Effects of a Supplement Containing Apoaequorin on Verbal Learning in Older Adults in the Community.

CONTEXT: The changes in verbal learning and working memory that often occur with aging may result in reduced social and intellectual interactions. These changes significantly affect an individual's quality of life. As humans age, the body's ability to regulate and maintain calcium levels is diminished. Pharmacological manipulation of the entry of free calcium (Ca2+) has been shown to be effective in increasing some aspects of cognitive function in the aged brain. Apoaequorin has been shown in laboratory studies to regulate levels of intracellular calcium in neuronal cells and to provide protection against ischemic cell death. OBJECTIVE: The study was designed to assess the effects of a supplement of apoaequorin on verbal learning and working memory. DESIGN: The current study, the Madison Memory Study, was a randomized, double-blind, placebo-controlled trial. SETTING: The study occurred in Madison, WI, USA. PARTICIPANTS: Participants were 218 community-dwelling adults, aged 40-91 y, with self-reported memory concerns. INTERVENTION: Participants were randomly assigned to receive either apoaequorin (apoaequorin group) or a matched placebo (control group) for 90 d. OUTCOME MEASURES: The study used quantitative, computerized tools for cognitive assessment the CogState International Shopping List (ISL) and the CogState ISL-Delayed Recall (ISL-DR). Scores from computerized cognitive tasks were measured at baseline and at several points during the 90-d study. RESULTS: No significant differences existed between the intervention and control groups in any parameter at baseline. The intervention group (apoaequorin group) showed a statistically significant improvement in verbal learning and recall on the ISL and the ISL-DR, respectively, during the 90-d study. Apoaequorin was tolerated very well in the study. CONCLUSIONS: The results indicated a strong relationship between apoaequorin and improvements on a quantitative measure of cognitive function, specifically verbal learning. The study found that apoaequorin is a well-tolerated supplement that improved cognitive function in aging adults. The results suggest potential utility for apoaequorin in addressing the declines in cognitive function associated with aging.

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.

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability.

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.

Expression of a synthetic porcine alpha-lactalbumin gene in the kernels of transgenic maize.

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine alpha-lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3' terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The alpha-lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine alpha-lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5' end of the synthetic porcine alpha-lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine alpha-lactalbumin accumulation in transgenic maize kernels.