ABSTRACT
We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.
ABSTRACT
While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.
Subject(s)
Methamphetamine , Methamphetamine/metabolism , Methamphetamine/pharmacology , Suprachiasmatic Nucleus/metabolism , Motor Activity , Circadian Rhythm/geneticsABSTRACT
Clock gene expression in most organs of the living body exhibits a diurnal rhythm synchronized with the external 24 h light-dark (LD) cycle via circadian pacemaker suprachiasmatic nucleus (SCN). Disturbances in clock gene expression due to desynchronization of clock gene expression of the external LD cycle are risk factors for developing various diseases. Measuring the in vivo clock genes expression rhythm for a long duration under LD conditions can greatly contribute to understand the pathogenic mechanism of the disease caused by the disturbance of the biological rhythm. However, it is presently difficult to continuously measure gene expression for a long duration under LD conditions. In present study, we succeeded in measuring Period1 (Per1) gene expression under LD conditions using ultraviolet (UV) light with filter cut the visible light range. In addition, we succeeded in measuring the kinetic change of liver Per1 gene expression during the process of desynchronization of behavioral rhythm from the LD cycle by chronic administration of methamphetamine (MAP). In the future, by using this system to measure clock gene expression rhythms of brain tissues such as SCN and peripheral tissues under LD conditions, it could contribute to understand the onset mechanism of diseases induced by the desynchronization mechanism of biological rhythm to the LD cycle.
ABSTRACT
The inhalation anesthetic sevoflurane reversibly suppresses Period2 (Per2) mRNA expression in the suprachiasmatic nucleus (SCN). However, a discrepancy exists in phase shifting of the Per2 expression rhythm between sevoflurane application in rats (in vivo application) and explants (ex vivo application). This investigation aimed to resolve this issue. First, tissues from the SCN, choroid plexus in the lateral ventricle (CP-LV), and choroid plexus in the fourth ventricle (CP-4V), which are robust circadian oscillators, and pineal gland (PG) tissue, which is a circadian influencer, were prepared from Per2::dLuc transgenic rats. Significant phase responses of bioluminescence rhythms for different preparation times were monitored in the four tissue explant types. Second, tissue explants were prepared from anesthetized rats immediately after sevoflurane treatment, and bioluminescence rhythms were compared with those from non-anesthetized rats at various preparation times. Regarding bioluminescence rhythm phases, in vivo application of sevoflurane induced phase shifts in CP-LV, CP-4V, and PG explants according to the times that rats were administered anesthesia and the explants were prepared. Phase shifts in these peripheral explants were withdrawn due to the recovery period after the anesthetic treatment, which suggests that peripheral tissues require the assistance of related tissues or organs to correct phase shifts. In contrast, no phase shifts were observed in SCN explants. These results indicated that SCN explants can independently correct bioluminescence rhythm phase. The bioluminescence intensity of explants was also decreased after in vivo sevoflurane application. The suppressive effects on SCN explants were withdrawn due to a recovery day after the anesthetic treatment. In contrast, the suppressive effects on the bioluminescence intensities of CP-LV, CP-4V, and PG explants remained at 30 days after anesthesia administration. These results suggest that anesthetic suppression is imprinted within the peripheral tissues.
ABSTRACT
To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.
Subject(s)
Gene Expression , Luciferases/genetics , Luminescent Measurements/methods , Period Circadian Proteins/genetics , Animals , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Hair/metabolism , Luciferases/metabolism , Luminescent Measurements/instrumentation , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/physiology , Movement/physiology , Period Circadian Proteins/metabolism , Reproducibility of Results , Scalp/metabolism , Skin/cytology , Skin/metabolism , Vibrissae/metabolismABSTRACT
Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Liver/metabolism , Olfactory Bulb/metabolism , Period Circadian Proteins/biosynthesis , Animals , Blood Glucose/drug effects , Body Weight/drug effects , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/physiopathology , Drinking/drug effects , Gene Expression , Hair/drug effects , Hair/metabolism , Locomotion , Mice, Inbred C57BL , Period Circadian Proteins/genetics , Periodicity , StreptozocinABSTRACT
Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l-Luciferin, which is synthesized using racemization of d-luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d-luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.
Subject(s)
Luciferases, Firefly , Luminescent Measurements , Animals , Benzothiazoles , Firefly Luciferin , Gene Expression , Luciferases, Firefly/genetics , MiceABSTRACT
The red alga Chondria armata is known to produce and contain a rich diversity of secondary metabolites, such as domoic acid-related alkaloids and triterpene polyethers. Our investigation on red alga C. armata from Kagoshima coast, Japan, resulted in the isolation of two new triterpene polyethers, bandokorols A (1) and B (2). The structures of these compounds were determined based on spectroscopic data such as infrared (FTIR), 1H-NMR, APT, 1H-1H-COSY, HSQC, HMBC, NOESY and FAB mass spectrometry (HRFABMS). The anticancer potentials of these compounds were tested against adult T-cell leukaemia (ATL), S1T cells and their IC50 values are reported here.
Subject(s)
Rhodophyta , Squalene , Japan , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular StructureABSTRACT
Members of the marine soft coral genus Xenia are rich in a diversity of diterpenes. A total of 199 terpenes consisting of 14 sesquiterpenes, 180 diterpenes, and 5 steroids have been reported to date. Xenicane diterpenes were reported to be the most common chemical skeleton biosynthesized by members of this genus. Most of the literature reported the chemical diversity of Xenia collected from the coral reefs in the South China Sea and the coastal waters of Taiwan. Although there was a brief review on the terpenoids of Xenia in 2015, the present review is a comprehensive overview of the structural diversity of secondary metabolites isolated from soft coral genus Xenia and their potent biological activity as reported between 1977 to 2019.
Subject(s)
Anthozoa/chemistry , Aquatic Organisms/chemistry , Terpenes/isolation & purification , Animals , Steroids/chemistry , Terpenes/chemistryABSTRACT
Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity and cancer. To understand the mechanism of regulating clock gene expression rhythms in vivo, multiple real time recording systems are required. In the present study, we developed a double recording system of Period1 expression rhythm in peripheral tissue (liver) and the brain. In peripheral tissue, quantification of gene expression in a steadily moving target was achieved by using a photomultiplier tube (PMT) attached to a tissue contact optical sensor (TCS). Using this technique, we were able to analyze circadian rhythms of clock gene expression over a prolonged period in the liver and olfactory bub (OB) of the brain. The present double recording system has no effect on behavioral activity or rhythm. Our novel system thus successfully quantifies clock gene expression in deep areas of the body in freely moving mice for a period sufficient to analyze circadian dynamics. In addition, our double recording system can be widely applied to many areas of biomedical research, as well as applications beyond medicine.
Subject(s)
Circadian Rhythm/physiology , Light Signal Transduction , Liver/physiology , Olfactory Bulb/physiology , Period Circadian Proteins/genetics , Suprachiasmatic Nucleus/physiology , Animals , Circadian Rhythm/radiation effects , Electrodes, Implanted , Gene Expression Regulation , Genes, Reporter , Light , Liver/radiation effects , Luciferases/genetics , Luciferases/metabolism , Mice , Mice, Transgenic , Movement/physiology , Olfactory Bulb/radiation effects , Optogenetics , Period Circadian Proteins/metabolism , Stereotaxic Techniques , Suprachiasmatic Nucleus/radiation effectsABSTRACT
Because the disruption of circadian clock gene is a risk factor in many diseases such as obesity and cancer, it is important to monitor and analyzed the expression of the rhythm of the clock gene throughout the body over a long period of time. Although we previously reported on a new gene expression analysis system tracking a target position on the body surface of freely moving mice, the experimental apparatus required a large space. We have therefore developed an in vivo recording system using a portable photomultiplier tube (PMT) system attached to an optical fibre. Directly connecting the target area with the device, we could easily measure the photon counts in a very small space. However, little information is known about the characteristics of optical fibres when exposed to twisting/looping in association with a moving mouse and the effect of the surface of optical fibre. In the present study, we report on the characteristics of optical fibres to detect gene expression rhythm in freely moving mice. Using this portable optical device directly connected with a target area, we were able to measure the circadian rhythm of clock gene expression over a prolonged period in freely moving mice in a small space.
Subject(s)
Olfactory Bulb , Suprachiasmatic Nucleus , Animals , Circadian Rhythm/genetics , Fiber Optic Technology , Gene Expression , MiceABSTRACT
Sour citrus are prized for their flavor and fragrance. This work identified the components of the peel oil of Hetsuka-daidai (Citrus sp. hetsukadaidai), a special sour citrus that is native to the southern part of the Osumi peninsula, Kagoshima, Japan. These compounds were compared to those identified from the peels of six other major sour citrus: lime (Citrus latifolia), lemon (Citrus limon), Yuzu (Citrus junos), Kabusu (Citrus aurantium), Kabosu (Citrus sphaerocarpa), and Sudachi (Citrus sudachi). Peel oil contents were analyzed for the duration of four months during harvest season to investigate the differences in peel oil/fragrance during ripening. These results could facilitate the development of preferred flavor and scent profiles using local species.
Subject(s)
Citrus/chemistry , Oils, Volatile/chemistry , Oils, Volatile/isolation & purification , Seasons , Gas Chromatography-Mass Spectrometry , Japan , Oils, Volatile/analysisABSTRACT
5-Fluorouracil (5-FU) is widely used in the treatment of various types of solid cancer. Our study showed that ribosomal protein L11 (RPL11) was a crucial factor affecting sensitivity of gastric cancer to 5-FU, implying that RPL11 expression is a potential biomarker for predicting 5-FU sensitivity. Kaplan-Meier survival analysis indicated that high RPL11 expression in gastric cancer patients treated with 5-FU was significantly associated with good prognosis. It was therefore investigated whether RPL11 affected the sensitivity of gastric cancer against 5-FU using four human gastric cancer cell lines, MKN45 (wild-type TP53 gene), NUGC4 (wild-type), MKN7 (mutated), and KE39 cells (mutated). In vitro assays demonstrated that RPL11 knockdown in gastric cancer cell lines carrying the TP53 wild-type gene attenuated 5-FU-induced cell growth suppression and activation of the P53 pathway, but not in cells carrying mutated TP53, suggesting that 5-FU suppresses tumor progression via RPL11-mediated activation of the P53 pathway in gastric cancer. The present study provides a potential therapeutic strategy for improving 5-FU resistance in gastric cancer by elevating RPL11 expression.
ABSTRACT
Circadian rhythms are a fundamental biological phenomena that control various physiological functions. The suprachiasmatic nucleus (SCN) is a master clock that integrates various peripheral clocks. Recently, the choroid plexus (CP) was reported to be one such peripheral clock, a circadian oscillator that might conversely affect the SCN. Hence, the principle aim of our study was to unravel the circadian oscillator within the CP. Quantitative PCR against rPer1, rPer2, and rBmal1 showed that CP in the lateral ventricle (CP-LV) and fourth ventricle (CP-4V) has a robust circadian oscillator. The phases of the CP oscillator are between those of the pineal gland (PG) and SCN. Bioluminescence monitoring of explants showed that the intrinsic circadian period of CP-LV and CP-4V was approximately 21 h, which is shorter than SCN and PG. It is possible that interaction between oscillators of the CP-LV, CP-4V, PG, and SCN ensures the SCN adopts a stable 24 h rhythm, with each of the regions having an intrinsic oscillator with different phases and periods. In situ hybridization analysis revealed that dusk-to-dawn variation of rPer2 expression was found in epithelial cells of the CP only. Furthermore, the CP circadian oscillator might control cerebrospinal fluid secretion. However, no dusk-to-dawn variation in expression of the water channel, aquaporin 1, was observed. Further investigations are needed to clarify the involvement of circadian rhythm on CP.
Subject(s)
Choroid Plexus/physiology , Circadian Rhythm , Animals , Aquaporin 1/analysis , Aquaporin 1/genetics , CLOCK Proteins/analysis , CLOCK Proteins/genetics , Choroid Plexus/ultrastructure , Gene Expression Regulation , Male , Period Circadian Proteins/analysis , Period Circadian Proteins/genetics , Rats , Rats, Transgenic , Rats, WistarABSTRACT
Gold and silver nanoparticles were prepared from the green tea and black tea extracts of the leaves of Camellia sinensis. The metal nanoparticle solutions were obtained by reacting HAuCl4 or AgNO3 aqueous solutions with aqueous NaHCO3 and tea leaf extracts, which were obtained from used tea leaves at low temperature, under ambient conditions. The nanoparticles were stable at room temperature and had a uniform particle size (Au: â¼10 nm, Ag: â¼30 nm). Nanoparticle-immobilized cotton cloths were then prepared, which displayed high antibacterial activity and a characteristic color, thereby showing potential application as antimicrobial pigments. This study provides a means of utilizing used tea leaves, which would otherwise be considered waste products.
Subject(s)
Anti-Bacterial Agents/chemistry , Camellia sinensis/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Plant Extracts/chemistry , Silver/chemistry , Anti-Bacterial Agents/pharmacology , Colony Count, Microbial , Coloring Agents/chemistry , Coloring Agents/pharmacology , Green Chemistry Technology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/growth & development , Metal Nanoparticles/ultrastructure , Particle Size , Plant Extracts/pharmacology , Plant Leaves/chemistry , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Waste ProductsABSTRACT
One new compound, 12-epi-9-deacetoxyxenicin (1) along with a hydroperoxide product, 12-epi-9-deacetoxy-8-hydroperoxyxenicin (2) and two known sesquiterpenoids (3-4) were isolated from a population of Bornean soft coral Xenia sp. The structures of these secondary metabolites were elucidated based on their spectroscopic data. Compounds 1 and 2 showed cytotoxic activity against ATL cell line, S1T. In addition, compound 3 exhibited hyphal inhibition of Lagenidium thermophilum.
Subject(s)
Anthozoa/chemistry , Diterpenes/isolation & purification , Animals , Cell Line, Tumor , Diterpenes/chemistry , Humans , Lagenidium/drug effects , Malaysia , Molecular StructureABSTRACT
A new xenicane diterpenoid, 15-deoxy-isoxeniolide-A (1) along with four known compounds 9-deoxy-isoxeniolide-A (2), isoxeniolide-A (3), xeniolide-A (4) and coraxeniolide-B (5) were isolated from the Bornean soft coral Xenia sp. The structures of these metabolites were elucidated on the basis of spectral analysis, NMR and HRESIMS. Compound 5 showed cytotoxic activity against ATL cell line, S1T.
Subject(s)
Anthozoa/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Diterpenes/chemistry , Animals , Anthozoa/metabolism , Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , Humans , Leukemia, T-Cell/drug therapy , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Molecular StructureABSTRACT
Preparation, characterization, and stability evaluation of gold nanoparticles stabilized by ω-sulfonylated alkylsulfanylaniline have been described. The particle solution was obtained by the spontaneous reaction of HAuCl4 and ω-sulfonylated alkylsulfanylaniline in boiling water. It showed a deep red color, owing to surface plasmon resonance of the resulting gold nanoparticles. The size and shape of the nanoparticles were pH-dependent, and pH 8 was found to be the most suitable condition to prepare stable nanoparticles with an average size of 11.2 ± 5.9 nm. The resulting particle solution was stable for a wide range of pH (3-13) and in phosphate-buffered saline (PBS) solution. The nanoparticles were storable as dried powder for at least two weeks, and were redispersible in water or PBS to give almost the same absorption spectra as the freshly prepared solution. Nanoparticle modification was achieved by simply adding thiol molecules to the particle solution.
Subject(s)
Chlorides/chemistry , Drug Stability , Gold Compounds/chemistry , Metal Nanoparticles , Buffers , Drug Storage , Hot Temperature , Hydrogen-Ion Concentration , Particle Size , Powders , Sodium Chloride , Solutions , Sulfhydryl Compounds/chemistry , Sulfuric Acid Esters/chemistry , Surface Plasmon Resonance , WaterABSTRACT
A total synthesis of (±)-hyptinin was achieved via a convergent route using the key phosphonate, cyclic ketone, and aryl Grignard components. The 1H and 13C NMR spectra of natural hyptinin did not agree with those of the synthesized compound. In particular, there were considerable differences between the signals assigned to the protons and carbons surrounding the lactone carbonyl group for the natural and synthesized compounds. The NMR data strongly suggested that the naturally occurring compound, hyptinin, was a structural isomer of the synthesized compound. The structure of the natural compound was eventually established as (+)-ß-apopicropodophyllin, based on the synthesis results.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Ketones/chemistry , Lactones/chemical synthesis , Podophyllin/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/chemistry , Lactones/chemistry , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Podophyllin/chemistry , Protons , StereoisomerismABSTRACT
Clock genes are expressed throughout the body, although how they oscillate in unrestrained animals is not known. Here, we show an in vivo imaging technique that enables long-term simultaneous imaging of multiple tissues. We use dual-focal 3D tracking and signal-intensity calibration to follow gene expression in a target area. We measure circadian rhythms of clock genes in the olfactory bulb, right and left ears and cortices, and the skin. In addition, the kinetic relationship between gene expression and physiological responses to experimental cues is monitored. Under stable conditions gene expression is in phase in all tissues. In response to a long-duration light pulse, the olfactory bulb shifts faster than other tissues. In Cry1(-/-) Cry2(-/-) arrhythmic mice circadian oscillation is absent in all tissues. Thus, our system successfully tracks circadian rhythms in clock genes in multiple tissues in unrestrained mice.