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The solidification kinetics of an alloy from its liquid state forms an underlying basis for microstructural engineering, wherein the state of thermodynamic equilibrium associated with the melt-grown crystal and the quenched amorphous solid denotes the two limits for crystallinity in the alloy synthesis. In this study, we report the implication of the crystalline state on the thermal and electrical transport properties of partially substituted Mn(Si

Single crystals of Ir2S3 (diiridium trisulfide) and Rh2S3 (dirhodium trisulfide) were grown in evacuated silica-glass tubes using a chemical transport method and their crystal structures were determined by single-crystal X-ray diffraction analysis. These compounds have a unique sesquisulfide structure in which pairs of face-sharing octahedra are linked into a three-dimensional structure by further edge- and vertex-sharing. Ir2S3 and Rh2S3 had similar unit-cell parameters and bond distances. The atomic displacement parameter (MSD: mean-square displacement) of each atom in Ir2S3 was considerably smaller than that in Rh2S3. The Debye temperatures (ΘD) estimated from the observed MSDs for the Ir, S1 and S2 sites in Ir2S3 were 259, 576 and 546 K, respectively, and those for Rh, S1 and S2 in Rh2S3 were 337, 533 and 530 K, respectively. The bulk Debye temperature for Ir2S3 kashinite (576 K) was found to rank among the higher values reported for many known sulfides. The bulk Debye temperature for Rh2S3 bowieite (533 K) was lower than that for Ir2S3 kashinite, which crystallizes in the early sequences of mineral crystallization differentiation from the primitive magma in the Earth's mantle.

Case A 39‐year‐old woman presented with a genital hemorrhage at 5 weeks of gestation after an artificial cycle double frozen‐thawed embryo transfer. She was diagnosed with a cervical heterotopic pregnancy. Although hormone supplementation was discontinued to terminate the pregnancy at 5 weeks of gestation, the intrauterine and cervical gestational sacs continued to develop. Outcome The cervical gestational sac was surgically removed and the intrauterine pregnancy continued uneventfully, except for vasa previa. At 36 weeks of gestation, the patient underwent a cesarean section and gave birth to a healthy female infant. At the delivery, massive bleeding occurred and a hysterectomy was performed due to total placenta accreta. Conclusion This case provides a novel example of a near‐term delivery after a cervical heterotopic pregnancy and emphasizes the need for intensive care, even after the successful management of a cervical pregnancy. Most importantly, the present case implies a possible link between hormonal withdrawal and abnormal placentation.

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.

Squamous cell carcinoma is frequently observed on the lateral eyebrow, one of the most difficult sites for one-stage reconstruction because of its location close to the eyelids. The reconstruction of defects by the single-stage flap is advantageous for elderly patients because of its good functional and aesthetic outcome. We surgically treated three cases of squamous cell carcinoma on this area and performed reconstruction with a simply designed bilobed flap, all of which resulted in favorable outcomes. We therefore report on the usefulness of this reconstructive method for the lateral eyebrow defects.

SummaryCord factor, also called trehalose-6,6′-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle is known to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4egfp reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity of TDM.

Purpose: The usefulness of dissolving microneedles (DMs) for local skin therapy by basic fibroblast growth factor (bFGF) was studied in rats. Methods: We prepared four kinds of bFGF-loaded DMs, approximately 500 μm length and 300 μm diameter at the bottom. Long-term stability and dissolution studies were performed by HPLC method. Pharmacokinetic and pharmacological evaluations were performed after administration of bFGF loaded DMs to rats. Results: The bFGF contents were 2.15 ± 0.07, 1.07 ± 0.04, 0.56 ± 0.07 and 0.12 ± 0.03 μg. The 100.2 ± 3.4%, 100.2 ± 3.3%, 99.3 ± 1.4% and 100.4 ± 3.0% of bFGF were recovered after 1, 3 and 6 months and 1 year incubation at 40°C. The bFGF was released from DMs within 5 min. In a pharmacokinetic study using 2.0 and 1.0 μg bFGF-loaded DMs, no systemic exposure of bFGF was detected. The initial bFGF concentrations in the rat skin tissue after administration of bFGF-loaded DMs to the hair-removed rat abdominal skin were 510.2 ± 20.1 ng/g wet weight for 2 μg bFGF DMs and 264.2 ± 56.5 ng/g wet weight for 1 μg DMs, declining slowly thereafter to 226.3 ± 33.5 and 105.1 ± 27.4 ng/g wet weight at 6 hr after administration. Good dose-dependency was observed. Pharmacological evaluation of bFGF-loaded DMs of 2.0, 1.0, 0.5, and 0.1 μg, in the wound healing rat model, all used DMs, but 0.1 μg DMs, showed good healing effects. Considered collectively, these results suggest the usefulness of bFGF-loaded DMs for local therapy of skin wound disease.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.

In order to develop melanoma-targeted in situ peptide vaccine immunotherapy, magnetite nanoparticles were conjugated with a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP). Magnetite nanoparticles introduced thermotherapy which caused non-apoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). NPrCAP was expected to develop a melanoma-targeted therapeutic drug because of its selective incorporation into melanoma cells and production of highly reactive free radicals, that result in not only oxidative stress but also apoptotic cell death by reacting with tyrosinase.

In this study, we regenerated skin and its appendages by transplanting cultured normal dermal fibroblasts, into which morphogen genes had been introduced. We cultured normal dermal fibroblasts obtained from Fisher 344 rats on the surface of hydroxyapatite beads, and then adsorbed them onto the surface of a collagen sponge, which was transplanted into a full-thickness skin defect prepared on the backs of rats. Before transplantation, genes were introduced into the dermal fibroblasts via adenovirus vector (ad)-bone morphogenetic protein 2 and ad-wingless int 3 genes in addition to fibroblast growth factor-2 protein. By Week 4, the appearance of follicle germs or primitive hair germs was observed only in the ad-bone morphogenetic protein 2+ad-wingless int 3 combined with the fibroblast growth factor-2 protein group. By Week 16, in that same group, hair follicles having mature pilosebaceous systems with equally spaced localization had formed in the ulcer wound.

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of alpha-smooth muscle actin (alpha-SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 microg) and/or fibroblasts (1 x 10(6) cells/cm(2)) applied to experimentally produced full-thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast-seeded collagen sponge (fibroblast-seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF-treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased alpha-SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in alpha-SMA-positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of alpha-SMA-positive fibroblastic cells and inhibits alpha-SMA expression in the treated wound, thus reducing wound contraction.

Polymerase chain reaction(PCR)を用いると帯状疱疹患者の末梢血リンパ球からも水痘帯状疱疹ウイルス(varicella-zoster virus: VZV)核酸が検出される。本研究では，札幌医大附属病院皮膚科に入院しビダラビン(アラセナ®A)600mg/日を5日間投与された帯状疱疹患者10例よりリンパ球を調製し，PCRによって末梢血リンパ球のVZV gene 29およびgene 62のDNAおよびRNAの検出を行った。検討した10例のうち基礎疾患のある重症帯状疱疹の3例と中等症の1例は補体結合反応値が128倍と高値を示し，さらにPCRによりVZV DNAおよびRNAが検出された。しかし，これら4症例とも入院7～10日後(第14病日)には末梢血リンパ球からウイルス核酸は検出されなかった。他の中等症の1例と軽症5例の末梢血リンパ球からはVZV gene 29あるいは62のDNAとRNAは検出されなかった。患部皮疹が潰瘍化した重症の1例を除き，いずれの症例も3週間以内に上皮化し2～4週間後の帯状疱疹関連神経痛はVASスコアで0/5あるいは1/5まで低下した。これらの結果より，重症例ではウイルス抗体価が上昇し末梢血リンパ球にVZV遺伝子が検出され少なくとも一部のウイルス遺伝子が発現しているが，ビダラビン600mg/日，5日間の投与によって発症2週間後には末梢血よりウイルスが駆逐されると考えられた。

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.

We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.

Among a large collection of nonsense (termination) suppressors of Saccharomyces cerevisiae, a few remained obscure for their molecular nature. Of those, a group of weak and recessive suppressors, sup111, sup112 and sup113, is of particular interest because of their dependency on [PSI+], a yeast prion. From the facts that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1 genes, respectively, and that some mutations in the UPF genes confer termination suppressor activity, we suspected that sup111, sup112 and sup113 would very well be mutant alleles of the UPF genes. We tested our speculation and found that sup113, sup111 and sup112 were in fact complemented with the wild-type alleles of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112, respectively, had point mutations. From these results, we conclude that sup111, sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and thus attribute suppressor activity of these mutations to defects in the NMD (nonsense-mediated mRNA decay) machinery.

By means of gene disruption analyses, Saccharomyces cerevisiae strain YPH499 was shown to have two, and only two, copies of ATP3 that encodes the γ-subunit of H+-dependent ATP synthase and locates on the right arm of chromosome II. Linkage analyses of the two distinguishably marked copies of ATP3 indicated that the distance between them was about 43 cM. Since YBR030W, an ORF proximal to ATP3 by a distance of 17 kbp, was also found to be duplicated, we marked them with two distinguishable nutritional markers, which were also distinguishable from those used for marking the two copies of ATP3, and achieved four-point linkage analyses; CEN2 marked with an appropriate nutritional marker gene was included as a reference point. And, the following linkage map was deduced: CEN2– [11 cM]–YBR030Wa– [8 cM]–ATP3a–[47 cM]–ATP3b– [55 cM]–YBR030Wb. From this map, we suspected that a segment spanning at least YBR030W–ATP3 would be inversely duplicated on the right arm of chromosome II. We then carried out chromosome fragmentation analyses, using several laboratory strains including YPH499, and obtained data in accord with our speculation for all strains, although the distance between the two copies of ATP3 varied from 48 kbp to 192 kbp among the strains examined. Copyright © 2005 John Wiley & Sons, Ltd.

We have previously reported that the HMRa-bearing restriction fragment of a rho degrees sir4-11 strain (HMLalpha-MATalpha-HMRa), which acts as an alpha-mater because of being rho degrees , changes its electrophoretic mobility when the strain mates with a certain group of a-mating strains (HMLalpha-MATa-HMRa). In this study, we found that the sir4-11 strain being rho degrees was not essential for this phenomenon and also that the altered form of the fragment contained HMRalpha in place of HMRa. Furthermore, we observed conversion of HMLa to HMLalpha in the cross in which a sir4-11 HMLa-MATalpha-HMRalpha strain was mated with a representative of the above-mentioned a-mating strain. In addition, when this a-mating strain was mated with a SIR(+) HMLalpha-MATa-HMRalpha strain, the resultant diploid was found to be HMLalpha/HMLalpha MATa/MATalphaHMRa/HMRalpha, indicating that conversion of MATa to MATalpha had taken place in the course of mating. From all these observations, we conclude that there is a group of S. cerevisiae strains that carries factor(s) that induces conversion of a mating-type cassette of the mating partner to alpha mating-type cassette and that this mating type cassette conversion takes place in all three mating type loci, HML, MAT and HMR, if the loci are in the non-silenced condition.

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.

Some strains of Saccharomyces cerevisiae have detectable activities of L -serine O-acetyltransferase (SATase) and O-acetyl- L -serine/O-acetyl- L -homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L -cysteine exclusively via cystathionine by cystathionine β-synthase and cystathionine γ-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L -cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L -cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L -cysteine. These results indicate that S. cerevisiae SATase cannot support L -cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L -cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L -cysteine.

Many researchers have reported the effects of cytokines on ulcers, especially on chronic ulcers. However, the long-term influences of cytokines on acute incisional wounds with respect to breaking strength, quality of the scar etc. have not been elucidated completely. In the present study, the breaking strength of scars produced by full thickness incisional wounds of the skin created on backs of rabbits were compared when the wounds were treated by conventional suturing or with basic fibroblast growth factor (bFGF) in addition to suturing. We administered bFGF to full thickness acute incisional wounds of the skin created on backs of rabbits. The breaking strengths of wounds treated by conventional suturing and those treated with bFGF soon after the operation were compared as were the conditions of the scars after long-term follow up of up to 7 weeks. They were also examined histopathologically. Administration of bFGF at the time of wound closure, especially 1.0 μg in amount per cm, increased the breaking strength significantly compared to the control group from 5 weeks after the operation. Moreover, histopathological examination revealed that there was rich vascularization soon after the operation and an arrangement of collagen fibers which lenticulated horizontally from the 5th week and later after the operation in the bFGF treated groups in a dose dependent manner. This study demonstrates that administration of bFGF at the time of wound closure significantly increased the breaking strength compared to the control group from 5 weeks after the operation. And the administration of bFGF also improved the quality of the scar, the condition of the scar by surface condition by inspection as well as the width of scar at histological evaluations. So we think this cytokine may be highly effective clinically. This may be especially true for patients whose wound healing process tends to be delayed such as aged patients or when high breaking strength is recommended even for patients with normal wound healing potential.

We cloned a DNA fragment from Saccharomyces cerevisiae that complemented the deficiency in high-affinity glutathione transport activity conferred by a gsh11 mutation, and found that the ORF responsible was YJL212c, which had already been designated as OPT1 and HGT1 by others. Northern analysis clearly demonstrated that this ORF, now referred to as OPT1/ HGT1/ GSH11, was induced by sulfur starvation and repressed by adding cysteine to the growth medium. Reporter gene assays showed that a segment spanning the region between positions -371 and -355 was essential for the regulation of this gene. A sequence of 9 nt, CCGCCACAC (from -364 to -356), in this region was shown to be required for protein binding, using an electrophoretic mobility shift assay. Based on these results, we propose that CCGCCACAC comprises the core of a cis-acting element involved in cysteine-responsive gene regulation in S. cerevisiae.

Hair growth after reconstructive surgery for auricular malformations including microtia and full-thickness skin grafts poses serious problems for patients. Currently, the author and his colleagues use a long-pulsed alexandrite laser system with a cooling device to remove hair from hairy regions before and after reconstructive surgery of auricular malformations. They achieve good results with approximately two to five treatments, and report the details here. Using their protocols there were no adverse reactions such as ulceration, scarring, pigmentation, or depigmentation. The use of this system may change the technique of auricle treatment because of the advantages it offers, such as low invasiveness, high effectiveness, and safety.

アクリレートシリコーングラフトコポリマーに粉体表面への吸着セグメントとしてピロリドン部分を導入した新しい分散剤を合成した。このグラフトコポリマーは, 無機粉体粒子表面全体に吸着層を形成し, 揮発性シリコーンオイル中への微粒子無機粉体の分散性が非常に優れていた。また, エステル油や有機紫外線吸収剤を含む混合分散媒中でも, 広い組成領域で分散安定化が実現した。さらに, このグラフトコポリマーを使用した塗布膜は耐水性に優れ, 耐水性試験後にも紫外線遮蔽効果を維持し, 可視光に対する透明性にも変化がないことが認められた。このグラフトコポリマーを分散剤として使用することにより, 従来よりも透明感があり, 耐水性に優れた紫外線防御化粧料の製剤化が可能となった。

Based on helical volume scan computed tomography (HVCT) data, it has been demonstrated previously that real-size models can be prepared with the binder-jet method with extremely high precision of various parts ranging from the outer structure of the cranio-maxillofacial bone to such fine parts as the cranial base and the orbital walls. The application of this method to clinical cases with large cranial bone defects has also been studied. The results showed the usefulness of the binder-jet method, employing starch for fixing, with which a model can be prepared faster and with less deformity caused by the weight of the material itself. Therefore, the binder-jet method is expected to attain greater clinical significance in this field.In this study the comparison between the shapes of implants before and after implantation revealed that the most important aspect of the design of hydroxyapatite (HAP) implants was determining the shapes of edges, especially its angles. That is, an implant needs very little trimming before being implanted to the patient's defective site if the shape of the edges of the implant can be designed and prepared to fix the incised bone surfaces properly. In addition, to obtain optimum results it was also shown that it is necessary to design optimal shapes of implants, taking into account thickness, porosity, and curvature, before processing the shapes of edges. It was found necessary to establish a technology and a method for producing HAP implants that perfectly fit cranial bone defects. For this purpose, it would be useful to make a template of the implants using the mirror-reversion and binder-jet methods. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res (Appl Biomater) 63: 312–318, 2002

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

We conducted this study to determine the clinical variables associated with the production of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 01_AE neutralizing human monoclonal antibodies (NhMAbs) using a hybridoma technique. This cross sectional study was performed in 20 asymptomatic HIV-1-infected Thais. Peripheral blood mononuclear cells (PBMCs) were obtained from each study participant and fused with SPYMEG cells. Culture supernatant collected from growing hybridomas was tested for neutralizing activity against HIV-1 CRF01_AE Env-recombinant viruses. Fifty hybridomas expressing anti-HIV-1 NhMAbs with strong neutralizing activity against at least 1 CRF01_AE Env-recombinant virus were found. A positive association between the numbers of hybridomas produced and the CD4 counts of study participants (p = 0.019) was observed. NhMAb-producing hybridomas with strong neutralizing activity were mostly found in participans diagnosed with HIV-1 infection within the previous 1 year. The HIV-1 viral load was not significantly correlated with the numbers of either established hybridomas or clones expressing anti-HIV-1 NhMAbs with strong neutralizing activity. To our knowledge, this is the first study of NhMAb-producing hybridomas obtained from HIV-1 CRF01_AE-infected populations identified by antibody binding to HIV-1 V3 loop peptide enzyme-linked immunosorbent assay (ELISA) or TRUGENE HIV-1 Genotyping Assay (HIV-1 pol sequence). It provides important criterion to slect study participants with high CD4 counts who produce large numbers of hybridoma clones. The results are valuable for further studies related to nurtalizing antibodies production and HIV-1 vaccine development.

We report here the production of transgenic quails using a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G). The retroviral vector was injected into laid quail embryos at the blastodermal stage, and the embryos were incubated to hatch to produce G(0) transgenic quails. Among 134 embryos subjected to viral injection, 37 hatched. The viral vector sequence was detected in the tissues of all G(0) quails. The germ-line transmission efficiency of G(0) quails mated with nontransgenic quails was more than 80% on average. Southern blot analysis revealed that the G(1) transgenic progeny had one to three copies of the transgene. The expression of vector-encoded neomycin-resistance gene under the control of the Rous sarcoma virus (RSV) promoter was observed in several tissues including heart and muscle of both G(1) and G(2) transgenic offspring. Due to the high frequency of germ-line transmission, this method may markedly facilitate the production of transgenic avian.

We devised a technique to fix the temporalis muscle to the transplanted hydroxyapatite implant by using a titanium plate, which is fixed to the hydroxyapatite ceramic implant by screws and achieves good clinical results. The size, shape, and curvature of the hydroxyapatite ceramic implants were determined according to full-scale models fabricated using the laser lithographic modeling method from computed tomography data. A titanium plate was then fixed with screws on the implant before implantation, and then the temporalis muscle was refixed to the holes at both ends of the plate. The application of this technique reduced the hospitalization time and achieved good results esthetically.

In this study, we established a delayed healing chronic type wound model in order to investigate the etiology of chronic wound healing, including wound contraction. Establishment of the model was important for clarification of the mechanism(s) of chronic wound healing and wound contraction and for use in evaluating therapeutic efficacy. A pedicled skin flap was raised beneath the panniculus carnosus membrane on the backs of mice, and after the loose connective tissue at the base of the flap was completely removed surgically, the flap was replaced and sutured. Seven days after surgery, a full-thickness defect measuring 1.5 x 1.5 cm was made in the center of the skin flap. At that time, a defect of the same size, including the panniculus carnosus membrane, was made in another group of mice as controls, and changes with time in wound area were compared between the two groups. The exudate retained on the wound surface was collected, and various cytokines contained in the exudate were measured. In the control group, the wound rapidly contracted and almost completely epithelialized and closed 21 days after surgery. On the other hand, the wound area was significantly larger in the delayed model than in the control animals during the observation period, revealing a delay in wound contraction. Transforming growth factor-beta, interleukin-1beta, and tumor necrosis factor-alpha in the exudates from the wound of the model were significantly higher than in those of the control group, whereas interleukin-6 was low in the model. From these results, it was concluded that this model could be a useful experimental system for studies on wound contraction as well as clarifying the mechanism of so called chronic type wounds.

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and development of pathological scarring. To examine the phenomenon of apoptosis and its involvement in the process of pathological scarring, we immunohistochemically quantified differential levels of expression of caspase-3 and -2, which are activated during apoptosis in vitro, in surgical resected scar tissues. We divided 33 cases of normally healed flat scars and 18 cases of pathological scars (15 cases of hypertrophic scars and 3 cases of keloid) into three groups (S1 =10 months' duration; S2 = 10 to 40 months' duration; and S3 =40 months' duration) according to the duration of scar. In all three groups examined, the semiquantitative scores for caspase-3 staining were significantly higher for the combination of hypertrophic scars and keloid as a group compared with normally healed flat scars, suggesting reduced cell survival and increased apoptotic cell death in hypertrophic scars and keloid. Apoptosis and caspase proteolytic activities were examined in vitro using two flat scar-derived fibroblast lines (FSFB-1 and -2) and two keloid-derived fibroblast lines (KFB-1 and -2). After 24 hours of serum deprivation, apoptotic cells were significantly increased in both KFB lines, whereas serum deprivation of FSFB-1 cells did not result in a significant increase in apoptotic cell number. After serum deprivation, significant increases in caspase-3 proteolytic activities were detected in both KFB lines compared with both FSFB lines. In contrast, no significant differences with caspase-8 activity were observed between similarly treated KFB and FSFB lines. Furthermore, serum deprivation-induced apoptosis of KFB-2 cells was significantly inhibited by the caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK), indicating that caspase-3 is important for serum deprivation-induced apoptosis in KFB-2 cells. Considering the role of caspase-3 as a key effector molecule in the execution of apoptotic stimuli, our results suggested that enhanced expression of caspase-3 in hypertrophic scars and keloid induces apoptosis of fibroblasts, which may play a role in the process of pathological scarring.

Our new surgical procedure with two triangular flaps for macrostomia repair allows us to achieve all three therapeutic goals, including formation of symmetric lips and commissures of the mouth, reconstruction of the orbicularis muscle of mouth to restore labial function, and reconstruction of the commissure of the mouth with a natural looking contour. Furthermore, the position of the commissure of the mouth can be adjusted intraoperatively according to the extent of macrostomia. As reported here, our method provides very satisfactory clinical results and is relatively easy to perform. Thus, we believe that our method can serve as a standard for the surgical treatment of macrostomia.

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine β-synthase.

Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O-acetylserine and O-acetylhomoserine sulphydrylase). They were defective in the high-affinity glutathione transport system, GSH-P1. Newly acquired mutations belonged to the same complementation group, gsh11. However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17. Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11. From this finding, we propose that the sulphate assimilation pathway acts as a sulphur-recycling system and that this function is especially vital to the cell when the supply of glutathione is limited.