Calcium dynamics in the epidermis play a crucial role in barrier homeostasis and keratinocyte differentiation. We have recently suggested that the electro-physiological responses of the keratinocyte represent the frontier of the skin sensory system for environmental stimuli. In the present study, we have evaluated the responses of proliferating and differentiated human keratinocytes to mechanical stress by measuring the intracellular calcium level. Before differentiation, mechanical stress induces a calcium wave over a limited area; this is completely blocked by apyrase, which degrades ATP. In the case of differentiated keratinocytes, the calcium wave propagates over a larger area. Application of apyrase does not completely inhibit this wave. Thus, in differentiated cells, the induction of calcium waves might involve not only ATP, but also another factor. Immunohistochemical studies indicate that connexins 26 and 43, both components of gap junctions, are expressed in the cell membrane of differentiated keratinocytes. Application of octanol or carbenxolone, which block gap junctions, significantly reduces calcium wave propagation in differentiated keratinocytes. Thus, signaling via gap junctions might be involved in the induction of calcium waves in response to mechanical stress at the upper layer of the epidermis.

We developed a novel bioassay system using a glass microchip and cultured cells. A microchamber for cell culture and microchannels for reactions and detection were fabricated on a Pyrex glass substrate by photolithography and wet etching techniques. Cell culture, chemical and enzymatic reactions, and detection were integrated into the microchip. To keep different temperatures locally in three areas of the microchip, we designed and fabricated a temperature control device. Nitric oxide released from macrophage-like cells stimulated by lipopolysaccharide was successfully monitored with the microchip, the temperature control device, and a thermal lens microscope. The total assay time was reduced from 24 to 4 h, and detection limit of NO was improved from 1 x 10(-6) to 7 x 10(-8) M compared with conventional methods. Moreover, the system could monitor a time course of the release, which is difficult to measure by conventional batch methods. We conclude that this system is promising for a rapid bioassay system with very small consumption of cells.

In the present study, we examined the relationships between plasma concentrations of risperidone and clinical responses, extrapyramidal symptoms, plasma levels of cotinine and caffeine, or cytochrome (cyp)2D6 genotypes. In addition, we also investigated the relationships between plasma levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) or homovanillic (HVA) acid and clinical responses to risperidone. One hundred and 36 patients (male/female: 58/78, age 37+/-13 years) who met DSM-IV criteria for schizophrenia, schizoaffective disorder, delusional disorder and brief psychotic disorder, and who were being treated with risperidone alone, were evaluated regarding their clinical improvement and extrapyramidal symptoms using the Positive and Negative Syndrome Scale (PANSS) and Simpson and Angus (SAS), respectively, and plasma levels of cotinine, caffeine, MHPG and HVA were analysed by high-performance liquid chromatography. The cyp2D6*5 and *10 alleles were identified using the polymerase chain reaction. There was a positive correlation between plasma levels of risperidone plus 9-hydroxyrisperidone (active moiety) and SAS scores, but not the PANSS. Pretreatment HVA levels in responders were higher than those in nonresponders. In addition, there was a negative correlation between changes in HVA levels and improvement in PANSS scores. There was no association between plasma levels of risperidone and plasma levels of cotinine or caffeine. Furthermore, there were no differences in the risperidone/9-hydroxyrisperidone ratio, clinical improvements and extrapyramidal symptoms among cyp2D6 genotypes. These results indicate that pretreatment HVA levels and plasma concentrations of active moiety might play a part in predicting the clinical response and occurrence of extrapyramidal symptoms, respectively, when treating patients with risperidone.