RESUMEN
A total of 100 patients were retrospectively analyzed with magnetic resonance imaging-ultrasonography (MRI-US) fusion biopsy(KOELIS, TRINITY®) at our institution between October 2019 and May 2020. The median patient age was 71 years, median prostate specific antigen (PSA) level was 7.4 ng/ml, and median PSA-density was 0.183 mg/ml. Sixty-one of the patients were positive for cancer ; 14 of them were positive by targeted biopsy only, 9 were positive by systematic biopsy only, and 38 were positive by both. Clinically significant prostate cancer (CPSC ; Gleason Score ≥3ï¼4 and % core ≥50%) was detected by target biopsies in 46 patients and by systematic biopsies in 33 patients. The positive core detection rate for CSPC was 32.5% for targeted biopsies and 7.0% for systematic biopsies(Pï¼0.0001), with a significantly higher rate for targeted biopsies. These results indicate that in MRI-US fusion biopsy, targeted biopsy has a higher detection rate for cancer and a significantly higher detection rate for clinically significant prostate cancer compared with systematic biopsy.
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Próstata , Neoplasias de la Próstata , Anciano , Humanos , Biopsia Guiada por Imagen/métodos , Imagen por Resonancia Magnética/métodos , Masculino , Clasificación del Tumor , Próstata/diagnóstico por imagen , Próstata/patología , Antígeno Prostático Específico , Neoplasias de la Próstata/patología , Estudios Retrospectivos , Ultrasonografía Intervencional/métodosRESUMEN
Orexin-mediated stimulation of orexin receptors 1/2 (OX[1/2]R) may stimulate the diaphragm and genioglossus muscle via activation of inspiratory neurons in the pre-Bötzinger complex, which are critical for the generation of inspiratory rhythm, and phrenic and hypoglossal motoneurons. Herein, we assessed the effects of OX2R-selective agonists TAK-925 (danavorexton) and OX-201 on respiratory function. In in vitro electrophysiologic analyses using rat medullary slices, danavorexton and OX-201 showed tendency and significant effect, respectively, in increasing the frequency of inspiratory synaptic currents of inspiratory neurons in the pre-Bötzinger complex. In rat medullary slices, both danavorexton and OX-201 significantly increased the frequency of inspiratory synaptic currents of hypoglossal motoneurons. Danavorexton and OX-201 also showed significant effect and tendency, respectively, in increasing the frequency of burst activity recorded from the cervical (C3-C5) ventral root, which contains axons of phrenic motoneurons, in in vitro electrophysiologic analyses from rat isolated brainstem-spinal cord preparations. Electromyogram recordings revealed that intravenous administration of OX-201 increased burst frequency of the diaphragm and burst amplitude of the genioglossus muscle in isoflurane- and urethane-anesthetized rats, respectively. In whole-body plethysmography analyses, oral administration of OX-201 increased respiratory activity in free-moving mice. Overall, these results suggest that OX2R-selective agonists enhance respiratory function via activation of the diaphragm and genioglossus muscle through stimulation of inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons. OX2R-selective agonists could be promising drugs for various conditions with respiratory dysfunction.
Asunto(s)
Diafragma , Nervio Hipogloso , Neuronas Motoras , Receptores de Orexina , Nervio Frénico , Animales , Diafragma/efectos de los fármacos , Diafragma/inervación , Diafragma/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Receptores de Orexina/agonistas , Receptores de Orexina/metabolismo , Ratas , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiología , Ratones , Masculino , Nervio Hipogloso/efectos de los fármacos , Nervio Hipogloso/fisiología , Ratas Sprague-Dawley , Inhalación , Bulbo Raquídeo/efectos de los fármacos , Bulbo Raquídeo/fisiología , Isoquinolinas , PiridinasRESUMEN
Orexin A (OX-A) and orexin B are neuropeptides produced in orexin neurons located in the lateral hypothalamus that exert multiple biological functions through the activation of orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) throughout the central nervous system. OX1R and OX2R have distinct functions: OX1R is involved in reward seeking, whereas OX2R has a pivotal role in sleep/wake regulation. OX2R-selective agonists are in development as novel therapeutic agents for the treatment of hypersomnia. However, their potential to induce orexin release, which may indirectly stimulate both OX1R and OX2R in vivo, is unclear. Herein, we assessed the effects of the OX2R-selective agonist TAK-994 on wakefulness and orexin release in monkeys. Oral administration of TAK-994 at 10 mg/kg in the beginning of the sleep phase (zeitgeber time [ZT] 12) significantly increased wakefulness time in monkeys but did not increase OX-A levels in monkey cisternal cerebrospinal fluid (CSF). Moreover, oral administration of TAK-994 (10 mg/kg) during the active phase (ZT1) did not increase OX-A levels in monkey CSF. These findings indicate that the OX2R agonist TAK-994 selectively activates OX2R in vivo and would not robustly induce spontaneous orexin release during the daytime or nighttime in monkeys.
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Receptores Acoplados a Proteínas G , Vigilia , Animales , Receptores de Orexina , Orexinas/farmacología , Macaca fascicularisRESUMEN
Orexin-A (OXA) and -B (OXB) are involved in the regulation of multiple physiological functions including the sleep-wake states; therefore, it is critical to monitor their levels under various conditions. Unfortunately, the widely used radioimmunoassay has insufficient specificity for OXA. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) has higher specificity for OXA, previously reported OXA levels in human cerebrospinal fluid (CSF) measured using this technique are still inconsistent. Moreover, to the best of our knowledge, OXB has not been detected in the CSF. In this study, we established a novel method for OXA and OXB measurement. We noticed that OXA and OXB in the CSF was sticky; thus, citric acid and Tween 80 were used to prevent their nonspecific binding. Then, highly specific and sensitive nanoflow liquid chromatography-high-resolution mass spectrometry (nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation of the diurnal fluctuations of OXA and OXB in cisternal and lumbar CSF samples from cynomolgus monkeys revealed a sharp increase in the early light period, followed by a gradual increase to the maximum levels at the end of the light period, and then a sharp drop to the minimum levels during the early dark period. OXB levels were lower than OXA levels in cisternal CSF. Although basal OXA levels in individual monkeys showed substantial variations, the ratios between the maximum and minimum OXA levels of each monkey were similar. Our method for accurate OXA and OXB measurement should help improve our knowledge of orexin biology.
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Ritmo Circadiano , Orexinas , Animales , Cromatografía Liquida , Macaca fascicularis/metabolismo , Orexinas/líquido cefalorraquídeo , Espectrometría de Masas en TándemRESUMEN
The selective activation of the muscarinic M1 receptor (M1R) may be a promising approach for treating cognitive impairment associated with cholinergic dysfunction. We previously reported that low cooperativity (α-value) is associated with a favorable cholinergic side effect profile of M1R positive allosteric modulators (M1 PAMs), as well as being a crucial factor for the cognitive improvement observed after combining M1 PAMs with donepezil, in rodents. In this study, we preclinically characterized TAK-071, a novel M1 PAM with low cooperativity (α-value = 199), as a new therapy for schizophrenia. We tested TAK-071 in the offspring of polyriboinosinic-polyribocytidylic acid-treated dams, which is a maternal immune activation model of schizophrenia. TAK-071 improved sociability deficits and working memory in this model. In a genetic mouse model of schizophrenia, miR-137 transgenic (Tg) mice, TAK-071 improved deficits in working memory, recognition memory, sociability, and sensorimotor gating. Patients with schizophrenia usually take several antipsychotics to treat positive symptoms. Thus, we also investigated the combined effects of TAK-071 with currently prescribed antipsychotics. Among the 10 antipsychotics tested, only olanzapine and quetiapine showed M1R antagonistic effects, which were counteracted by TAK-071 at possible effective concentrations for cognitive improvement in vitro. Moreover, haloperidol did not affect the ability of TAK-071 to improve working memory in miR-137 Tg mice, suggesting a low risk of losing efficacy when combined with dopamine D2 receptor antagonists. In conclusion, TAK-071 can exert beneficial effects on social behavior and cognitive function and could be a new therapy for schizophrenia.
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Antipsicóticos , Disfunción Cognitiva , Agonistas Muscarínicos , Receptor Muscarínico M1 , Esquizofrenia , Animales , Humanos , Ratones , Regulación Alostérica/efectos de los fármacos , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Células CHO , Cognición/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/genética , Cricetulus , Modelos Animales de Enfermedad , Haloperidol/administración & dosificación , Memoria a Corto Plazo/efectos de los fármacos , Ratones Transgénicos , MicroARNs/genética , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Olanzapina/administración & dosificación , Fumarato de Quetiapina/administración & dosificación , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/metabolismo , Proteínas Recombinantes/metabolismo , Esquizofrenia/complicaciones , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Conducta SocialRESUMEN
The N-methyl-d-aspartate receptor antagonist, ketamine, exhibits rapid and sustained antidepressant activity in patients with treatment-resistant depression (TRD), but its use is associated with psychotomimetic side effects. Evidence has suggested that the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors followed by activation of the mechanistic target of rapamycin (mTOR) signaling pathway and production of brain derived neurotrophic factor (BDNF) protein may underlie the antidepressant efficacy of ketamine. In this study, we characterized the antidepressant-like effects of TAK-653, a novel AMPA receptor potentiator with virtually no agonistic activity. In rat primary cortical neurons, TAK-653 significantly increased phosphorylated and activated forms of mTOR and p70S6 kinase and their upstream regulators Akt and extracellular signal-regulated kinase (ERK). TAK-653 also significantly increased BDNF protein levels in rat primary cortical neurons. Ketamine at 30 mg/kg, i.p. produced antidepressant-like effects in the reduction of submissive behavior model (RSBM) in rats. Ketamine's antidepressant-like effect was blocked by pretreatment with the AMPA receptor antagonist NBQX at 10 mg/kg, i.p., indicating the essential role of AMPA receptor activation in the antidepressant-like effect of ketamine. Consistent with this finding, a sub-chronic administration of TAK-653 for 6 days produced significant antidepressant-like effect in the rat RSBM. Unlike ketamine, however, TAK-653 did not induce a hyperlocomotor response in rats, which is a behavioral index associated with psychotomimetic side effects in humans. TAK-653 may be a promising drug for the treatment of major depressive disorders including TRD with the potential for an improved safety profile compared with ketamine.
Asunto(s)
Antidepresivos/farmacología , Trastorno Depresivo Mayor/tratamiento farmacológico , Receptores AMPA/metabolismo , Tiadiazinas/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trastorno Depresivo Mayor/metabolismo , Trastorno Depresivo Resistente al Tratamiento/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Ketamina/farmacología , Masculino , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tiadiazinas/químicaRESUMEN
The Quaternary Kurobegawa Granite, central Japan, is not only the youngest known granitic pluton exposed on the Earth's surface, it is one of few localities where both Quaternary volcanics and related plutons are well exposed. Here, we present new zircon U-Pb ages together with whole rock and mineral geochemical data, revealing that the Kurobegawa Granite is a resurgent pluton that was emplaced following the caldera-forming eruption of the Jiigatake Volcanics at 1.55 ± 0.09 Ma. Following the eruption, the remnant magma chamber progressively cooled forming the voluminous Kurobegawa pluton in the upper crust (~ 6 km depth) until ~ 0.7 Ma when resurgence caused rapid uplift and erosion in the region. This is the first study to document the detailed spatiotemporal evolution of resurgent pluton for a Quaternary caldera system. Our new findings may contribute significantly to understanding the fate of active caldera systems that can produce supereruptions.
RESUMEN
Persistent epigenetic dysregulation may underlie the pathophysiology of neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here, we show that the inhibition of lysine-specific demethylase 1 (LSD1) enzyme activity normalizes aberrant epigenetic control of gene expression in neurodevelopmental disorders. Maternal exposure to valproate or poly I:C caused sustained dysregulation of gene expression in the brain and ASD-like social and cognitive deficits after birth in rodents. Unexpectedly, a specific inhibitor of LSD1 enzyme activity, 5-((1R,2R)-2-((cyclopropylmethyl)amino)cyclopropyl)-N-(tetrahydro-2H-pyran-4-yl)thiophene-3-carboxamide hydrochloride (TAK-418), almost completely normalized the dysregulated gene expression in the brain and ameliorated some ASD-like behaviors in these models. The genes modulated by TAK-418 were almost completely different across the models and their ages. These results suggest that LSD1 enzyme activity may stabilize the aberrant epigenetic machinery in neurodevelopmental disorders, and the inhibition of LSD1 enzyme activity may be the master key to recover gene expression homeostasis. TAK-418 may benefit patients with neurodevelopmental disorders.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/genética , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Femenino , Histona Demetilasas/metabolismo , HumanosRESUMEN
BACKGROUND: Fibroblast growth factors (FGFs) are promising agents with which to treat problems of skin and hair. But their inability to penetrate into the skin due to their large size and hydrophilic nature prevents their topical application as effective cosmetic ingredients. AIMS: To identify small peptide(s) with FGF-like activity and epidermis permeability. METHODS: Several peptides deduced from our earlier studies were tested for their ability to promote keratinocyte growth and to activate FGF receptors (FGFRs). Permeability was assessed using HPLC after derivatization. RESULTS: A dipeptide, prolyl-isoleucine (Pro-Ile), not only stimulated growth of human keratinocytes, it also moderately activated FGFR3c and FGFR4, and activated FGFR1c to a lesser extent. This receptor specificity of Pro-Ile is similar to that of FGF18. The activity of Pro-Ile toward FGFR/BaF3 cells was enhanced by heparin and was inhibited by an FGFR inhibitor, PD173074. Pro-Ile enhanced the activity of 5 ng/mL FGF18, but suppressed the activity of 50 ng/mL FGF18 toward FGFR3c and FGFR4. Pro-Ile was found to permeate through validated model human epidermis. CONCLUSIONS: These results indicate that the dipeptide Pro-Ile acts as a partial agonist/antagonist for FGFR signaling, that it has receptor specificity similar to FGF18, and that it is able to penetrate into the model epidermis. Because FGFs expressed in the cutaneous system are physiological regulators, these results suggest the potential utility of this peptide as a topically applicable cosmetic ingredient for the regulation of skin physiology, hair growth, and wound healing.
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Cosméticos/farmacología , Dipéptidos/farmacología , Epidermis/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Línea Celular , Humanos , Queratinocitos , Ratones , Permeabilidad , Transducción de SeñalRESUMEN
Axon guidance molecules trigger a cascade of local signal in growth cones and evoke various morphologic responses, including axon attraction, repulsion, elongation, and retraction. However, little is known about whether subcellular compartments, other than axonal growth cones, control axon outgrowth. We found that in isolated dentate granule cells, local application of glutamate to the somatodendritic areas, but not the axon itself, induced rapid axon retraction, during which a calcium wave propagated from the somata to the axon terminals. The calcium wave and axon retraction were both inhibited by blockade of voltage-sensitive calcium channels and intracellular calcium dynamics. A combination of perisomatic application of calcium ionophore and depolarizing current injection induced axonal calcium sweep and axon retraction. Thus, perisomatic environments can modulate axon behavior through long-range intracellular communication.
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Axones/metabolismo , Axones/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Neuronas/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Comunicación Celular , Células Cultivadas , Dendritas/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/fisiología , Hipocampo/citología , Ionóforos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Wistar , Espermina/análogos & derivados , Espermina/farmacologíaRESUMEN
BACKGROUND: The biological activities claimed for placental extract (PE) in its medical and cosmetic applications are largely assumed to be the combined effects of its various signaling molecules and nutritional constituents. But there are considerable uncertainties about this assumption. AIMS: To determine the specific biological activity of PE at a molecular level. METHODS: Fibroblast growth factor (FGF) activity was assessed based on the ability to induce proliferation of FGF receptor (FGFR)-overexpressing BaF3 cells. RESULTS: Porcine PE (PPE), an ingredient in numerous cosmetics, activated proliferation of BaF3 cells overexpressing FGFR subtypes 1c, 2c, 2b, 3c, or 4, that is, all the major FGFR subtypes. The effect was suppressed largely or partially when the cells were treated with a FGFR inhibitor PD173074, and the FGFR-negative BaF3 parent cells exhibited minimal growth promotion as compared to the FGFR-expressing BaF3 cells. The high (>10 kDa) and low (<3 kDa) molecular weight fractions of PPE were effective activators of FGFR signaling. PPE was found to contain sulfated glycosaminoglycans, including heparin/heparan sulfate and chondroitin sulfate, which serve as both structural stabilizers of FGFs and indispensable cofactors for FGF-FGFR signaling. CONCLUSIONS: These results indicate that PPE is capable of evoking FGF signaling in cells via FGFRs. Given that recombinant FGFs have proven useful for medical/cosmetic purposes, our results suggest that the medical/cosmetic utility of PPE is provided at least partly through the activation of FGF signaling in epidermal, dermal, and subdermal tissues.
Asunto(s)
Cosméticos/uso terapéutico , Factores de Crecimiento de Fibroblastos/uso terapéutico , Extractos Placentarios/uso terapéutico , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Envejecimiento de la Piel/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Heparitina Sulfato/farmacología , Humanos , Unión Proteica , Sensibilidad y Especificidad , PorcinosRESUMEN
Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we established an experimental system that can simultaneously monitor the behaviors of axonal and dendritic outgrowth. Our previous study shows that axons and dendrites respond differentially to manipulated cyclic adenosine monophosphate signaling, but we report here that cyclic guanosine monophosphate exerts similar effects on axons and dendrites; that is, both axonal and dendritic growth cones collapsed after activation of cyclic guanosine monophosphate signaling. In addition, nitric oxide donor-induced growth-cone collapse was prevented by the inhibition of cyclic guanosine monophosphate signaling, and this effect again did not differ between axons and dendrites. Thus, unlike cyclic adenosine monophosphate, cyclic guanosine monophosphate modulates extending axons and dendrites in a similar manner.
Asunto(s)
GMP Cíclico/metabolismo , Giro Dentado/citología , Conos de Crecimiento/fisiología , Neuronas/citología , Óxido Nítrico/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Conos de Crecimiento/efectos de los fármacos , Inmunohistoquímica/métodos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Donantes de Óxido Nítrico/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Faloidina/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
A series of benzofuran derivatives with neuroprotective activity in collaboration with IGF-1 was discovered using a newly developed cell-based assay involving primary neural cells prepared from rat hippocampal and cerebral cortical tissues. A structure-activity relationship study identified compound 8 as exhibiting potent activity and brain penetrability. An in vitro pharmacological study demonstrated that although IGF-1 and 8 individually exhibited the neuroprotective effect, the latter acted in collaboration with IGF-1 to enhance neuroprotective activity.
Asunto(s)
Benzofuranos/farmacología , Descubrimiento de Drogas , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Benzofuranos/síntesis química , Benzofuranos/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Estructura Molecular , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Ratas , Relación Estructura-ActividadRESUMEN
The Ca2+ influx controlled by intracellular Ca2+ stores, called store-operated Ca2+ entry (SOC), occurs in various eukaryotic cells, but whether CNS neurons are endowed with SOC capability and how they may operate have been contentious issues. Using Ca2+ imaging, we present evidence for the presence of SOC in cultured hippocampal pyramidal neurons. Depletion of internal Ca2+ stores by thapsigargin caused intracellular Ca2+ elevation, which was prevented by SOC channel inhibitors 2-aminoethoxydiphenyl borate (2-APB), SKF96365, and La3+. Interestingly, these inhibitors also accelerated the decay of NMDA-induced Ca2+ transients without affecting their peak amplitude. In addition, SOC channel inhibitors attenuated tetanus-induced dendritic Ca2+ accumulation and long-term potentiation at Schaffer collateral-CA1 synapses in hippocampal slice preparations. These data suggest a novel link between ionotropic receptor-activated SOC and neuroplasticity.
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Calcio/metabolismo , Potenciación a Largo Plazo , Células Piramidales/metabolismo , Células Piramidales/fisiología , Animales , Compuestos de Boro/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Células Cultivadas , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Giro Dentado/fisiología , Potenciales Postsinápticos Excitadores , Imidazoles/farmacología , Transporte Iónico , Lantano/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The present study demonstrates that ultrasound irradiation can facilitate bacteria-mediated gene delivery (bactofection). Escherichia coli modified with avidin were employed as a vehicle for delivery of the green fluorescent protein (GFP) gene, a model heterologous gene, into the breast cancer cell line MCF-7. Avidin-mediated binding of E. coli to MCF-7 cells enhanced the internalization of E. coli by approximately 17%, irrespective of the use of ultrasound irradiation. Furthermore, the use of ultrasound irradiation increased the internalization by approximately 5%, irrespective of the presence of avidin on the E. coli cell surface. The percentages of GFP-expressing MCF-7 cells at 24h after bactofection were below 0.5% and 2% for the case with only avidin-modification of E. coli cell surface and only ultrasound irradiation, respectively. However, combining avidin modification with the ultrasound treatment increased this value to 8%. Thus, the use of avidin-modified bacteria in conjunction with ultrasound irradiation has potential as an effective strategy for tumor-targeted bactofection.
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Escherichia coli/genética , Escherichia coli/metabolismo , Transfección/métodos , Ultrasonido , Avidina/metabolismo , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/genética , Humanos , Células MCF-7 , Plásmidos/genéticaRESUMEN
An image analyzing method was developed to evaluate in situ bioluminescence expression, without exposing the culture sample to the ambient oxygen atmosphere. Using this method, we investigated the effect of dissolved oxygen concentration on bioluminescence from an obligate anaerobe Bifidobacterium longum expressing bacterial luciferase which catalyzes an oxygen-requiring bioluminescent reaction.
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Bacterias Anaerobias/efectos de los fármacos , Bacterias Anaerobias/metabolismo , Bifidobacterium/efectos de los fármacos , Bifidobacterium/metabolismo , Mediciones Luminiscentes/métodos , Oxígeno/farmacología , Bacterias Anaerobias/genética , Bifidobacterium/genética , Luciferasas/genética , Luciferasas/metabolismo , Oxígeno/metabolismoRESUMEN
Although the quest for Earth's oldest rock is of great importance, identifying the youngest exposed pluton on Earth is also of interest. A pluton is a body of intrusive igneous rock that crystallized from slowly cooling magma at depths of several kilometers beneath the surface of the Earth. Therefore, the youngest exposed pluton represents the most recent tectonic uplift and highest exhumation. The youngest exposed pluton reported to date is the Takidani Granodiorite (~ 1.4 Ma) in the Hida Mountain Range of central Japan. Using LA-ICP-MS and SHRIMP U-Pb zircon dating methods, this study demonstrates that the Kurobegawa Granite, also situated in the Hida Mountain Range, is as young as ~ 0.8 Ma. In addition, data indicate multiple intrusion episodes in this pluton since 10 Ma with a ~ 2-million-year period of quiescence; hence, a future intrusion event is likely within 1 million years.
RESUMEN
Responses to environmental stimuli are mediated by the activation and inactivation of various signalling proteins. However, the temporal dynamics of these events in living animals are not well understood. Here we show real-time imaging of the activity of the key regulator of the MAP kinase pathway, Ras, in living Caenorhabditis elegans and that Ras is transiently activated within a few seconds in olfactory neurons in response to increase in the concentration of odorants. This fast activation of Ras is dependent on the olfactory signalling pathway and Ras guanyl nucleotide-releasing protein (RasGRP). A negative feedback loop then quickly leads to Ras inactivation despite the continued presence of the odorant. Phenotypes of Ras mutants suggest this rapid activation and inactivation of Ras is important for regulation of interneuron activities and olfactory behaviours. Our results reveal novel kinetics and biological implication of transient activation of Ras in olfactory systems.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Genes ras , Vías Olfatorias/fisiología , Activación Transcripcional , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Retroalimentación Fisiológica , Guanilato Ciclasa/metabolismo , Interneuronas/fisiología , Proteínas de la Membrana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas Receptoras Olfatorias/fisiología , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
Although primary cultures of neurons are essential methods for cell biological and pharmacological researches, many animals must be sacrificed for each experiment. Here we introduce a novel system to cryopreserve hippocampal granule cells (GCs) prepared from postnatal rats. Being thawed after as long as 60 days of cryopreservation, GCs expressed the mature neuronal marker MAP-2 and elongated single tau-1-positive axons and multiple tau-1-negative dendrites. These properties closely resembled intact GCs in primary cultures, providing the advantage of being able to repeatedly prepare stable cultures with a single sacrifice of animals.
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Criopreservación/métodos , Hipocampo/citología , Animales , Anticuerpos Monoclonales/análisis , Axones/metabolismo , Células Cultivadas , Dendritas/metabolismo , Expresión Génica , Proteínas Asociadas a Microtúbulos/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we succeeded in simultaneously monitoring the behaviors of axonal and dendritic outgrowth. Our previous study shows that cAMP contributes differentially to Sema3F-induced responses of axons and dendrites, but we report here that the cGMP modulation does not have such a striking axo-dendritic difference. Treatment with Sema3F induced collapse of about 90% growth cones, and pretreatment with 1 muM LY83583, an inhibitor of soluble guanylyl cyclase, partially alleviated the collapse of both axons and dendrites. Thus, unlike cAMP, cGMP modulates axonal and dendritic extension in a similar manner.