various body glands/organs that severely compromises their function. Particularly affected are  

CH12 RECOMBINANT BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 

Some 70 per cent of all cystic fibrosis patients exhibit a specific three-base-pair deletion in the gene, which results in the loss of a single amino acid (phenylalanine 508) from its final polypeptide product. Other cystic fibrosis patients display various other mutations in the same gene. 

The gene product is termed cystic fibrosis transmembrane conductance regulator (CFTR), and it codes for a chloride ion channel. It may also carry out additional (as yet undetermined) functions. 

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Kabsch, W., et al. Atomic structure of the actin DNAse I complex. Nature 347 37-44, 1990. 

Fike most enzymes (see Chapter 14), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), while others are specific for RNA (the RNases). Still... 

DNase I (pancreas) DNA a Preferably between Py and Pu nicks dsDNA, creating 3 -OH ends... 

ISG20 IFN-stimulated gene product of 20kDA A member of the DEDD exonuclease superfamily with RNAse and DNAse activity... 

DNase 1 j Under appropriate conditions, produces j single-stranded nicks in DNA. Nick translation mapping of hypersensitive sites mapping protein-DNA interactions. 

Footprinting DNA with protein bound is resistant to digestion by DNase enzymes. When a sequencing reaction is performed using such DNA, a protected area, representing the footprint of the bound protein, will be detected. 

E. Physical Methods and Analytical Techniques.—Nucleotide maps of enzymic digests of DNA have been obtained using the same ionophoretic techniques as have been developed for RNA digests. Pancreatic DNase and Neurospora crassa endonuclease produce very similar maps with E. coli DNA but this technique still awaits the discovery of specific DNases. 

They may produce extracellular enzymes, which attack the substrate without the need for transport into the cell, for example, cellulase, DNAse, or gelatinase. 

Earlier studies using thermal denaturation analysis and spectrophotomet-ric titration with TxA T and CxC-C" containing DNA triplexes showed that coralyne binds strongly to these triplexes by intercalation and does not exhibit a significant sequence-selectivity [222]. In a later study by Morau Allen et al. [217], employing DNase footprinting, thermal denaturation analysis, UV-visible spectrophotometric titrations, circular dichroism and NMR spectroscopy, showed that coralyne is fully intercalated into TxA T triplex DNA whereas in C GxC triplex, it is partially intercalated due to electrostatic repulsion between the cationic alkaloid and the protonated cytosine [217]. Kepler et al. [223] demonstrated that coralyne intercalated to parallel triplex DNA but did not intercalate to antiparallel triplex DNA. Recently Hud and coworkers [219,224] demonstrated that duplex poly(dA) poly(dT) is trans-... 

Dornase alfa (Pulmozyme ) is a recombinant human (rh) DNase that selectively cleaves extracellular deoxyribonucleic acid (DNA). This DNA is released during neutrophil degradation and contributes to the high viscosity of CF sputum. Nebulization of dornase alfa 2.5 mg once or twice daily improves daily pulmonary symptoms and function, reduces pulmonary exacerbations, and improves quality of life.16 N-acetylcysteine and hypertonic saline are other mucolytic agents that are occasionally used however, they are not preferred agents due to a greater incidence of bronchospasm and unpleasant odor and taste.5... 

Pulomozyme (DNAse) Solution for inhalation via nebulizer Store at 2-8°C protect from light lmg/mL (2.5 mL) 0.15mg/mL CaCl2, 8.77mg/mL NaCl... 

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

The histone core protects the DNA bound to the nucleosome from digestion by pan-creatic deoxyribonuclease (DNase) I or micrococcal nuclease. Nucleases, however, will cleave the linker DNA that connects the nucleosome subunits to one another. 

The Fur protein from E. coli was isolated in one step due to its high affinity for metal-chelate columns loaded with zinc. In DNase footprinting experiments, the Fur protein was shown to bind DNA in the promoter region of several iron-regulated genes. The consensus sequence, called the Fur box, is GATAATGATAATCATT ATC. In vitro binding is dependent on the divalent cations Co2+ Mn2+ /s Cd2+ Cu2+ at 150 iM, while Fe2+ seemed to be less active at this concentration, probably due to oxidation to Fe3+ (De Lorenzo et al., 1987). The unspecificity for divalent metals observed in vitro shows that the cells have to select the ions transported carefully and have to balance their active concentrations. In addition, it is a caveat for the experimenter to test a hypothesis on metal-ion specificity not only in vitro, but also in vivo. 

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