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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.

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.

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.

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.

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 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.

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.

To investigate the molecular mechanism of the melatonin rhythm in the mammalian retina, we examined the temporal mRNA expression pattern of arylalkylamine (serotonin) N -acetyltransferase (AA-NAT), the rate-limiting enzyme in melatonin synthesis in the rat retina. Rat AA-NAT mRNA was detected exclusively in the retinal photoreceptors in the outer nuclear layer—low during the day and increased more than threefold at night. The nocturnal AA-NAT expression in rat retina was also confirmed by RNase protection and the AA-NAT enzymatic activity. This is the first report to localize the site of AA-NAT mRNA circadian expression in mammalian photoreceptor cells.

This paper describes the localization of newly identified visual pigment opsins in the tiered retina of the Japanese yellow swallowtail Papilio xuthus. We first cloned three cDNAs encoding visual pigment opsins, PxRh1, PxRh2 and PxRh3, and then carried out histological in situ hybridization to localize their mRNAs in the retina. By combining the present data with our previous electrophysiological results, we concluded that both PxRh1 and PxRh2 correspond to visual pigments expressed in photoreceptor cells sensitive in the green wavelength region (green receptors), whereas PxRh3 corresponds to a pigment in red receptors. The in situ hybridization studies showed that some photoreceptor cells express two opsin mRNAs. In the ventral half of the eye, all green receptors in the distal tier were labelled by both PxRh1 and PxRh2 probes. The labelling by the PxRh2 and PxRh3 probes was detected throughout the eye in the proximal tier; in 18 % of ommatidia, the probes labelled the same photoreceptor cell. These results suggest that the possible co-localization of two different visual pigments will broaden the sensitivity spectrum of the photoreceptor cells.

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.

Northern blot analysis revealed the circadian expression of rat period homologue (rPer2) mRNA in peripheral mononuclear leukocytes that have no neuronal connections. The mRNA expression in the suprachiasmatic nucleus (SCN), peripheral mononuclear leukocytes, and other peripheral tissues exhibited prominent, synchronous circadian oscillation with peaks at early night. As we have previously shown the peripheral circadian expression of rPer2 mRNA is abolished in SCN-lesioned rats [Sakamoto, K., Nagase, T. Fukui, H., Horikawa, K., Okada, T., Tanaka, H., Sato, K., Miyake, Y., Ohara, O., Kako, K. and Ishida, N., J. Biol. Chem., 273 (1998) 27039–27042], our findings suggest that some humoral signals, driven by the SCN, mediate the circadian expression of mammalian per homologues in peripheral tissues.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5 � -end single-pass sequences, cDNAs having the signal sequence were selected by PSORT (‘signal sequence trap’). We also applied ‘secretion or membrane protein-related keyword trap’ based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165 (80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by ‘keyword trap’ preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

DNA encoding a fragment of putative arylalkylamine N-acetyltransferase (NAT) of the American cockroach, Periplaneta americana, was amplified by PCR with degenerate primers based on the two peptides previously purified from the testicular system of this species. A full clone was obtained by RACE-PCR. The clone consisted of 89 bp 5'-UTR, 753-bp open reading frame and 712-bp 3'-UTR. The amino acid sequence of 251 residues deduced from this ORF corresponded to the predicted molecular mass of 28.5 kDa. The predicted amino acid sequence had an overall identity of 35% with NAT1 and 27% with NAT2 of Drosophila melanogaster, respectively. Structural analysis revealed that NAT from P. americana contained two motifs characteristic of the NAT superfamily and three conserved regions (C/c-1, D/c-1, D/c-2) distinguishing aaNAT subfamily. Northern blot analysis showed that the mRNA of approximately 1.5 kb was transcribed at a high level in the testicular system, and corresponded to the length of the cDNA, i.e., 1,554 bp. Significant levels of NAT transcript were also detected in the midgut, ovary and the accessory glands and at much lower levels in the fat body and brain. Southern blot analysis suggested the presence of a single copy of the cloned gene in the genome.

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

To investigate the molecular mechanisms in photo-entrainment of circadian rhythms, expression patterns of mouse period1 (mPer1) and mPer2 mRNAs were examined in the suprachiasmatic nucleus (SCN) and skin under a complete light–dark (LD) cycle and a corresponding skeleton photoperiod (SPP). Under the SPP, the robust daily expressions of mPer1 and mPer2 were maintained in the SCN and in the skin. A slower rise time for mPer1 mRNA expression was observed in the SPP compared with that in the LD condition both in the SCN and in the skin, although the expression pattern of mPer2 was not significantly different between the two lighting conditions. The results suggest that the expressions of mPer1 and mPer2 mRNAs are differentially regulated not only in the SCN but also in peripheral tissues. Furthermore, we propose that the persistent expression of mPer2 mRNA is required for the entrainment to the SPP.

To investigate the functional involvement of the pineal gland in circadian expression of the rat period homolog gene (rPer2) in the suprachiasmatic nucleus (SCN) and peripheral tissues, we performed Northern blot analysis in tissues from pinealectomized rats. The ectomy did not have any significant effects on rPer2 mRNA expression patterns both in a daily light-dark condition and in a constant darkness. These results suggest that the rhythmic secretion of pineal melatonin is not essential for the circadian expression of clock genes in the SCN and other peripheral tissues of rats.

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.

The primary structures of two opsins from the brachyuran crab Hemigrapsus sanguineus were deduced from the cDNA nucleotide sequences. Both deduced proteins were composed of 377 amino acid residues and included residues highly conserved in visual pigments of other species, and the proteins were 75 % identical to each other. The distribution of opsin transcripts in the compound eye, determined by in situ hybridization, suggested that the mRNAs of the two opsins were expressed simultaneously in all of the seven retinular cells (R1–R7) forming the main rhabdom in each ommatidium. Two different visual pigments may be present in one photoreceptor cell in this brachyuran crab. The spectral sensitivity of the compound eye was also determined by recording the electroretinogram. The compound eye was maximally sensitive at about 480 nm. These and previous findings suggest that both opsins of this brachyuran crab produce visual pigments with maximal absorption in the blue-green region of the spectrum. Evidence is presented that crustaceans possess multiple pigment systems for vision.

To evaluate the possible contribution of atrial natriuretic peptide (ANP) to the development of glomerular hyperfiltration, we examined the effect of non-peptide competitive antagonist for biological receptors of ANP, HS-142-1, on glomerular filtration rate (GFR) and renal plasma flow (RPF) in diabetic rats. Increased GFR and RPF in diabetic rats were significantly ameliorated by the injection of HS-142-1, while blood pressure remained unchanged. Urinary cyclic GMP excretion was significantly higher in diabetic rats than in control rats and HS-142-1 decreased urinary cGMP excretion significantly. These results indicate that atrial natriuretic peptide contributes to the development of glomerular hyperfiltration and hyperperfusion in diabetes and HS-142-1 might be useful in the treatment of them.