Localization of sphingomyelinase in lesional skin of atopic dermatitis patients

Because it has been suggested that the majority of the activity hydrolysing [N-methyl-14C] sphingomyelin is due to sphingomyelin acylase in the lesional skins of atopic dermatitis (AD), in this study we used immunologic techniques to localize and quantitate sphingomyelinase in AD lesional and normal skin. A polyclonal antibody raised against a synthetic polypeptide corresponding to a portion of the amino acid sequence deduced from the cDNA of human acid sphingomyelinase, cross-reacted with a 58 kDa, pI 5.8 human epidermal protein in an immunoblot analysis. The 58 kDa protein-rich fraction, partially purified by immunoprecipitation, converted [N-methyl-14C]-sphingomyelin to 14C-phosphorylcholine and ceramides. The reaction products were immunohistochemically observed in the intercellular domain from the upper spinous cell layer to the upper stratum corneum cell layers in the lesional skin of AD patients. Immunoelectron-microscopically, gold particles appeared to be concentrated in the intercellular domains of the granular-upper stratum corneum cells in the lesional skin of AD patients. The total amount of the 58 kDa protein in a 7 mm2 area of the skin was measured by quantitative immunoblot analysis; and was slightly increased in the lesional skin samples [3.5 +/- 0.3 microg per 7 mm2 (n = 7)], as compared with the nonlesional skin samples of AD patients [2.8 +/- 0.19 microg per 7 mm2 (n = 10)] and with the normal skin samples [2.7 +/- 0.22 microg per 7 mm2 (n = 10)]. This difference (between the lesional skin of AD and the nonlesional skin of AD or the normal control) was significant (nonpaired student's t test, p < 0.05).     

Shift-multiplexed microhologram fabrication with photoisomeric chromophores

Microholographic memory is an attractive storage system for its capability to hold high-density data and for its access time. Using a photochromic chromophore (diarylethene)-doped recording medium can give rise to microholographic memory's durability and contrast. In addition, it is possible to increase the microholographic memory's density by shift-multiplexed recording, since a hologram pit is constructed in a small area. The microhologram was fabricated in the diarylethene-based sample with two counterpropagating focused beams. Also, surface images and cross-sectional images scanned by a confocal microscope indicated that shift-multiplexed recording was achieved in high contrast.     

Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

Occurrence of heterogeneity of N-linked oligosaccharides attached to sycamore (Acer pseudoplatanus L.) laccase after excretion

The N-linked oligosaccharide moieties of sycamore (Acer pseudoplatanus L.) laccase are known to be highly heterogeneous. We confirmed that this oligosaccharide heterogeneity was caused not only during the oligosaccharide biosynthesis in Golgi apparatus, but also after the excretion of laccase protein into a culture medium. The culture medium for the sycamore cells (Acer pseudoplatanus L.) contained beta-galactosidase, alpha-L-fucosidase, beta-N-acetylglucosaminidase, alpha-mannosidase and beta-xylosidase activities. We showed that the largest sugar chain in laccase, oligosaccharide F, [formula: see text] was degraded to [formula: see text] by a crude exoglycosidase mixture in the culture medium.     

Accumulative gene integration into a pre-determined site using Cre/loxP

Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.     

Japanese cedar pollinosis in children in our allergy clinic

Recently, it is pointed out that the incidence of Japanese cedar pollinosis has increased in children. We studied on the rate of sensitization and the onset of the disease in children under sixteen who visited allergy clinic in the Department of Otorhinolaryngology in Mie University Hospital. The percentage of positive rate of skin test and IgE antibodies of house dust mite was about 80 to 90% in 1981, 1991, and 1996. However, the percentage of positive rate of skin test and IgE antibodies of Japanese cedar pollen was 43% and 26%, respectively in 1981, but both increased to 58% in 1996. Most of Japanese cedar pollinosis patients also had house dust mite allergy. Among 115 pediatric patients who visited our allergy clinics in the past seven years, 32.2% were allergic to house dust mite alone, 8.6% were allergic to Japanese cedar pollen alone, 40.9% were allergic to both, and 6.1% were allergic to house dust mite, Japanese cedar pollen, and orchard grass pollen. 68% of the total 115 patients were boys, but significantly more girls had the pollinosis. 17.4% of those who are sensitized to the pollen were asymptomatic during the pollen season. Thus, it was confirmed that the rate of children sensitized to the pollen has apparently increased for the past twenty years. We should take care of those children who are sensitized to the pollen but asymptomatic during the pollen season.     

Nasal secretion analysis in allergic rhinitis, cystic fibrosis, and nonallergic fibromyalgia/chronic fatigue syndrome subjects

Vascular surgery can be safely performed in approximately 60% of patients with advanced peripheral vascular disease, because of the high frequency of concomitant coronary artery disease and consequent increased risk of perioperative cardiac complications. The aim of this study was to validate the hypothesis that endovascular revascularization could be safely applied to high-cardiac-risk patients with a lower incidence of perioperative cardiac complications. One hundred and fourteen patients with peripheral vascular disease referred for revascularization underwent preoperatively a clinical and echocardiographic evaluation, at rest and under dipyridamole stress test, to assess the cardiac risk. Patients with high clinical score (according to Goldman and Detsky), or low left ventricular ejection fraction at rest, or positive dipyridamole stress test, were considered at high cardiac risk. To record adverse cardiac events, all patients were monitored during surgery, postoperatively, and followed up for 18 months after hospital discharge. Forty-eight patients (42%) were found to be at high cardiac risk. In this high-cardiac-risk group, endovascular surgery was performed in 37/48 patients (77%) (group A), while the remaining 11/48 patients (23%) were bypassed with open surgery (group B). Postoperative cardiac complications occurred in 16% of patients in group A and in 45% of patients in group B with two deaths (p < 0.05). At follow-up, 51% of patients in group A and 44% of patients in group B had suffered late cardiac events (p=ns), with 10 deaths in group A and three deaths in group B (p=ns). Limb salvage rate was similar in the two groups (95% group A, 100% group B; p=ns). These data show that high-cardiac-risk patients with limb-threatening ischemia have significantly less perioperative cardiac complications when treated by endovascular procedures instead of bypass surgery. Follow-up data on cardiac events confirm the severity of concomitant coronary artery disease in patients with peripheral vascular disease.