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Biomedical subjects

Atsushi Yamada

Publications and source records attributed to Atsushi Yamada.

At least 19 recordsLinked to original sources
PubMed2006

Peroxisome proliferator-activated receptor alpha activation during pregnancy severely impairs mammary lobuloalveolar development in mice.

To identify the potential functions of peroxisome proliferator-activated receptor alpha (PPARalpha) in skin development, transgenic mice were generated to target constitutively activated PPARalpha (VP16PPARalpha) to the stratified epithelia by use of the keratin K5 promoter. In addition to marked alterations in epidermal development, the transgenic mice had a severe defect in lactation during pregnancy resulting in 100% pup mortality. In this study, the alteration of mammary gland development in these transgenic mice was investigated. The results showed that expression of the VP16PPARalpha transgene during pregnancy resulted in impaired development of lobuloalveoli, which is associated with reduced proliferation and increased apoptosis of mammary epithelia. Mammary epithelia from transgenic mice also showed a significant reduction in the expression of beta-catenin and a down-regulation of one of its target genes, cyclin D1, which is thought to be required for lobuloalveolar development. Furthermore, upon PPARalpha ligand treatment, similar effects on lobuloalveolar development were observed in wild-type mice, but not in PPARalpha-null mice. These findings suggest that PPARalpha activation has a marked influence in mammary lobuloalveolar development.

Animals↗
PubMed2006

A surface hopping method for chemical reaction dynamics in solution described by diabatic representation: an analysis of tunneling and thermal activation.

We present a surface hopping method for chemical reaction in solution based on diabatic representation, where quantum mechanical time evolution of the vibrational state of the reacting nuclei as well as the reaction-related electronic state of the system are traced simultaneously together with the classical motion of the solvent. The method is effective in describing the system where decoherence between reactant and product states is rapid. The diabatic representation can also give a clear picture for the reaction mechanism, e.g., thermal activation mechanism and a tunneling one. An idea of molecular orbital theory has been applied to evaluate the solvent contribution to the electronic coupling which determines the rate of reactive transition between the reactant and product potential surfaces. We applied the method to a model system which can describe complex chemical reaction of the real system. Two numerical examples are presented in order to demonstrate the applicability of the present method, where the first example traces a chemical reaction proceeded by thermal activation mechanism and the second examines tunneling mechanism mimicking a proton transfer reaction.

Journal Article↗
PubMed2006

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

Cell Extracts↗
PubMed2006

Epithelial cells and adipose cells both have their own temporal profile in 72-kd heat-shock protein expression determining their tolerance for ischaemia.

Seventy-two-kd heat-shock protein (HSP72) is one of the stress markers induced in cells under stress, such as in the case of ischaemia. Recent studies have suggested that HSP72 is a 'molecular chaperone' to protect cells from various kinds of stress, and that the temporal profile of HSP72 induction is related to ischaemic vulnerability. In this study, we attempted to analyse the temporal profiles of HSP72 induction in epithelial and adipose cells in skin flaps after various periods of transient ischaemia, and we investigated the reason why there were differences in ischaemic tolerance between these cells. We used the abdominal skin flap of Wister rats, which were divided into three groups: the sham control group (n=27), the 2-h ischaemia group (n=25), and the 8-h ischaemia group (n=25). At periods of 8, 24, 48, 96 h, and 7 days after reperfusion, we examined them for any histological changes and performed immunostaining for HSP72 (n=5, each time point). Two animals in the sham control group were sacrificed to harvest the samples immediately after the skin flaps were elevated. As a result, the epithelial cells in all groups revealed positive for HSP72 through the time course, regardless of the ischaemic stresses, and they were alive at 7 days. In the adipose cells, the cells in the sham control group revealed no immunoreactivity after the reperfusion, and they had no change at 7 days. In the 2-h ischaemia group, the adipose cells gradually increased the reactivity for HSP72; consequently they survived beyond 7 days. In the 8-h ischaemia group, the reactivity for HSP72 gradually decreased; consequently they played out a delayed cell death at 7 days. We concluded that these differences of HSP72 expression were related to the cellular vulnerability to ischaemia.

Adipocytes↗
PubMed2006

Alterations in skin and stratified epithelia by constitutively activated PPARalpha.

Peroxisome proliferator-activated receptor (PPAR)alpha is a pleiotropic regulator in many cell types and has recently been implicated in skin homeostasis. To determine the role of PPARalpha in skin physiology, transgenic mice were generated using the tetracycline Tet-off regulatory system to target constitutively activated PPARalpha to the epidermis and other stratified epithelia by the bovine keratin K5 promoter. Expression of the transgene during early development resulted in postnatal lethality within 2 days after birth. A thin epidermis, few hair follicles, and abnormal development of the tongue were observed in neonatal transgenic mice. Early mortality was not observed when transgenic PPARalpha expression was diminished by administration of doxycycline (dox) to the mothers. The alterations noted in neonatal mice were not observed in adult mice upon re-expression of the PPARalpha transgene by withdrawing dox. Attenuated hyperplasia of interfollicular epidermis after topical application of the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was observed in adult mice expressing the PPARalpha transgene. In addition, expression of the PPARalpha transgene in mammary gland during pregnancy resulted in abnormal development of this organ and impaired lactation. Further investigations using primary keratinocytes revealed that expression of the transgene in keratinocytes resulted in increased differentiation and decreased proliferation, which may contribute to the observed phenotype in the transgenic mice. Thus, these results indicate that PPARalpha plays an important role in the development of stratified epithelia including skin, tongue, and mammary gland.

Animals↗
PubMed2006

Induction of heat shock proteins in a rat composite tissue allotransplantation model of acute rejection.

BACKGROUND: Clinical cases of composite tissue allotransplantation have been reported, but the exact mechanisms of allograft rejection remain unclear. The current study examined the possible involvement of heat shock proteins in major histocompatibility complex-compatible minor-mismatch composite tissue allotransplantation rejection. METHODS: Allotransplantation (Fisher 344 to Lewis) and isotransplantation (Lewis to Lewis) of rat lower extremities were performed. Temporal profiles of 70-kDa and 60-kDa heat shock proteins (heat shock protein 70 and heat shock protein 60) were conducted by quantitative Western blot analysis and immunohistochemistry. Analyses were performed immediately after reperfusion (day 0) and on postoperative days 1, 3, 7, and 12 (n = 5 for quantitative Western blot analysis and n = 5 for immunohistochemistry). RESULTS: The expression levels of the inducible forms of heat shock protein 70 and heat shock protein 60 were uniformly and significantly augmented until postoperative day 7 in the allografts, compared with the isografts (p < 0.001 for each protein). Immunoreactivities to heat shock protein 70 and heat shock protein 60 in keratinocytes, hair follicular cells, and endothelial cells of the subdermal vascular plexus were enhanced in the allografts early on postoperative day 1. Positive staining of hair follicles extended to deeper parts of the hair follicles and hair bulbs in the allografts on postoperative day 3. Although dendritic cells were positive for both heat shock protein 70 and heat shock protein 60, infiltrating lymphocytes were positive only for heat shock protein 60. CONCLUSIONS: These results suggest the possible involvement of heat shock proteins in the process of major histocompatibility complex-compatible minor-mismatch composite tissue allotransplantation rejection. Thus, modulation of the function of heat shock proteins might be an important therapeutic option for improving the outcome of minor-mismatch allografts.

Animals↗
PubMed2005

Induction of uteroglobin-related protein 2 (Ugrp2) gene expression by the Th2 cytokines IL-4 and IL-13.

Uteroglobin-related proteins 1 and 2 (UGRP1 and -2) are thought to play important roles in inflammation and immunologic responses in the lung. In this study we demonstrate that IL-4 and IL-13 enhance Ugrp2 gene expression in the mouse transformed Clara cell line, mtCC, in a time- and dose-dependent manner. Addition of actinomycin D abrogated the IL-4- and IL-13-induced increase of Ugrp2 expression, demonstrating that this increase occurs at the transcriptional level. When mtCC cells were pretreated with IFN-gamma before the addition of IL-4 or IL-13, IL-4- and 13-induced Ugrp2 mRNA increase was markedly decreased. IL-4 and IL-13 induced phosphorylation of STAT6 in mtCC cells, which binds to the proximal STAT-binding element (SBE) in the Ugrp2 gene promoter, leading to transcriptional activation of this gene. Mutations of the proximal SBE abrogated the binding of activated STAT6 to this site and the IL-4-induced increase in Ugrp2 gene promoter activity. IFN-gamma-activated STAT1 binds to the same SBE in the Ugrp2 gene promoter to which STAT6 binds and decreases the binding of STAT6 to this site. Furthermore, an IL-4-induced increase in Ugrp2 expression was not observed in primary cultures of lung cells derived from STAT6-deficient mice. These results indicate that Ugrp2 expression is enhanced by IL-4 and IL-13 through STAT6 binding to the proximal SBE located in the Ugrp2 gene promoter.

Animals↗
PubMed2005

Induction of uteroglobin-related protein 2 (Ugrp2) expression by EGF and TGFalpha.

Uteroglobin-related protein 2 (UGRP2) is thought to play a role in inflammation and/or epithelial cell differentiation in the lung. Induction of Ugrp2 mRNA expression by epidermal growth factor (EGF) and transforming growth factor alpha was examined using mouse transformed lung Clara cell-derived mtCC cells. The EGF-induced increase of Ugrp2 occurred at the transcriptional level that required the EGF receptor and the activation of the ERK-MAPK and phosphoinositide-3 kinase pathways.

Animals↗
PubMed2005

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins.

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Animals↗
PubMed2005

Choice of flaps for breast reconstruction.

The optimal method for breast reconstruction should be safe, reliable, and accessible for every patient, and it should display little or no donor-site morbidity. After comparing mammary implants it has been found that autogenous breast reconstruction can create a ptotic, soft, symmetrical breast mound. The transverse rectus abdominis musculocutaneous flap (TRAM) remains the most popular method for autogenous reconstruction. Modern trends in breast reconstruction using the TRAM flap have promoted adequate blood supply to the flap while minimizing donor-site defects in the anterior abdominal wall. The pedicled TRAM flap remains one of the most frequently used flaps, but the indirect blood supply in this flap has required many modifications and refinements. Such modifications have included the bipedicled TRAM flap, the free TRAM flap, and the supercharged TRAM flap. To avoid donor-site morbidities, the muscle-sparing free TRAM, deep inferior epigastric perforator flap (DIEP), and superficial inferior epigastric artery (SIEA) flap were introduced. The DIEP perforator flap requires meticulous technique but offers proven reliability and a low rate of complications. As surgeons become more comfortable with harvesting DIEP flaps, the frequency of usage seems likely to increase. The latissimus dorsi musculocutaneous flap, gluteus maximus musculocutaneous flap, and others may be selected when these modifications of free TRAM flap are unavailable or unusable.

Breast Neoplasms↗
PubMed2005

Rigid external distraction using skeletal anchorage to cleft maxilla united with alveolar bone grafting.

OBJECTIVE: Documentation of the application of maxillary distraction osteogenesis using rigid external distraction (RED) with skeletal anchorage combined with predistraction alveolar bone grafting (ABG) in cleft maxilla. DESIGN: Case report. PATIENT: A patient with numerous congenital missing teeth and severe maxillary deficiency related to complete bilateral cleft lip and palate with large alveolar bone defect. INTERVENTION: The patient received preoperative orthodontic treatment, predistraction ABG, and maxillary distraction osteogenesis using RED with skeletal anchorage. RESULTS: Predistraction ABG completely united the cleft maxilla. The united maxilla was successfully advanced by the RED system with skeletal anchorage, despite unsound dentition with numerous congenital missing teeth. CONCLUSION: The present study demonstrates that the combination of predistraction ABG and RED system with skeletal anchorage is effective for the treatment of severe maxillary deficiency related to complete bilateral cleft lip and palate with large bone defect and numerous congenital missing teeth.

Adolescent↗
PubMed2005

[Donor site choice for free flaps in head and neck reconstruction after tumor surgery].

OBJECTIVE: To choose suitable free flaps for reconstructing head and neck defects caused by tumor resection. METHODS: A retrospective analyses was made in 86 cases of head and neck defects treated with four kinds of free flaps between January 1999 and January 2002. The head and neck defects were caused by tumor resection. The locations were oral cavity (n=32), hypopharynx (n=27), mandible (n=12), skull base (n=5), scalp and skin (n=6) and midface (n=4). The donor sites of free flaps included the rectus abdominis (n=32), anterolateral thigh (n=10), jejunum (n=25), fibula (n=11), latissimus dorsi (n=4), forearm (n=3) and scapula (n=1). The sizes of the cutaneous/musculocutaneous flaps ranged from 4 cm x 5 cm to 14 cm x 24 cm. The lengths of the fibula were 4-16 cm, of jejunum 9-20 cm. RESULTS: The overall free flap success rate was 92% (79/86). Of 32 oral cavity defects, 22 were reconstructed by rectus abdominis (69%) and 10 by anterolateral thigh flaps (31%). Of 27 hypopharyngeal defects, 25 were restored by jejunum flaps (93%). Eleven of 12 mandibular defects were reconstructed by fibula flaps (92%). Four of 5 defects of skull base were reconstructed by rectus abdominis flaps (80%). The free flaps of rectus abdominis, anterolateral thigh, jejunum and fibula were most frequently used, accounting for 91% (78/86) of all flaps in head and neck defect reconstruction. CONCLUSION: Although head and neck defects represent a complicated spectrum of subsites and loss, these four free flaps can manage most reconstruction problems.

Adult↗
PubMed2004

Significant association between nonsyndromic oral clefts and arylhydrocarbon receptor nuclear translocator (ARNT).

The etiology of nonsyndromic oral clefts (cleft lip, cleft palate, or cleft lip and palate) is still controversial, but is considered to involve both genetic and environmental factors. One of suspected environmental factors is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) found in tobacco, herbicides, contaminated soil, and food. TCDD administered during organogenesis in mice causes a high incidence of CP in fetuses. There is ample evidence that aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and cytochrome P450 1A1 (CYP1A1) are involved in TCDD metabolism. We assessed whether there is any association in the Japanese population of nonsyndromic oral clefts with single nucleotide polymorphisms (SNPs) in the AHR, ARNT, and CYP1A1 genes using transmission disequilibrium test (TDT) and case-control study. We identified and investigated three SNPs in ARNT; 567G/C (V189V), IVS12-19T/G, and 2117C/T (P706L). Two amino acid substitutions, R554L in AHR and I462V in CYP1A1, were also investigated. In the TDT, the C allele of ARNT 567G/C was preferentially transmitted to patients (P = 0.033). When a haplotype consisting of 567G/C and IVS12-19T/G in ARNT was considered, the preferential transmission of the CT (567C-IVS12-19T) haplotype was observed (P = 0.0012). In a case-control study, a significant association of IVS12-19T/G in ARNT was observed (P = 0.021). The SNPs studied in AHR and CYP1A1 were not associated with the disease. Our results suggest that ARNT is involved in the development of nonsyndromic oral clefts in the Japanese population.

Adult↗
PubMed2004

Role of protein in the primary step of the photoreaction of yellow protein.

We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment.

Bacterial Proteins↗
PubMed2004

Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction.

Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing.

Bacterial Proteins↗
PubMed2004

Association between nonsyndromic cleft lip with or without cleft palate and the glutamic acid decarboxylase 67 gene in the Japanese population.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is one of the most common craniofacial malformations. Both genetic and environmental factors are involved in the pathogenesis. In addition to its role as an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) synthesized by glutamic acid decarboxylase (GAD) is presumed to play a role in normal embryonic, especially facial, development. This notion has been substantiated by the fact that Gad67 knockout mice have been shown to have cleft palate. We hypothesized that GAD67 may be involved in the development of NSCLP and investigated the possible association between the GAD67 gene (GAD67) and NSCLP in Japanese patients. We screened 50 probands for single nucleotide polymorphisms (SNPs) in GAD67 using denaturing high performance liquid chromatography (DHPLC) and found seven SNPs. Since two SNPs showed complete linkage disequilibrium (LD) to the other SNPs, we constructed a 5-locus haplotype of GAD67. The frequency distribution of the haplotype differed between NSCLP patients and controls (P = 0.0028). The frequency of -445A, -292A, -147G, 111C, and IVS9-39T haplotype in the NSCLP patients was significantly lower than that in controls (P = 0.00098). In a transmission disequilibrium test (TDT) in 99 parent-offspring trios, we found -445C, -292C, -147G, 111C, and IVS9-39C haplotype was preferentially transmitted to the patients with cleft lip and palate (P = 0.0077). Our data suggest that GAD67 is involved in the pathogenesis of NSCLP in the Japanese population.

Asian People↗
PubMed2004

Neurovascular free-muscle transfer for the treatment of established facial paralysis following ablative surgery in the parotid region.

Neurovascular free-muscle transfer for facial reanimation was performed as a secondary reconstructive procedure for 45 patients with facial paralysis resulting from ablative surgery in the parotid region. This intervention differs from neurovascular free-muscle transfer for treatment of established facial paralysis resulting from conditions such as congenital dysfunction, unresolved Bell palsy, Hunt syndrome, or intracranial morbidity, with difficulties including selection of recipient vessels and nerves, and requirements for soft-tissue augmentation. This article describes the authors' operative procedure for neurovascular free-muscle transfer after ablative surgery in the parotid region. Gracilis muscle (n = 24) or latissimus dorsi muscle (n = 21) was used for transfer. With gracilis transfer, recipient vessels comprised the superficial temporal vessels in 12 patients and the facial vessels in 12. For latissimus dorsi transfer, recipient vessels comprised the facial vessels in 16 patients and the superior thyroid artery and superior thyroid or internal jugular vein in four. Facial vessels on the contralateral side were used with interpositional graft of radial vessels in the remaining patient with latissimus dorsi transfer. Cross-face nerve grafting was performed before muscle transfer in 22 patients undergoing gracilis transfer. In the remaining two gracilis patients, the ipsilateral facial nerve stump was used as the primary recipient nerve. Dermal fat flap overlying the gracilis muscle was used for cheek augmentation in one patient. In the other 23 patients, only the gracilis muscle was used. With latissimus dorsi transfer, the ipsilateral facial nerve stump was used as the recipient nerve in three patients, and a cross-face nerve graft was selected as the recipient nerve in six. The contralateral facial nerve was selected as the recipient nerve in 12 patients, and a thoracodorsal nerve from the latissimus dorsi muscle segment was crossed through the upper lip to the primary recipient branches. A soft-tissue flap was transferred simultaneously with the latissimus muscle segment in three patients. Contraction of grafted muscle was not observed in two patients with gracilis transfer and in three patients with latissimus dorsi transfer. In one patient with gracilis transfer and one patient with latissimus dorsi transfer, acquired muscle contraction was excessive, resulting in unnatural smile animation. The recipient nerves for both of these patients were the ipsilateral facial nerve stumps, which were dissected by opening the facial nerve canal in the mastoid process. From the standpoint of operative technique, the one-stage transfer for latissimus dorsi muscle appears superior. Namely, a combined soft-tissue flap can provide sufficient augmentation for depression of the parotid region following wide resection. A long vascular stalk of thoracodorsal vessels is also useful for anastomosis, with recipient vessels available after extensive ablation and neck dissection.

Adolescent↗