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Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) comprise the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyx mori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis, dorsolateral protocerebrum, and central complex of the brain. SNAP-25 was restricted to neurons in the pars intercerebralis and the central complex of the brain. Syntaxin 1A was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. SNAP-25 colocalizes with VAMP2 in the central complex and Syntaxin 1A colocalizes with bombyxin, which is an insect neuropeptide. These findings suggest that SNARE proteins play important roles in membrane trafficking in the insect brain.

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

Sleep disturbances can contribute to cognitive decline and neuropsychiatric disorders. However, the underlying mechanisms of these processes are poorly understood. The present study evaluated the effects of a chronic sleep disorder (CSD) on long-term memory formation and anxiety-like behavior in our originally established mouse model of psychophysiological stress-induced CSD characterized by disrupted circadian rhythms of wheel-running activity and sleep-wake cycles. Model mice are continuously exposed to mild stress imposed by perpetually staying on a running-wheel to avoid water. The findings of novel object recognition (NORT) and open field (OFT) tests showed that CSD impaired recognition memory and elicited anxiety-like behavior, respectively. These results suggested that the CSD impaired cognitive function and emotional status. Thus, this CSD model could be useful for studying the underlying mechanisms of neurobehavioral difficulties caused by sleep disorders. We then examined the hippocampal mRNA expression of genes associated with learning and memory, and anxiety and depression. The CSD increased the mRNA expression of Crhr1, Ngf and Phlpp1, and suppressed that of Ace, Egr2 and Slc6a4. Based on the functions of these genes, we inferred that the increase in Crhr1 mRNA was associated with the pathogenesis of psychiatric conditions, whereas mRNA levels of the other five genes were directed towards symptom relief. Upregulating hippocampal Crhr1 expression might contribute in part to the activation of corticotropin-releasing hormone (CRH)-CRH receptor1 signaling that mediates CSD-evoked mental disorders.

Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal (MG), which can cause cellular damage. The glyoxalase (GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO2 concentrations, where increased photosynthesis promotes the MG production, GLX1 and GLX2 activities in A. thaliana increased and the expression of AtGLX1-2 and AtGLX2-5 was enhanced. On the basis of these findings and the phylogeny of GLX in oxygenic phototrophs, we propose that the GLX system scavenges MG produced in chloroplasts during photosynthesis.

Acute or chronic effects of consuming or skipping breakfast on cognitive performance in humans are controversial. To evaluate the effects of chronically skipping breakfast (SB) on hippocampus-dependent long-term memory formation, we examined hippocampal gene expression and applied the novel object recognition test (NORT) after two weeks of repeated fasting for six hours from lights off to mimic SB in mice. We also examined the effects of SB on circadian rhythms of locomotor activity, food intake, core body temperature (CBT) and sleep-wake cycles. Skipping breakfast slightly but significantly decreased total daily food intake without affecting body weight gain. Locomotor activity and CBT significantly decreased during the fasting period under SB. The degree of fasting-dependent CBT reduction gradually increased and then became stabilized after four days of SB. Electroencephalographic data revealed that repeated SB significantly decreased the duration of wakefulness and increased that of rapid eye movement (REM) and of non-REM (NREM) sleep during the period of SB. Furthermore, total daily amounts of wakefulness and NREM sleep were significantly decreased and increased, respectively, under SB, suggesting that SB disrupts sleep homeostasis. Skipping breakfast significantly suppressed mRNA expression of the memory-related genes, Camk2a, Fkbp5, Gadd45b, Gria1, Sirt1 and Tet1 in the hippocampus. Recognition memory assessed by NORT was impaired by SB in accordance with the gene expression profiles. These findings suggested that chronic SB causes dysregulated CBT, sleep-wake cycles and hippocampal gene expression, which results in impaired long-term memory formation.

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause- and nondiapause-egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Kruppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Kruppel homolog 1 is an early juvenile hormone-inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.

This study investigated daily changes in susceptibility to insecticides in the silkworm Bombyx mori (Lepidoptera: Bombycidae). The daily rhythm of permethrin susceptibility in fifth instar larvae of the p63-silkworm strain was significant, with the lowest mortality rate during the late photophase under a 12-h light–12-h dark cycle. The rhythms were similar whether permethrin was intraperitoneally injected into hemolymph or topically applied to the dorsal abdomen. Drug intake through the cuticle probably did not significantly contribute to the organization of this susceptibility rhythm. We also found that the daily susceptibility profiles depended on insecticide type and silkworm strain. Temporal susceptibility variations might be attributable to the drug metabolic system. To our knowledge, this is the first description of an insecticide susceptibility rhythm in the larval stage of lepidopteran insects. Specifically timed delivery of insecticides might enable more efficient control of pest caterpillars tha...

The braconid parasitoid wasp that attacks exposed lepidopteran larvae, Meteorus pulchricornis (Wesmael), is a common natural enemy of pest caterpillars and may serve as a biological control agent. Recently spreading nighttime lighting for the control of noctuid moths may affect the behavior of the parasitoid if it is nocturnal, but little is known about its daily activity patterns. This study investigated the locomotor activity of M. pulchricornis under various light conditions. Over a daily 16-h light:8-h dim light cycle (16L8Dim), the wasps were inactive during most of the photophase (16L), gradually becoming active late in the photophase, with activity levels peaking just after the switch to the scotophase (8Dim). Subsequently, during the scotophase, activity decreased to a moderate level and continued at that level until the switch to the photophase. In addition, after transfer from daily light/dim light cycles (16L8Dim), daily fluctuations in activity persisted for at least 1 day under continuous light or dim light conditions. This is the first report describing the nocturnal activity rhythm of parasitoid wasps and the suppression of their activity under strong light conditions.

Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal (MG), which can cause cellular damage. The glyoxalase (GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO

Rab proteins are low-molecular weight (20–25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori.

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available.

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.

The present study investigated the functional involvement of melatonin and dopamine in photoperiodism to terminate pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Diapause in this long-day (short-night) species is maintained during long nights and can be terminated by exposure to a short-night photoperiod. We observed the effects of melatonin and dopamine and their receptor antagonists on diapause pupae. Melatonin and flupentixol, a dopamine receptor antagonist, terminated pupal diapause even under long-night photoperiods. Dopamine and luzindole, a melatonin receptor antagonist, retarded adult emergence during short nights, whereas melatonin advanced the timing of adult emergence under the short-night photoperiod in a manner dependent on the number of injections. The results of the day-length extension experiment indicated that a change in the photoperiod was immediately detected as mRNA expression of the rate-limiting enzyme of melatonin production. These findings suggest that the melatonin pathway transmits information on the photoperiod to terminate the pupal diapause of A. pernyi. The melatonin pathway also inhibited the dopamine production system, and the dopamine pathway inhibited the melatonin production system. We propose an insect model of the photoperiodic counter driven by mutual inhibition between the melatonin and dopamine pathways.

In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), embryonic diapause is under maternal control and the decision between diapause and direct development in bivoltine strains depends on environmental factors such as temperature and photoperiod experienced by the preceding generation. We reared ten bivoltine silkworm strains (c10, g32, k06, n25, p21, p22, p24, p44, p50 and p63) under various thermal and light conditions and examined the incidence of embryonic diapause in the next generation to identify strains in which the incidence of diapause can be controlled within the range of 0 to 100% by adjusting a single ambient parameter. Some strains were clearly destined towards either diapause or direct development. The diapause incidence in the c10, p22 and p50 strains was controlled by temperature during the egg stage (0% at 18°C and 100% at 25°C), that in the p50 strain was also determined to be dependent on illumination during the egg stage (0% under continuous darkness and 100% under continuous illumination), and photoperiod during the larval stage regulated diapause in p44 and p50 (0% and 100% under long-day and short-day photoperiod, respectively), when all other external parameters remained constant under each experimental condition. These diapause-controllable silkworm strains might serve as model systems for studies of insect diapause, especially for the differential screening of related factors.

Insects have both a circadian oscillatory system in peripheral tissues and a master clock in the central nervous system. However, the distribution of oscillator cells in the digestive system that is indispensable for nutrient intake has not been described in detail. This study immunohistochemically investigates cellular localization of the clock protein PERIOD in the larval midgut of the silkworm Bombyx mori (Lepidoptera: Bombycidae). PERIOD-like immunoreactivity was not uniformly distributed and was located exclusively in goblet cells of the midgut epithelium. The primary role of goblet cells is the active transport of potassium from hemolymph into the gut lumen. However, these cells might function as a peripheral clock that regulates the daily rhythms of physiological activities in the midgut of silkworm larvae.

In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause- and nondiapause-egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Krüppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Krüppel homolog 1 is an early juvenile hormone-inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.

Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,β-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night.

Object: For ectothermic insects, thermal tolerance is one of the most essential properties for survival in ambient environments. However, the molecular mechanisms involved in thermal tolerance in the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) have not been fully elucidated. The present study investigated mechanisms that provide heat tolerance in embryos of Bombyx mori . Materials and Methods: Eggs of the bivoltine silkworm strain p50 were exposed to different temperatures, to determine the lethal threshold temperature and to assess the effects of mild and low temperature incubation on tolerance to heat shock and on embryonic protein profiles. Protein levels were measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: When eggs were exposed to transient heat shock for one hour, the lethal threshold temperature was between 47.0°C and 48.0°C. However, exposure to 40.0°C for four hours, substantially elevated tolerance to the threshold heat shock and simultaneously increased levels of 70 and 27 kDa proteins in eggs, whereas exposure to 35.0°C did not. Exposure to 10.0°C for four hours lowered heat tolerance and did not alter the expression of 70 and 27 kDa proteins. Conclusion: The present findings indicated that hardening silkworm eggs at mild temperatures increases heat tolerance in embryos. To our knowledge this is the first observation of heat hardening in silkworm embryos. These putative heat-shock proteins of 70 and 27 kDa might be involved in the effect of mild temperature hardening on heat tolerance. Cold stress might deprive embryos of energy reserves available for protection against heat damage. Protective mechanisms against heat and cold stress are probably different in this species. Further studies of the molecular mechanisms of heat tolerance should provide insight into the development of novel, high-yield silkworm strains in tropical environments.

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24-hour rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This circadian clock is responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate and hormone release. The retinal circadian clock was the first extra-SCN circadian oscillator to be discovered in mammals and several studies have now demonstrated that many of the physiological, cellular and molecular rhythms that are present within the retina are under the control of a retinal circadian clock, or more likely a network of hierarchically organized circadian clocks that are present within this tissue. BioEssays 30:624-633, 2008. (c) 2008 Wiley Periodicals, Inc.

Previous studies have shown that, in the Royal College of Surgeon rat, circadian rhythms in the retinal dopaminergic and melatonergic systems are still present after the photoreceptors have degenerated, thus demonstrating that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors. The aim of the present study was to investigate the pattern of expression of the clock genes in the retina of the Royal College of Surgeons rat under different lighting conditions. Expression of clock genes was investigated in the retina of normal and dystrophic Royal College of Surgeons rats under 12 h of light/12 h of dark (LD), constant darkness (DD) and constant light (LL) using Real Time Quantitative RT-PCR. Our data indicate that, in control animals, Period1 , Period2 , Cryptochrome1 , Cryptochrome2 , Clock , Rora , Rev-Erbα and Npas2 mRNA levels showed a significant variation over the sampling period in LD cycles and in DD, whereas Bmal1 mRNA did not show any significant variation. In LL, the transcripts for Per1 , Per2 , Clock and Rev-Erbα showed significant temporal variations. In the dystrophic retina, only Per1 and Per2 mRNA levels showed a temporal variation over the 20-h period. Our work indicates that degeneration of the photoreceptor cells dramatically affected the expression levels and patterns of many clock genes. Finally, the present study suggests that investigating the expression pattern of clock genes using the whole retina or animals with photoreceptor degeneration may not provide any definitive answers about the working of the retinal circadian clock system.

Influence of Ni on the kinetics of the reactive diffusion between Au and Sn was experimentally studied at solid-state temperatures. Binary Sn-Ni alloys with Ni concentrations of 1, 3 and 5 mass% were used to prepare sandwich (Sn-Ni)/Au/(Sn-Ni) diffusion couples by a diffusion bonding technique. The diffusion couples were isothermally annealed at temperatures of T= 433, 453 and 473 K for various times in an oil bath with silicone oil. After annealing, AuNiSn 8 , AuSn 4 , AuSn 2 and AuSn compound layers were observed to form at the (Sn-Ni)/Au interface in the diffusion couple. The total thickness l of the compound layers monotonically increases with increasing annealing time t according to the equation l = k(t/t 0 ) n , where t 0 is unit time, 1 s. The exponent takes values between n = 0.29 and 0.37 under the present annealing conditions. Such values of n < 0.5 indicate that the grain boundary diffusion contributes to the rate-controlling process and the grain growth occurs at certain rates. The higher the Ni concentration of the Sn-Ni alloy is, the faster the overall growth of the compound layers occurs. This means that Ni is an accelerator for the reactive diffusion between Au and Sn at solid-state temperatures. The acceleration effect of Ni becomes more remarkable at higher annealing temperatures. Such influence of Ni on the kinetics is mainly attributed to the dependencies of the growth rate of the AuNiSn 8 layer on the composition of the Sn-Ni alloy and the annealing temperature.

The kinetics of the reactive diffusion between Pd and Sn at solid-state temperatures was experimentally examined using Sn/Pd/Sn diffusion couples prepared by a diffusion bonding technique. The diffusion couples were isothermally annealed at temperatures of T = 433, 453 and 473 K for various times up to 300 h in an oil bath with silicone oil. After annealing, PdSn 4 , PdSn 3 and PdSn 2 compound layers were observed at T = 433 K, but only PdSn 4 and PdSn 3 layers were recognized at T = 453 and 473 K. The total thickness l of the Pd–Sn compound layers is mathematically described as a power function of the annealing time t as follows: l = k ( t / t 0 ) n , where t 0 is unit time, 1 s. The diameter d of the columnar grain in the compound layers is also expressed as a power function of the annealing time t : d = k d ( t / t 0 ) p . At T = 433–473 K, n is close to 0.5, and p takes values of 0.14–0.17. Such values of p indicate that the grain growth occurs at certain rates in the compound layers. Nevertheless, the total thickness l is almost proportional to the square root of the annealing time t . Thus, it is concluded that the volume diffusion is the rate-controlling process of the reactive diffusion between Pd and Sn.

The solid-state reactive diffusion between Ni and Sn was experimentally studied using Sn/Ni/Sn diffusion couples. The diffusion couples were prepared by a diffusion bonding technique and then annealed at temperatures between T = 433 and 473 K for various times in an oil bath with silicone oil. At the Ni/Sn interface in the annealed diffusion couple, a rather wavy layer of Ni3Sn4 was observed at T = 453 and 473 K, but granular particles of Ni3Sn4 were recognized at T = 433 K. The average thickness l of the Ni3Sn4 layer monotonically increases with increasing annealing time t according to the equation l = k(t/t0)n, where t0 is unit time, 1 s. The experimental results provide n = 0.46 and 0.41 at T = 453 and 473 K, respectively. The values of n smaller than 0.5 indicate that the grain boundary diffusion contributes to the rate-controlling process and the grain growth occurs at certain rates. Under the present annealing conditions, the thickness of the Ni3Sn4 layer is more than one order of magnitude smaller than the total thickness of the AuSn4, AuSn2 and AuSn layers formed by the reactive diffusion between Au and Sn. This indicates that the Ni layer is an effective inhibitor against the reactive diffusion between the Sn-base solder and Au/Ni/Cu multilayer conductor alloys during annealing at solid-state temperatures after complete dissolution of the Au layer into the molten solder.

The kinetics of the reactive diffusion between Cu and Sn at solid-state temperatures was experimentally examined using Sn/Cu/Sn diffusion couples prepared by a diffusion bonding technique. The diffusion couples were isothermally annealed at temperatures between T = 433 and 473 K for various times in an oil bath with silicone oil. Due to annealing, compound layers consisting of Cu 3 Sn and Cu 6 Sn 5 are formed at the Cu/Sn interface. In most of the annealed diffusion couples, grains of CuSnO exist in the Cu 6 Sn 5 layer. However, the CuSnO grain scarcely influences the growth behavior of the compound layers. The thickness of the Cu 6 Sn 5 layer is around twice greater than that of the Cu 3 Sn layer. The difference between their thicknesses slightly increases with increasing annealing temperature. At a constant temperature, the ratio of the thicknesses remains constant independent of the annealing time. The total thickness l of the compound layers monotonically increases with increasing annealing time t according to equation l = k ( t / t 0 ) n , where t 0 is unit time, 1 s. The observations provide n = 0.37, 0.43 and 0.50 at T = 433, 453 and 473 K, respectively. The value n = 0.5 indicates that the volume diffusion is the rate-controlling process for the reactive diffusion at T = 473 K. As the annealing temperature decreases, the grain boundary diffusion will contribute to the rate-controlling process. Thus, the values of n smaller than 0.5 yield that there is the contribution of the grain boundary diffusion and the grain growth occurs at certain rates at T = 433 and 453 K. Since the ratio between the thicknesses of the compound layers is kept constant during isothermal annealing, the same rate-controlling process is expected to work in both compound layers.

The kinetics of the solid-state reactive diffusion between binary Au-Ag alloys and Sn was experimentally examined using Sn/ Au 0 . 7 5 Ag 0 . 2 5 /Sn and Sn/Au 0 . 5 Ag 0 . 5 /Sn diffusion couples. The diffusion couples were prepared by a diffusion bonding technique and then isothermally annealed at temperatures of T = 393,433 and 473 K for various times up to 1272 h in an oil bath with silicone oil. Under the present experimental conditions, AuSn 4 and AuSn 2 compound layers were observed after annealing. Furthermore, fine particles of Ag 3 Sn were rather uniformly distributed in the Au-Sn compound layers. The total thickness I of the Au-Sn compound layers is expressed as a power function of the annealing time t as follows: l = k(t/t 0 ) n , where to is unit time, 1 s. Here, the exponent takes values of n = 0.34-0.40. The mean interdistance r of the Ag 3 Sn particles is also described as a power function of t: r = k r (t/t 0 ) p , where p = 0.28-0.43. Assuming that the interdistance r varies in proportion to the grain size of the Au-Ag compound during annealing, the rate-controlling process of the reactive diffusion was estimated. If the grain boundary diffusion across the Au-Sn compound layers is the only rate-controlling process, the values of n calculated from the equation n = (1 - p)/2 become smaller than the experimental values of n = 0.34-0.40. Consequently, both the volume diffusion and the grain boundary diffusion should contribute to the rate-controlling process of the reactive diffusion.

The reactive diffusion at solid-state temperatures was experimentally studied for the binary Ag-Sn system. Sn/Ag/Sn diffusion couples were prepared by a diffusion bonding technique and then annealed at temperatures between T ¼ 433 and 473 K for various periods in an oil bath with silicone oil. After annealing, layers of the " (Ag3Sn) andcompounds were observed to form at the Sn/Ag interface. Furthermore, a thin layer alloyed with Sn was produced into the Ag specimen from the � /Ag interface owing to diffusion induced recrystallization (DIR). The thickness is about one order of magnitude greater for the " layer than for theand DIR layers. The total thickness l of the " andlayers is described as a function of the annealing time t by the equation l ¼ kðt=t0Þ n . Here, t0 is unit time, 1 s. The exponent n is 0.40, 0.38 and 0.36 at T ¼ 433, 453 and 473 K, respectively. If the reactive diffusion is controlled by the volume diffusion, n is equal to 0.5. However, n is actually smaller than 0.5 at all the annealing temperatures. This indicates that the grain boundary diffusion contributes to the reactive diffusion and the grain growth occurs at certain rates. As the annealing temperature decreases, the contribution of the grain boundary diffusion should be more remarkable, but the grain growth will become sluggish. This is the reason why the value of n increases with decreasing annealing temperature.

The reactive diffusion between Au and Sn was experimentally studied at solid-state temperatures using Sn/Au/Sn diffusion couples prepared by a diffusion bonding technique. The diffusion couples were annealed at temperatures of T = 393 and 473 K for various times in an oil bath with silicone oil. After annealing, compound layers composed of AuSn4, AuSn2 and AuSn were recognized to form at the Au/Sn interface. The thickness of the AuSn4 layer is about six and four times greater than those of the AuSn2 and AuSn layers at T = 393 and 473 K, respectively. The ratio of the thicknesses of the compound layers is kept constant independently of the annealing time. The total thickness l of the compound layers is described as a function of the annealing time t by the equation l = k(t/t0)n, where t0 is unit time, 1 s. The exponent n is nearly equal to 1/2 at T = 393 K but takes a value between 1/4 and 1/2 at T = 473 K. Such an intermediate value of n at T = 473 K indicates that the grain boundary diffusion contributes to the reactive diffusion and the grain growth occurs at certain rates. As the annealing temperature decreases, the contribution of the grain boundary diffusion should become more remarkable, but the grain growth will slow down. Consequently, n becomes close to 1/2 at T = 393 K. According to the constancy of the ratio of the thicknesses, it is concluded that the same rate-controlling process works in the AuSn4, AuSn2 and AuSn layers at a constant annealing temperature.

The kinetics of the reactive diffusion between Au and Sn was experimentally studied in a metallographical manner. Sn/Au/Sn diffusion couples were prepared by a solid-state diffusion bonding technique and then annealed at 433 K for various times between 1 and 127 h (3.6 × 103 and 4.57 × 105 s) in an oil bath with silicone oil. Due to annealing, intermetallic compound layers consisting of AuSn4, AuSn2 and AuSn are produced at the Au/Sn interface in the diffusion couple. The thickness of the AuSn4 layer is four times greater than those of the AuSn2 and AuSn layers. The ratio of the thicknesses of the compound layers remains constant during annealing. The total thickness l of the compound layers increases with annealing time t. Such a relationship is mathematically described by the equation l = k(t/t0)n, where t0 is unity time, 1 s. From the experimental results, the proportionality coefficient and the exponent are determined to be k = 2.7 × 10−7 m and n = 0.42, respectively, by a least-squares method. The exponent n is slightly smaller than 0.5. This implies that the grain boundary diffusion partially contributes to the rate-controlling process for the growth of the compound layers.

Cycle (Bmal1) is one of the circadian clock genes and the key regulator of the circadian system in many organisms, encoding a bHLH– PAS transcription factor. In the present study, we cloned cycle homolog (BmCyc) in Bombyx mori. We performed polymerase chain reaction with degenerated primers deduced from the conserved amino acid sequences of mammalian BMAL1 and Drosophila CYCLE. Then the partial clone obtained was used as a probe for screening a cDNA library constructed from pupal brains of B. mori. BmCyc is 5703 nucleotides long and encodes 700 amino acid residues. The BmCYC has bHLH, PAS A and PAS B domains, and the sequence identities for these domains are 85, 60 and 50%, to Drosophila CYCLE (dCYC) and 69, 58 and 50%, to human BMAL1 (hBMAL1), respectively. The deduced amino acid sequence of BmCYC is 37% identical to that of dCYC and 28% to hBMAL1. Northern blot analysis demonstrates that BmCyc gene was expressed in all the tissues tested, which were head, fat body, silk glands, and midgut. Also no significant day, night time-specific difference in expression of BmCyc gene in the head was detected.

To investigate the functional involvement of carotenoid in the insect circadian rhythm, we observed the effect of carotenoid depletion on the hatching patterns of the silkworm under several light conditions. When eggs were transferred from continuous light (LL) to continuous darkness (DD), an overt hatching rhythm was initiated. The first hatching peak, which was observed at 13.2 h after the transfer in the carotenoid-depleted silkworm, was reduced remarkably in both control groups (carotenoid-rich and carotenoid-depleted but with lutein supplementation), though subsequent peaks occurred with similar timing. Under LD 4 : 20, LD 12 : 12 and LD 20 :4, hatching patterns depended on the dietary carotenoid and the light intensity of the photophase. At a low light intensity, carotenoid depletion suppressed hatching as a masking effect just after light-on. Under LD 4 : 20 at a low light illumination, hatchings in the carotenoid-depleted silkworm were observed during scotophase, but few larvae hatched during scot...

In this work an approach for rapid prototyping and manufacturing of metallic as well as ceramic parts is presented. By using Mold Shape Deposition Manufacturing (MoldSDM), a wax mold is fabricated which is filled with a slurry containing the final part material in powder form. The wax mold is manufactured by first decomposing the desired part geometry into machinable compacts. In a next step the mold is built up in a series of layers by machining either the wax or a sacrificial support material. The surface quality of the final mold is only limited by the abilities of the CNC machine. After casting a ceramic or metallic slurry, the wax mold is removed and the obtained green part debinded and sintered. In this work the mechanical and microstructural properties of the parts obtained by this method are presented. The materials which have been investigated are silicon nitride and stainless steels. Due to the high mold quality the process is suitable for a variety of applications in rapid prototyping and manufacturing where parts made of engineering materials are required.

Recent studies have suggested that peripheral tissues in mammals have an autonomous circadian oscillator driven by negative feedback loops consisting of periodical expression of clock genes. In the present study we investigated the mechanism that regulates circadian rhythms in mammalian peripheral tissues, and observed developmental aspects in circadian expression of clock genes in the heart of postnatal rats. Daily expression patterns of clock genes (rPer1, rPer2 and BMAL1) and a clock-controlled gene Dbp were examined by Northern blot analysis. Circadian expression of rPer1, BMAL1 and Dbp mRNAs started between postnatal day 2 (P2) and P5, but rPer2 mRNA did not show rhythmicity until P14. Rhythmic expression of other genes in the heart occurred even in the absence of rhythmic rPer2 expression. These findings suggest that in the rat heart, rhythmic expression of rPer2 was not essential to generate circadian rhythmicity at the early postnatal stage. Judging from mRNA rhythms in the heart, it was probably after P20 that rats established the mature circadian system controlling peripheral rhythms.

To investigate the mechanism that controls circadian rhythms in mammalian peripheral tissues, we housed mice in short days (6 h light: 18 h dark) or long days (18 h light: 6 h dark) and examined the rhythmic expression patterns of the mammalian clock genes mPer1, mPer2 and mPer3 and a clock-controlled gene Dbp in the mouse heart. Northern blot analyses showed that peak levels of mPer1 mRNA expression in long days were about 50 % higher than those in short days. On the contrary the amplitude of the mPer2 mRNA peak in long days was about 25 % lower than that in short days. We could not find any effect of photoperiod on either the amplitude or waveform of the rhythms of mPer3 and Dbp mRNAs. Photoperiod differentially affected the expression of three mPer genes even in a peripheral tissue of mice.

To elucidate the scavenging systems of sugar- and lipid-derived reactive carbonyls (RCs) in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803), we selected proteins from S. 6803 based on amino-acid (AA) sequence similarities with proteins from Arabidopsis thaliana, and characterized the properties of the GST-fusion proteins expressed. Slr0942 catalyzed the aldo-keto reductase (AKR) reaction scavenging mainly sugar-derived RCs, methylglyoxal (MG). Slr1192 is the medium-chain dehydrogenase/redutase (MDR). It catalyzed the AKR reaction scavenging several lipid-derived RCs, acrolein, propionaldehyde, and crotonaldehyde. Slr0315 is a short-chain dehydrogenase/redutase (SDR), and it catalyzed only the reduction of MG in the AKR reaction. Slr0381 catalyzed the conversion of hemithioacetal to S-lactoylglutahione (SLG) in the glyoxalase (GLX) 1 reaction. Sll1019 catalyzed the conversion of SLG to glutathione and lactate in the GLX2 reaction. GLX1 and GLX2 compose the glyoxalase system, which scavenges MG. These enzymes contribute to scavenging sugar- and lipid-derived RCs as scavenging systems.

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.

In Arabidopsis thaliana, the aldo-keto reductase (AKR) family includes four enzymes (The AKR4C subfamily: AKR4C8, AKR4C9, AKR4C10, and AKR4C11). AKR4C8 and AKR4C9 might detoxify sugar-derived reactive carbonyls (RCs). We analyzed AKR4C10 and AKR4C11, and compared the enzymatic functions of the four enzymes. Modeling of protein structures based on the known structure of AKR4C9 found an (α/β)8-barrel motif in all four enzymes. Loop structures (A, B, and C) which determine substrate specificity, differed among the four. Both AKR4C10 and AKR4C11 reduced methylglyoxal. AKR4C10 reduced triose phosphates, dihydroxyacetone phosphate (DHAP), and glyceraldehydes 3-phosphate (GAP), the most efficiently of all the AKR4Cs. Acrolein, a lipid-derived RC, inactivated the four enzymes to different degrees. Expression of the AKR4C genes was induced under high-[CO2] and high light, when photosynthesis was enhanced and photosynthates accumulated in the cells. These results suggest that the AKR4C subfamily contributes to the detoxification of sugar-derived RCs in plants.

To investigate the molecular mechanism that regulates circadian rhythms in mammalian peripheral tissues, we examined the phase shifts evoked by light exposure in the circadian mRNA expression rhythms of mammalian Period genes (mPer1, mPer2 and mPer3) and a clock-controlled gene Dbp, in the mouse heart, by Northern blot analysis. The light pulse did not induce any acute mRNA expression of mPer in the heart, but the pulse gave rise to phase shifts in the circadian mRNA rhythms. On the first day after the exposure, only mPer1 mRNA showed a phase shift, whereas obvious phase shifts were not observed in the rhythms of mPer2, mPer3 and Dbp mRNAs. On the second day, phase shifts occurred to a similar extent in the mRNA rhythms of all four genes examined. The rhythm of mPer1 mRNA shifted fastest among those of the three mPers. Therefore mPer1 seems to play an important role in phase resetting of mammalian peripheral oscillators. Immediate responses to light pulses in mRNA expression of mPers may not be required for phase shifting of peripheral circadian oscillators. Our findings suggest that mammals require more than one day to have peripheral oscillators entrained to a new daily schedule.

The primary structure of the cone-rod homeobox protein (CRX), a member of the OTX-like homeobox protein family, in the rat was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 299 amino acid residues with motifs conserved in mammalian CRXs, and was 98% identical to mouse CRX. Northern blot analysis showed that Crx mRNA levels in the rat retina were constant under a daily light-dark cycle. These findings suggest that the expression mechanism of rat Crx in the retina was different from that in the pineal, where Crx mRNA exhibits a daily expression rhythm.

In the present study, methods in which the debonding is detected in the adhesively bonded joint constructed with aluminum and pultruded GFRP (PULGFRP) plates using an optical fiber have been investigated. The proposed detecting methods for the debonding in the bonded joints utilize the breakage of the embedded optical fiber caused by the damage such as cracks. In the present study, two detecting methods for the debonding in which the situations of the embedded optical fiber in the bonded joint were different were adopted, and their ability of detecting the debonding were examined. Further, the effects of embedding the optical fibers in the bonded joints on the strength of the joints were investigated, and the ability of the detection of the debonding in the joints whose adhesives were in undercure was also studied. From these results, the applicability of these detecting methods to the health-monitoring system were investigated. As the conclusions it was obtained that one of the two methods could be applied to the health-monitoring system as the detecting method for the debonding in the Al-PULGFRP adhesively bonded joints.

BMAL1 is a putative transcription factor which is involved in circadian rhythm generation in Drosophila. Northern blot analysis was performed to investigate the expression of rat BMAL1 mRNA in the suprachiasmatic nucleus (SCN) and peripheral tissues. In the SCN, circadian expression of BMAL1 mRNA which reaches its peak level at the time of dark-light transition was observed, and the expression pattern was antiphase to those of two period (per) homologues, rPer1 and rPer2. However, no circadian oscillation for rat Clock mRNA was detected. The circadian expression of BMAL1 mRNA was also observed in peripheral tissues such as brain (excluding the SCN), eye, heart, kidney, and lung. The amplitudes of BMAL1 and rPer2 mRNA expression levels were correlated between the different tissues, suggesting that the circadian expression of BMAL1 mRNA plays an important role in generating the circadian expression of per homologue genes in mammals.