Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Safety assessment of Apoaequorin, a protein preparation: subchronic toxicity study in rats.

Apoaequorin, a calcium-binding protein originally isolated from jellyfish is available commercially as a dietary supplement. The objective of the present study was to investigate potential adverse effects, if any, of Apoaequorin, a recombinant protein preparation, in rats following subchronic administration. For this study, Sprague-Dawley (Hsd:SD) rats (10/sex/group) were administered via oral gavage 0 (control), 92.6, 462.9, and 926.0mg/kg body weight (bw)/day of Apoaequorin preparation, for 90 days. The corresponding amount of Apoaequorin protein was 0, 66.7, 333.3 and 666.7 mg/kg bw/day, respectively. Administration of the Apoaequorin preparation did not result in any mortality. There were no clinical or ophthalmological signs, body weight, body weight gain, food consumption, food efficiency, clinical pathology or histopathological changes attributable to administration of Apoaequorin. Any changes noted were incidental and in agreement with those historically observed in the age and strain of rats used in this study. Based on the results of this study, the No Observed-Adverse-Effect Level (NOAEL) for Apoaequorin was determined as 666.7 mg/kg bw/day, the highest dose tested.

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats.

Lactoferrin and lysozyme are important proteins of the human innate immune system. These proteins are found in breast milk and have been associated with improved infant health. Recombinant human apo-lactoferrin (apo-rhLF), 1800 and 180mg/kg bw/day, and recombinant human lysozyme (rhLZ), 360 and 36mg/kg bw/day, were orally administered to Wistar rats for 28 days. Apo-rhLF and rhLZ were expressed in rice grain, extracted, purified; the lactoferrin was iron desaturated. The animals were examined for evidence of toxicity; there were no deaths and in-life physical signs were normal. Transient differences in mean food consumption occurred in high dose apo-rhLF and low dose LZ females at week three. There were no biologically significant differences in hematological or clinical chemistry parameters. Necropsy results were normal and microscopic evaluation showed no treatment related changes in animals dosed with 1800mg/kg/day apo-rhLF or 360mg/kg/day rhLZ. The results of the 28-day oral administration demonstrate a lack of toxicity of apo-rhLF and rhLZ in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weight and pathology. The no observed adverse effect level (NOAEL) is greater than 1800mg/kg/day for apo-rhLF and 360mg/kg/day for rhLZ.

Epitope spread is not critical for the relapse and progression of MOG 8-21 induced EAE in Biozzi ABH mice.

Emerging autoimmunity (epitope-spreading) generated as a consequence of myelin damage is suggested to underlie the relapses in multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG 8-21) induces relapsing EAE in ABH mice characterized by broadening of the autoimmune reportoire. Despite epitope spreading tolerance to the priming antigen, but not emerging epitope reactivities, resulted in long-term inhibition of clinical relapse. In contrast, spinal cord homogenate induced EAE was dominated by a proteolipid protein (PLP 56-70) autoreactivity despite the plethora of CNS antigens in the immunogen. This data suggests that during relapsing-remitting demyelinating disease the pathogenic process is dominated by the initiating antigen, with only a minor role played by emerging T-cell populations. These findings may have important implications for the efficacy of antigen-based immune therapies in autoimmune disorders.

Delineation of the minimal encephalitogenic epitope of proteolipid protein peptide(91-110) and critical residues required for induction of EAE in HLA-DR3 transgenic mice.

Previously, we have reported that proteolipid protein (PLP) peptide 91-110 can induce experimental autoimmune encephalomyelitis (EAE) in HLA-DR3 transgenic (tg) mice. Here we, report that residues spanning 97-108 are the minimal epitope required for induction of EAE in DR3 mice. Utilizing a series of alanine-substituted peptides, positions 99, 101, 102, 103, 104, and 106 are identified as residues necessary for an immune response. Further analysis indicated that amino acid isoleucine (99), aspartate (102) and lysine (104) are anchor residues facilitating binding to HLA-DR3 molecules. These results may have applications in the future design of peptide based immunotherapy.

Role of zinc in the structure and toxic activity of botulinum neurotoxin.

Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.

Hemoglobin exacerbates the ocular inflammatory response to endotoxin.

BACKGROUND: There is a clinical impression that bleeding into sites of inflammation exacerbates the inflammatory response. It has been hypothesized that hemoglobinic iron (Fe) contributes to this response by catalyzing free radical reactions. In the present study, the effects of autologous hemoglobin on the inflammatory response to endotoxin was determined. In addition, the possible contributions of Fe to this response was assessed by co-injection of either transferrin or desferrioxamine. METHODS: A mild ocular inflammation was induced in rabbits by intravitreal injection of 0.25 ng endotoxin. In some animals apotransferrin, hemoglobin, hemoglobin + apotransferrin or hemoglobin + desferrioxamine were co-injected. Twenty-four hours later, anterior uveitis was quantified by slit-lamp examination and determination of protein concentration and infiltration of white cells into the aqueous humor. RESULTS: Co-injection of autologous hemoglobin with endotoxin greatly exacerbated the ocular inflammatory response to endotoxin, especially the infiltration of white cells, which was increased 15-fold. Both apotransferrin, which binds Fe at high affinity, and desferrioxamine, which chelates Fe, greatly decreased the cellular response to the co-injection. CONCLUSIONS: It is likely that hemoglobinic Fe is responsible for the increased infiltration of white cells caused by the co-injection of autologous hemaglobin and endotoxin.

Purification and properties of peditoxin and the structure of its prosthetic group, pedoxin, from the sea urchin Toxopneustes pileolus (Lamarck).

A protein toxin, termed peditoxin, containing an active prosthetic group was isolated and purified from the globiferous pedicellariae of the sea urchin Toxopneustes pileolus (Lamarck). The prosthetic group, called pedoxin, is a small molecular mass substance (206 daltons) with an empirical formula of C10H10N2O3 and is comprised of a heterocyclic lactone structure formed from pyridoxal and glycine. Administered subcutaneously or intramuscularly to mice in sublethal doses, pedoxin markedly reduced basal body temperature and led to sedation and anesthetic coma accompanied by muscular relaxation. At higher doses, it leads to convulsion and death (LD50 200 mg/kg). The apoprotein, a cytochrome b-like heme protein called pedin (molecular mass, 10,000 daltons) is itself not toxic, but the purified holoprotein is extremely toxic, causing anaphylaxis-like shock and death in experimental animals at low doses (LD50 70 micrograms/kg). Small amounts of the prosthetic group added to holoprotein preparations caused the toxicity to be greatly enhanced, suggesting that holoprotein preparations also contain apoprotein capable of being activated by the low molecular weight toxin. The structure of pedoxin was determined to be 3-hydroxy-2-methyl-5-methoxy-4-pyridineformyl-glycyliden ester and was confirmed by total synthesis.

Further evaluation of the pertussis component vaccine produced by apoceruloplasmin affinity chromatography.

The heat-treated apoceruloplasmin (Apocp) is a useful protein as an affinity ligand for the purification of pertussis toxin (PT). The amounts of Apocp in the purified antigens or the pertussis component vaccine were determined. Anti-Apocp antibodies were not detected by the passive cutaneous anaphylaxis (PCA) test in rats. No anti-Apocp antibody was detected after hyperimmunization of rabbits with the vaccine. Apocp was not detected in PT and filamentous hemagglutinin (FHA) by ELISA using rabbit anti-Apocp IgG. In the experiments using 125I-labelled Apocp, 125I-Apocp was not detected in either PT or FHA which were purified by 125I-labeled Apocp-Sepharose, DEAE Sepharose, and cellulose sulfate chromatography. The contents of human DNA were also determined to be less than 10 pg per 1 mg of Apocp, by the dot-blot hybridization method using the 32P-labeled DNA probe of Alu sequence. In the tests for the presence of inapparent viruses, HBs antigen and HTLV-III antibody, no contamination was found in either the Apocp or in the vaccine. Large amounts of various viruses, which were intentionally added to the Apocp (spiking test), were completely inactivated by heating at 65 degrees C for 18 hr. Both the Apocp and the vaccine passed the general pharmacology and acute toxicity tests. From these results, the heat-treated Apocp was considered to be a suitable affinity ligand for the purification of the antigens for the pertussis component vaccine.

[The cytotoxicity of oxidized lipoproteins]. / Etude de la cytotoxicité des lipoprotéines oxydées.

The peroxidation of low-density lipoproteins (LDL) and their subsequent cytotoxicity is believed to be involved in the atherogenesis. The aim of this work was to determine the possible involvement of lipid peroxides in the cytotoxicity of lipoproteins. We report a new experimental model system constituted by lipoproteins treated by short-UV radiations which result in a major lipid peroxidation without functional alteration of apoproteins. UV radiations induced the following lipid modifications: the content of polyunsaturated fatty acids decreased considerably in all lipid classes; the level of natural antioxidants, i.e. vitamin E and carotene, dropped dramatically; conjugated dienes and thiobarbituric acid reactive substances (TBARS) were notably increased; apoproteins from LDL exhibited little structural modification but no functional alteration. The cytotoxicity was studied in lymphoblastoid cell lines established from lymphocytes derived from normal subjects or from patients with familial hypercholesterolemia. High density lipoproteins (HDL) were shown to inhibit the cytotoxicity induced by low doses of peroxidized LDL; the protective effect of HDL was complete up to the ratio ApoB/ApoA close to 1. In addition, vitamin E and catechin, two well known antioxidants, blocked the cytotoxicity of peroxidized LDL. These results corroborate the possibility of the synergic effect of HDL and antioxidant molecules for the protection of cells against oxidized LDL that are incorporated through the LDL-receptor pathway.

Auromomycin-induced DNA damage and repair in human leukemic lymphoblasts (CCRF-CEM cells).

An alkaline elution procedure was used to study the nature of DNA damage induced by auromomycin, an antitumor protein, in human leukemic lymphoblasts (CCRF-CEM cells). The filter elution of drug-treated cells at pH 12.2 and 9.6 showed induction of both single and double strand DNA breaks. The DNA strand scission activities were linear in relation to drug concentration. The frequency of single strand breaks was higher than that of the double strand breaks. Protein-associated DNA single strand breaks were also detected in alkaline elution of drug-treated cells when a proteinase K digestion step was included in the assay protocol. The auromomycin-induced single strand breaks were repaired to almost completion within 8 h of postincubation of DNA-damaged cells whereas the repair of double strand breaks was not detected.