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Sericin, which is derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori), functions as a glue to fix fibers of developing cocoons. Although sericin has potential ...

This multicentre, randomized, comparative, and investigator-masked crossover clinical trial sought to compare the efficacy and tolerability of fixed-combinations 0.1% brimonidine/0.5% timolol (BTFC) versus 1% dorzolamide/0.5% timolol (DTFC) as adjunctive therapies to prostaglandin analogues. A total of 110 patients with open-angle glaucoma or ocular hypertension previously treated with prostaglandin analogue monotherapy were randomized to receive either BTFC or DTFC as adjunctive therapy for eight weeks. These patients were then crossed over to the alternative treatment arm for another eight weeks. The intraocular pressure (IOP) reduction (primary outcome), adverse event occurrence, ocular discomfort after instillation, and patient preference (secondary outcomes) were recorded through patients’ interviews. BTFC instillation for eight weeks resulted in IOP reduction by 3.55 mmHg, demonstrating non-inferiority to DTFC instillation (3.60 mmHg; P

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor‐7 (FGF‐7) fused with the polyhedron‐encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF‐7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF‐7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF‐7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF‐7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF‐7‐polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.

Sericins are derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori) and act as a glue to fix fibers of the developing cocoon. Although sericin has potential uses ...

Ecdysteroids are mainly synthesised in the prothoracic glands, which regulate larval moulting and metamorphosis in insects. After degeneration of the prothoracic gland during pupal–adult metamorphosis, the ovary becomes the source of ecdysteroids in adult females. Here, we demonstrated that the high ecdysteroid level after adult eclosion decreased in the first few days in the eclosed adult red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The ecdysteroids in the freshly eclosed adult might originate from pupae and/or ecdysteroidogenic cells present in the anterior part of the adult body. The ecdysteroid in the haemolymph at the very beginning of adult stage may be important for ovarian development. While ecdysteroid levels decreased in the haemolymph, the ovaries acquired the ability to produce ecdysteroids with increased enzyme gene expression.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

We investigated the effects of nanosized TiO 2 particles on the death of mouse leukemia L1210 cells. TiO 2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO 2 ) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO 2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO 2 particles. TiO 2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO 2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO 2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.

Recently, our group discovered an alternative titanium dioxide (TiO(2)) activation method that uses ultrasound irradiation (US/TiO(2)) instead of ultraviolet irradiation. The pre-S1/S2 protein from hepatitis B virus, which recognizes liver cells, was immobilized to the surface of TiO(2) nanoparticles using an amino-coupling method. The ability of the protein-modified TiO(2) nanoparticles to recognize liver cells was confirmed by surface plasmon resonance analysis and immuno-staining analyses. After uptake of TiO(2) nanoparticles by HepG2 cancer cells, the cells were injured using this US/TiO(2) method; significant cell injury was observed at an ultrasound irradiation intensity of 0.4 W/cm(2). Together with these results, this strategy could be applied to new cell injuring systems that use ultrasound irradiation in place of photodynamic therapy in the near future.

An ultrasonic system with TiO2 particles was utilized for disinfection of Escherichia coli (E. coli) under condition of darkness. The mechanism of disinfection was studied by investigating the cell lysis and degradation of DNA leaked through the cell membrane. The degradation of DNA was enhanced remarkably in the presence of TiO2 particles. The effects of Al2O3 particles on the degradation of DNA were also examined, and these particles were found to be less effective than TiO2 particles at enhancing degradation. Compared with TiO2 addition case, the ultrasonic irradiation with no particles afforded 5% degradation, while the addition of Al2O3 induced approximately 54% degradation under the conditions used. The role of hydroxyl radicals in this process was also investigated by examining the effects of a hydroxyl radical scavenger, glutathione. A significant suppression of DNA leakage through the cell membrane was observed in the solutions containing glutathione, and the suppressive effects were dose-dependent. These results indicate that hydroxyl radicals may play a primary role in the degradation of DNA as well as cell lysis.

During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period. © 2006 Zoological Society of Japan.

The Algerian Government enacted the Microcredit Law in 1999 and started the microcredit program as a poverty and unemployment reduction policy in cooperation with two banks; Banque Nationale de l'Algerie and Banque El-Khalifa. But soon afterwards, this program became deadlocked because of the low repayment rates and the bankruptcy of the latter bank. This report examines the causes of failure by investigating the legislative contents of the Microcredit Law and analyzing the practical process of the program. Also by comparing the governmental microcredit program with that of an NGO, the merits and demerits are clarified as well as some administrative problems and its inefficiency involved in the microcredit programs in Algeria.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), prevents metamorphosis in larvae and stimulates vitellogenesis in adult females. Whether the same JH signaling pathway regulates both processes is presently unknown. Here, we employ the robust JH response during reproduction and development of the linden bug, Pyrrhocoris apterus, to compare the function of key JH-signaling genes encoding the JH receptor, Methoprene-tolerant (Met), its binding partner Taiman (Tai), and a JH-inducible protein, Kruppel-homolog 1 (Kr-h1). RNA interference (RNAi) with Met or Tai, but not Kr-h1, blocked ovarian development and suppressed vitellogenin gene expression in the fat body of females raised under reproduction-inducing conditions. Loss of Met and Tai matched the effects of CA ablation or the natural absence of JH during reproductive diapause. Stimulation of vitellogenesis by treatment of diapausing females with a JH mimic methoprene also required both Met and Tai in the fat body, whereas Kr-h1 RNAi had no effect. Therefore, the Met-Tai complex likely functions as a JH receptor during vitellogenesis. In contrast to Met and Kr-h1 that are both required for JH to prevent precocious metamorphosis in P. apterus larvae, removal of Tai disrupted larval ecdysis without causing premature adult development. Our results show that while Met operates during metamorphosis in larvae and reproduction in adult females, its partner Tai is only required for the latter. The diverse functions of JH thus likely rely on a common receptor whose actions are modulated by distinct components.

http://www.pnas.org/; International audience; Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors.

婦人の自覚症状訴え率と, 年齢, 職業, 間食の頻度, コーヒーの摂取量, 加工食品・調理済み食品の利用頻度, 外食の回数, 食生活の改善, 食物摂取得点の8要因25カテゴリーとの関連について, 林の数量化理論I類により, 多変量解析を行い, 次のような結果を得た。1) 自覚症状訴え率との関連性の強い要因は, 間食の頻度, 加工食品・調理済み食品の利用頻度, 食生活の改善などである。2) 自覚症状訴え率を高めるカテゴリーは, 間食を毎日する, 食物摂取得点が低い, 家で内職・アルバイトをする, 加工食品・調理済み食品を普通程度に利用するなどである。3) 自覚症状訴え率を低めるカテゴリーは, 年齢50歳以上, 加工食品・調理済み食品の利用頻度が少ない, 食生活は今のままでよいなどである。

Drosophila egg production depends upon the nutritional available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. We have shown that expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone induces and suppresses the apoptosis, respectively. In this study, we investigated which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. We measured ecdysteroid levels in the whole body, ovaries and haemolymph samples by RIA and found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. We suggest there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake.

The prothoracic glands of early last (fifth) instar larvae of the silkworm are inactive with regard to ecdysteroidogenesis and unresponsive to prothoracicotropic hormone (PTTH) [J. Insect Physiol. 31 (1985) 455]. In an attempt to elucidate the hormonal mechanisms that cause the inactivity, we compared the effects of PTTH, dibutyryl cyclic AMP (dbcAMP), a cAMP phosphodiesterase inhibitor (IBMX), juvenile hormone analogue (JHA) and 20-hydroxyecdysone (20E) on secretory activity of the third, fourth and fifth instar glands. Among the factors examined, feedback inhibition by 20E was indicated to be the most likely factor. Inhibition was moderate in the third and early fourth instars while 20E strongly inhibited the glands of middle fourth instar larvae. The inhibitory effect of 20E was reduced by removal of the brain and corpora allata. Once the glands were suppressed by 20E to the degree of exhibiting neither secretory activity nor responsiveness to PTTH, dbcAMP or IBMX did not elicit ecdysone secretion at all. Thus the feedback inhibition may shut down ecdysteroidogenesis although it is obscure whether it affects the intracellular transductory cascade from the PTTH receptor through cAMP. Taken together, this evidence suggests that inactivity of the gland in the early fifth instar is brought about by feedback inhibition of the glands by 20E occurring in the late fourth instar, and that this inactivity is maintained by the juvenile hormone found in the early fifth instar.

To establish a preventive measures against excessive intake of salt, sensation of saltiness was investigated for residents in Nagoya city and its vicinity. The result was statistically treated using chai-square test and the following conclusions were obtained.1) The higher the concentration of the soup stock, the more intensely the saltiness is felt.2) Comparison between the sexes revealed that females preferred less salty taste.3) Comparison among different age groups indicated that the young males preferred salty taste.4) As for the relation between the health condition and the sensitivity to saltiness, healthy subjects were more sensitive.5) As for the relation between the degree of hunger and the sensitivity, males were more sensitive to saltiness when they were suitably hungry. The sensititivity of females are hardly influenced by the degree of hunger.

This study is a part of Research Project entitled "Cultural fusion and cultural conflict in the Gulf" sponsored by the Ministry of Education of Japan. The field-work was carried out in Dubai, The United Arab Emirates (U.A.E.) during July and August, 1987. U.A.E., like the other Gulf countries, has undergone drastic socio-economical changes since oil exploitation started. The huge oil money income has brought not only economic changes but also socio-structural and cultural changes due mainly to the number of foreign migrant workers. They have been immigrating to this area in response to the needs of manpower in the process of socio-economic development. In this paper, the topic of cultural fusion and cultural conflict is surveyed by focusing on the migrant workers in Dubai. Interviews with the migrant workers were conducted and their motivation for immigration to U.A.E., their impressions about life in Dubai, their daily problems, and their perspectives for the future were examined. After carrying out this preliminary research, we came to a tentative counclusion contrary to our original supposition. In spite of the co-existence of many different ethnic groups of migrant workers in Dubai, cultural fusion and cultural conflict are not occuring as much as we had expected. For the foreign migrant workers, economic and working conditions and creation of a new social-network in Dubai are the central issues of concern and have greater importance than cultural assimilation, such as learning the Islamic way of life or the Arabic language. The creation of a social network, in a way, functions as a strategy for resolving their daily problems and for adaptation to the life in U.A.E. without major modification of their own cultual background.

塩味覚と9つの要因すなわち, 性, 年齢, 職業, 居住地域, 最近の健康状態, 官能検査日の健康状態, 空腹感, 病気, 用いた試料のだし濃度との関連について, 林の数量化理論第2類により多変量解析を行った。得られた結論は次のようである。1) だし濃度, 年齢, 性, 職業の4つの要因は, 塩味評価と密接に関連することがわかった。i) だし濃度の濃いほうが塩味を強く感じる。ii) 高年齢になるほど, うす塩味嗜好が強い。iii) 男性は女性より濃い塩味嗜好が強い。iv) うす塩味に慣れる訓練を受けた栄養士養成施設校の学生は, うす塩味嗜好が強い。2) しかしながら, 今回の調査方法によって得られた塩味評価と要因との相関は, 各被験者の属する群を判別するには不十分であった。このことから, 結果を改良するためには, 今回採用した以外の要因も含めて検討する必要があると思われる。