No abstract

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 +/- 1.4 ng/ml (mean +/- s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50-100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC50 approximately 100-200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50-100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes.

Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proUlacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-4acZ fusion gene at low copy number showed that the plasmidencoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced some proU expression, but other salts were ineffective, thereby ruling out ionic strength as the stimulatory signal. High concentrations of sucrose, trehalose, or glycine betaine did not induce proU expression in vitro either, eliminating osmolarity per se as the stimulus. Reconstitution in a cell-free system rules out osmoregulatory mechanisms that depend on turgor, transmembrane signaling, or trans-acting regulators synthesized after osmotic upshock.

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (؉1 to ؉6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a ϳ20-fold increase in potency relative to wild type for the ؉3 hyperactive variant Q9R/ E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was ϳ100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of ϳ400-fold over wild type for the ؉2 variant E13R/N74K. The ؉3 enzymes have ϳ4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the ؉6 enzyme has ϳ30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

No abstract

Enzyme replacement therapy for lysosomal storage disorders depends on efficient uptake of recombinant enzyme into the tissues of patients. This uptake is mediated by oligosaccharide receptors including the cation-independent mannose 6-phosphate receptor and the mannose receptor. We have sought to exploit alternative receptor systems that are independent of glycosylation but allow for efficient delivery to the lysosome. Fusions of the human lysosomal enzymes ␣-L-iduronidase or acid ␣-glucosidase with the receptor-associated protein were efficiently endocytosed by lysosomal storage disorder patient fibroblasts, rat C6 glioma cells, mouse C2C12 myoblasts, and recombinant Chinese hamster ovary cells expressing individual members of the low-density lipoprotein receptor family. Uptake of the fusions exceeded that of phosphorylated enzyme in all cases, often by an order of magnitude or greater. Uptake was specifically mediated by members of the low-density lipoprotein receptor protein family and was followed by delivery of the fusions to the lysosome. The advantages of the lipoprotein receptor system over oligosaccharide receptor systems include more efficient cellular delivery and the potential for transcytosis of ligands across tight endothelia, including the blood-brain barrier.Enzyme replacement therapy for lysosomal storage disorders relies on the efficient delivery of recombinant enzyme to all of the affected tissues of the patient (1). Uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself and corresponding oligosaccharide receptors on target cells. Examples include the binding of phosphorylated high-mannose oligosaccharides on ␣-L-iduronidase by the cation-independent mannose 6-phosphate receptor (MPR) 1 and binding of high-mannose oligosaccharides on glucocerebrosidase by the mannose receptor (2, 3). The former system is the basis for treatment of patients with Hurler syndrome, the latter for Gaucher syndrome. Factors that limit uptake by these systems include the extent to which the recombinant enzyme is modified with the necessary oligosaccharides and the density of the oligosaccharide receptors on different tissues. The absence of quality control mechanisms for oligosaccharide modifications in the secretory pathway can lead to poorly modified recombinant enzymes. This deficiency has been addressed, with varying success, by post-secretory modification of recombinant enzymes with glycosidases or glycosyltransferases (4). Another solution to the problem is to utilize alternative uptake mechanisms that rely on proteinbased, rather than oligosaccharide-based, targeting determinants. Some studies have focused on the use of small, basic peptides, like human immunodeficiency virus TAT, for this purpose (5, 6). Others have demonstrated the feasibility of this approach using insulin-like growth factor-2, a peptide ligand for the MPR (7). The low density lipoprotein receptor (LDLR) family is one of the most widely distributed and intensively utili...

Background and Purpose-Although alteplase, a recombinant tissue plasminogen activator (tPA), is structurally identical to endogenous tPA and therefore should not induce allergy, single cases of acute hypersensitivity reactions have been reported. Until now, specific antibodies against alteplase were not detected in blood samples obtained in these patients. Case Description-We report an anaphylactic reaction in a 70-year-old white female who was treated with intravenous alteplase for thrombolysis of acute ischemic stroke 160 minutes after onset of a right-sided hemiparesis. Thirty minutes after infusion of alteplase had been started, the patient suffered acute severe sinus tachycardia and hypotension, followed by cyanosis and loss of consciousness. The alteplase infusion was stopped, and following antiallergic therapy, tachycardia and hypotension resolved within 1 hour. The hemiparesis remained unaltered, but additional harm resulting from the hemodynamic complication was not observed. Serum samples analyzed with a radioimmunoprecipitation assay were negative for total antibodies to alteplase, but in a subsequent ELISA, both samples were positive for IgE antibodies to alteplase. Conclusions-The detection of specific IgE antibodies reactive with alteplase in this patient could provide the first evidence of an anaphylactic-type reaction to alteplase in man. Because previous exposure to alteplase can be excluded, the results suggest that this patient had preexisting antibodies that were cross-reactive with one or more epitopes of alteplase and therefore precipitated the anaphylactic-type reaction.

The responses of mulberry (Morus alba L.) on exposure to different concentrations of cadmium (Cd) were studied. Plant growth and nutritional quality of the leaves were affected by soil-applied Cd. Reduction in the above parameters were obvious at the increasing concentration (above 20 mg=g) of Cd tested, although most of the parameters showed an increase at the initial concentrations (5-20 mg=g). The nutritional quality of the leaves (total protein, carbohydrate, and total chlorophyll) exhibited a declining trend with an increase in the test concentration of Cd. However, free aminoacid and total nitrogen JOURNAL OF PLANT NUTRITION, 25(4), 689-700 (2002) 689 content of the leaves showed an increase. Crude fibre content on the other hand showed an initial declining trend followed by an increase; however, compared to control no increase was observed.