Shigenobu Kasai

Proliferation assay on a silicon chip applicable for tumors extirpated from mammalians

We describe a novel anticancer drug sensitivity assay on a silicon chip applicable for tumors extirpated from in vivo mammalians. Human promyelocytic leukemia (HL‐60) cells were subcutaneously (s.c.) inoculated in SCID mice, then removed 31 days after the inoculation. The cells were embedded in a small volume (18 nL) of a collagen‐gel matrix on a pyramid‐shaped silicon microstructure for further cultivation. The respiration activity of the cells on the chip was measured by scanning electrochemical microscopy (SECM). The proliferation behavior was continuously monitored for 6 days. It seemed that the proliferation rate of the cells removed from the mice was lower than that cultured in a flask and conformed to that in mice. The effects of cisplatin (CDDP) and etoposide (VP‐16) on the HL‐60 cultured in vivo were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the SECM‐based assay is appropriate for biopsy specimens in a relatively short‐time evaluation.

The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats.

ABSTRACT  To evaluate the role of vitamin B12 on spermatogenesis, the effects of dietary vitamin B12 deficiency on sperm maturation in developing rat fetuses and young growing rats were examined. The vitamin B12‐defi‐cient diet was given to all the animals for three different periods: whole period (gestation to mature), gestation period (gestation to weaning), or immature period (3–12 weeks postnatal). Sperm examination revealed that the sperm count was markedly lower in male progeny (F1) that were vitamin B12‐deficient during the whole period. In addition, a significantly higher number of abnormal sperm, such as tailless and amorphous sperm, was observed. In male rats that were vitamin B12‐deficient during the immature period, the incidence of abnormal sperms was 14.4% and 4.8% for tailless and short tail, respectively. The motion rates, such as path velocity and straight line velocity, were decreased to 20–40% of the control value in rats that were vitamin B12‐deficient both during the whole and gestation periods. However, no effects of vitamin B12 deficiency on sperm motility were observed during the immature and mature periods. From these findings, we suggest that dietary vitamin B12 deficiency during pregnancy may induce irreversible damage in the germ cells of embryos and affect the maturation of spermatozoa.

Maternal vitamin B12 deficiency affects spermatogenesis at the embryonic and immature stages in rats

ABSTRACT  To evaluate the role of cobalamin (Cbl) on spermatogenesis, the effect of dietary vitamin B12 deficiency on early spermatogenesis was histologically investigated in male fetuses and newborns in the first filial generation (F1 males) of rats. There was no difference in the number of gonocytes and supporting cells of Sertoli in the gonad in male fetuses on day 16 of gestation and in the testes in F1 males at 0 days of age between vitamin B12‐deficient (VB12‐D) and vitamin B12‐supplemented (VB12‐S) groups. However, at 21 days of age, a decreased number of spermatogonia and no spermatocytes were observed in the VB12‐D group. Numerous TUNEL positive cells were located among spermatocytes of the spermatogenic epithelium. The ultrastructural features examined using transmission electron microscopy were considered to be indicative of apoptosis. The incidence of seminiferous tubules having apoptotic cells was 51.5% in the VB12‐D group. At 60 days of age, aplasia of the spermatids and spermatozoa was detected in the VB12‐D group. In the connective tissue between the seminiferous tubules, many interstitial Leydig cells and blood vessels were observed in the VB12‐D group, as compared with the VB12‐S group. These changes produced by vitamin B12 deficiency can be reversed by providing a VB12‐S diet after weaning at 21 days of age. From these findings, such a vitamin B12 deficiency during gestation and lactation could affect the germ cells and especially damage spermatocytes in F1 male rats, which indicates that Cbl may be an essential constituent in the meiosis of spermatogenesis.

Electrochemical monitoring of hydrogen peroxide released from leucocytes on horseradish peroxidase redox polymer coated electrode chip

We developed an electrochemical-sensing device for continuous monitoring extracellular hydrogen peroxide (H(2)O(2)). The device consists of an indium-tin-oxide electrode coated with osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) and a poly-dimethyl siloxane well to house the cells on the chip. Granulocyte-like differentiated HL-60 cells were accommodated in the well and stimulated with phorbol 12-myristate 13-acetate (PMA), which triggered the generation of H(2)O(2). The extracellular H(2)O(2) released from the cells was enzymatically reduced at the Os-HRP-modified electrode chip using Os(II) as an electron donor, resulting in reduction current responses by the device. The reduction current increased immediately upon PMA stimulation and this current transient was similar to that obtained by conventional chemiluminescence assays using sodium luminol. Apocynin, an inhibitor of NADPH oxidase activation, eliminated both the electrochemical and chemiluminescence signals. On the other hand, superoxide dismutase (SOD) increased the amperometric signals and catalase (CAT) decreased, whereas SOD decreased luminescence emission and CAT did not. These results were in accordance with the expected reaction mechanism, and strongly indicate that this new electrochemical-sensing device successfully detects extracellular H(2)O(2) production.

Generation Mechanism of Radical Species by Tyrosine-Tyrosinase Reaction

Alleviated melanin formation in the skin through inhibition of tyrosine-tyrosinase reaction is one of the major targets of cosmetics for whitening ability. Since melanin has a pivotal role for photoprotection, there are pros and cons of inhibition of melanin formation. This study applying electron spin resonance (ESR)-spin trapping method revealed that •H and •OH are generated through tyrosine-tyrosinase reaction. When deuterium water was used instead of H2O, the signal of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-H (a spin adduct of DMPO and •H) greatly decreased, whilst DMPO-OH (a spin adduct of DMPO and •OH) did not. Thus, it is suggested that •H was derived from H2O, and •OH through oxidative catalytic process of tyrosine to dopaquinone. Our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting •OH generation via the enzyme reaction.

Mapping peroxidase in plant tissues by scanning electrochemical microscopy

Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H(2)O(2) to increase FMA(+) concentration. Two-dimensional reduction current profiles for FMA(+) produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles.

Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device

All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques.

Detection of hydrogen peroxide in Photosystem II (PSII) using catalytic amperometric biosensor

Hydrogen peroxide (H2O2) is known to be generated in Photosystem II (PSII) via enzymatic and non-enzymatic pathways. Detection of H2O2 by different spectroscopic techniques has been explored, however its sensitive detection has always been a challenge in photosynthetic research. During the recent past, fluorescence probes such as Amplex Red (AR) has been used but is known to either lack specificity or limitation with respect to the minimum detection limit of H2O2. We have employed an electrochemical biosensor for real time monitoring of H2O2 generation at the level of sub-cellular organelles. The electrochemical biosensor comprises of counter electrode and working electrodes. The counter electrode is a platinum plate, while the working electrode is a mediator based catalytic amperometric biosensor device developed by the coating of a carbon electrode with osmium-horseradish peroxidase which acts as H2O2 detection sensor. In the current study, generation and kinetic behavior of H2O2 in PSII membranes have been studied under light illumination. Electrochemical detection of H2O2 using the catalytic amperometric biosensor device is claimed to serve as a promising technique for detection of H2O2 in photosynthetic cells and subcellular structures including PSII or thylakoid membranes. It can also provide a precise information on qualitative determination of H2O2 and thus can be widely used in photosynthetic research.

A Reactive Oxygen/Nitrogen Species Sensor Fabricated from an Electrode Modified with a Polymerized Iron Porphyrin and a Polymer Electrolyte Membrane

We have developed an electrochemical reactive oxygen/nitrogen species sensor that can detect superoxide anion radicals (O2-•) and nitric oxide (NO). The reactive oxygen/nitrogen species sensor was fabricated by surface modification of an electrode with polymerized iron tetrakis(3-thienyl)porphyrin (FeT3ThP), and it can detect either O2-• or NO by switching the applied potential. Furthermore, we fabricated a sensor with improved selectivity by coating a Nafion® film onto the poly(FeT3ThP)-modified electrode. An interference current caused by NO2- was seen for the poly(FeT3ThP)-modified electrode, while the interference current was significantly reduced at the Nafion®/poly(FeT3ThP)-modified electrode, leading to improved selectivity for NO detection. The current response at the Nafion®/poly(FeT3ThP)-modified electrode exhibited good linearity in the O2-• and NO concentration ranges 1.3 - 4.1, and 0.5 - 10 μM, respectively. The Nafion®/poly(FeT3ThP)-modified and poly(FeT3ThP)-modified electrodes are highly versatile, because these electrodes can detect either O2-• or NO by switching the applied potential. Since the Nafion®/poly(FeT3ThP)-modified and poly(FeT3ThP)-modified electrodes contain no bio-derived compounds, the reactive oxygen/nitrogen species sensor should be safe even when it is used in vivo.

Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell

Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials.