Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis

Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.     

Photo-rechargeability of film electrodes prepared by pulsed laser deposition

The film electrodes of TiO₂, which have two functions of opto-electric conversion and storage of electrochemical energy, were prepared by pulsed laser deposition. To investigate the relationship between the photo-rechargeability and the surface structure, the TiO₂ film electrodes with different surface morphology were prepared by changing the inert gas pressure during the deposition. The photo-charged quantity was found to be proportional to the third power of the rms of surface roughness. The results suggest that not only the surface but also the interstices between crystal grains near the surface contribute to the photo-charging with low photo-emf.     

Prostaglandin E receptor subtypes in mouse osteoblastic cell line

PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.     

Halo congenital nevus undergoing spontaneous regression. Involvement of T-cell immunity in involution and presence of circulating anti-nevus cell IgM antibodies

BACKGROUND: Halo congenital nevus is a condition in which halo formation is associated with congenital nevocellular nevi. Although several theories have been proposed, the immunologic mechanisms of halo formation and concomitant nevus regression still remain unclear. We presented immunologic findings in a case of halo congenital nevus with unique histologic location of inflammatory cells. OBSERVATIONS: Histologically, the present case of halo congenital nevus undergoing spontaneous regression showed a marked inflammatory infiltrate with remnants of original nevus cell nests. In the infiltrating T cells, CD8+ cells outnumbered CD4+ cells and the infiltrate of natural killer cells was not substantial. Direct and indirect immunofluorescence studies demonstrated the presence of IgM antibodies against nevus cells as well as melanoma cells and cultured melanocytes in the patient's serum. CONCLUSIONS: Our findings suggest that both T-cell-mediated immunity and IgM antibodies may be involved in the regression of halo congenital nevus. However, it is important to point out that our results may simply be epiphenomena and not directly responsible for the destruction of nevus cells.     

Preparation of plastic spherical microlenses by use of a fluoropolymer stencil and oil-bath heating

A new method for fabricating plastic spherical microlenses was developed, which allowed self-alignment of lenses and self-organized formation of a spherical shape. First a low-surface-energy fluoropolymer thin film was deposited and patterned as a stencil. Then photosensitive phenol resin was patterned on it as the lens material. Finally the resin was annealed in an oil bath to form a sphere. The molten phenol resin spontaneously formed a sphere and positioned itself in the center of the fluoropolymer ring pattern as a result of the difference of surface free energy and the equivalently zero-gravity condition in the oil bath. When a light-emitting-diode printer head was loaded with spherical microlenses, its optical output increased by 1 order of magnitude.     

Validity of sleep log compared with actigraphic sleep-wake state

OBJECTIVE: To evaluate the efficacy and complications of external cephalic version in term (37 or more weeks) gestation. STUDY DESIGN: A case series from February 1990 until December 1994 studied 160 patients with term singleton breech presentation. External cephalic version (ECV) with prophylactic tocolysis was the method used. RESULTS: ECV was successful in 67% of the cases (107/160) overall, in 56% of the nulliparous (57/102) and 86% of the multiparous women (50/58). About 90% of those with successful ECV (96/107) had a vaginal delivery with a vertex presentation of the fetus. Emergency Caesarean section due to fetal distress was not required during or immediately after the procedure. No perinatal mortality or babies with an Apgar score less than 7 at 5 min were observed. CONCLUSIONS: ECV in term pregnancy seems to be useful and it is safe both for the mother and the fetus. It should be performed at term, with close monitoring of the fetus, and in an environment able to provide an emergency Caesarean section.     

Serotonin 5-HT4 receptor agonistic activity of the optical isomers of (+/-)-4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro-2,3- dihydro-2-methylbenzo[b]furan-7-carboxamide

OBJECTIVE: To compare the influence of incongruent (asymmetric) follicular development on treatment outcome in IVF-ET and GIFT cycles. DESIGN: A retrospective comparative study. SETTING: Tertiary referral center for infertility. PATIENT(S): Five hundred forty-three consecutive assisted reproduction cycles (428 IVF-ET and 115 GIFT) in 422 infertile patients. INTERVENTION(S): Controlled ovarian hyperstimulation (COH) and IVF-ET or GIFT. MAIN OUTCOME MEASURE(S): The incongruity ratio as a parameter of the asymmetry in follicular development and pregnancy rate (PR). RESULT(S): For GIFT cycles, the PRs were 37.8% and 15.7% in cycles with congruent and incongruent follicular development, respectively. However, for IVF-ET cycles, the PR was not affected by incongruent follicular development: 28.2% and 29.0%, respectively. An inverse relationship was observed between the degree of incongruity and the estimated probability of pregnancy in GIFT cycles but not in IVF-ET cycles. Neither the side of the dominant ovary nor the degree of incongruity were consistent in consecutive cycles. CONCLUSION(S): Incongruent follicular development during COH has a significantly negative influence on the outcome of GIFT cycles but not on the outcome of IVF-ET cycles. The reason for this difference is not clear. We recommend considering IVF-ET instead of GIFT if incongruent follicular development occurs.     

Three-dimensional organization of collagen fibrils during corneal stromal wound healing after excimer laser keratectomy

PURPOSE: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze. SETTING: University of Tokyo Hospital, Tokyo, Japan. METHODS: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microscopy after re-embedding. RESULTS: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased. The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy. CONCLUSION: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.     

Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.