Pseudovitamin B12 and factor S are the predominant corrinoid compounds in edible cricket products.

In this study, we determined the vitamin B12 content of commercially-available edible insect products using a bioassay based on Lactobacillus delbrueckii ATCC 7830. Although the vitamin content of giant water bug, bee larva, grasshopper, and weaver ant products was low, we found that diving beetle and cricket products contained relatively high amounts of vitamin B12 (approximately 89.5 and 65.8 µg/100 g dry weight, respectively). In the cricket products most widely circulated as foods, specific corrinoid (vitamin B12) compounds were extracted and identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Despite the bioassay detecting high vitamin B12 content (approximately 50-75 µg/100 g dry weight) in these cricket products, UPLC-MS/MS analysis indicated that pseudovitamin B12 and 2-methylmercaptoadenyl cobamide (also known as factor S) were actually the predominant corrinoid compounds (~74% and ~21%, respectively), with authentic vitamin B12 making up only 5% of total corrinoids.

Development of evaluation system for bioactive substances using human artificial chromosome-mediated osteocalcin gene expression.

Bioactive substances in daily food and supplements are expected to prevent various lifestyle-related diseases. Recently, many evaluation systems for bioactive substances were developed with cell lines integrated with green fluorescence protein (GFP) reporter gene. To evaluate osteogensis activity in functional food, we developed a novel cell line that reports osteocalcin gene expression using the human artificial chromosome (HAC) vector. HAC vectors are able to avoid various problems in usual plasmid vector such as difficulty in control of transgene copy number. HAC is transmitted to cells as an independent chromosome from host chromosomes, and expresses transgenes depending on host cell circumstances. We established Chinese hamster ovary cell lines that carried GFP gene regulated by osteocalcin gene promoter on the HAC. Expression of GFP was responded to vitamin D(3) [1alpha,25(OH)(2)D(3)]. Furthermore, we constructed HAC vector bearing tandem repeats of reporter gene unit, to enhance intensity of gene expression. GFP expression in these reporter cells is related to the copy number of reporter gene units. Using the evaluation system for bioactive substances, we could show osteogenic activity in some fish oils.

Mutagenic radioadaptation in a human lymphoblastoid cell line.

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.

Mutation induction in cultured human cells after low-dose and low-dose-rate gamma-ray irradiation: detection by LOH analysis.

To study the genetic effects of low-doses and low-dose-rate ionizing radiation (IR), human lymphoblastoid TK6 cells were exposed to 30 mGy of gamma-rays at a dose-rate of 1.2 mGy/hr. The frequency of early mutations (EMs) in the thymidine kinase (TK) gene locus was determined to be 1.7 x 10(-6), or 1.9-fold higher than the level seen in unirradated controls. These mutations were analyzed with a loss of heterozygosity (LOH) detection system, a methodology which has been shown to be sensitive to the effects of radiation. Among the 15 EMs observed after IR exposure, 8 were small interstitial-deletion events restricted to the TK gene locus. However, this specific type of event was not found in unirradiated controls. Although these results were observed under the limited conditions, they strongly suggest that the LOH detection system can be used for estimating the genetic effects of a low-dose IR exposure delivered at a low-dose-rate.

Elevation of plasma membrane permeability on laser irradiation of extracellular latex particles.

In this report, we describe a laser-latex combination system that enables membrane-impermeable molecules to penetrate cell membranes. Laser light (Q-switched Nd:YAG laser, 532.5 nm) was used to irradiate a mixture of commercial latex particles (blue dyed, 1 micro m in diameter) and mouse fibrosarcoma (Meth-A) cells. After irradiation, membrane permeability was evaluated by flow cytometric assaying using propidium iodide (PI) and fluorescein diacetate (FDA). The proportion of permeabilized-resealed cells was affected by changes in the light intensity (approximately 780 mW/cm(2)), the irradiation time (approximately 240 s), and/or the particle concentration (approximately 10(9) particles/ml). The permeability persisted up to 20 min after light irradiation. Near the sites of individual particles, the permeability of the cell membrane is modified, probably due to localized temperature changes. These results suggest that this laser-induced permeabilization strategy constitutes a new means of delivering exogenous materials into living cells.

Enhancement of cytotoxic effect of bleomycin with transient permeabilization of plasma membrane by laser-induced multiple stress waves in vitro.

We investigated the effect of multiple stress waves with peak stress of less than 3 MPa on chemosensitivity of HeLa cells adhered on plastic. HeLa cells exposed to stress waves retained more than 95% of the viability found in untreated cells. The scanning electron microscopy of cells exposed to stress waves showed ruffling microvilli, indicating a change in the cell surface morphology. The cytotoxicity of bleomycin (BLM) on HeLa cells was enhanced by the stress waves exposure. Our findings demonstrated that the low-intensity stress wave would allow to deliver the BLM molecules into cytoplasm by repetition exposure.