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1.
Plant Biotechnol J ; 22(5): 1325-1334, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38213067

RESUMEN

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.


Asunto(s)
Brassica , Infertilidad Masculina , Masculino , Humanos , Brassica/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Fitomejoramiento , Mitocondrias/genética , Fertilidad/genética , Infertilidad Vegetal/genética
2.
Mol Biol Rep ; 49(9): 8785-8799, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-35809181

RESUMEN

BACKGROUND: Cestrum species present large genomes (2 C = ~ 24 pg), a high occurrence of B chromosomes and great diversity in heterochromatin bands. Despite this diversity, karyotypes maintain the chromosome number 2n = 16 (except when they present B chromosomes), and a relative similarity in chromosome morphology and symmetry. To deepen our knowledge of the Cestrum genome composition, low-coverage sequencing data of C. strigilatum and C. elegans were compared, including cytogenomic analyses of seven species. METHODS AND RESULTS: Bioinformatics analyses showed retrotransposons comprising more than 70% of the repetitive fraction, followed by DNA transposons (~ 17%), but FISH assays using retrotransposon probes revealed inconspicuous and scattered signals. The four satellite DNA families here analyzed represented approximately 2.48% of the C. strigilatum dataset, and these sequences were used as probes in FISH assays. Hybridization signals were colocalized with all AT- and GC-rich sequences associated with heterochromatin, including AT-rich Cold-Sensitive Regions (CSRs). Although satellite probes hybridized in almost all tested species, a satDNA family named CsSat49 was highlighted because it predominates in centromeric regions. CONCLUSIONS: Data suggest that the satDNA fraction is conserved in the genus, although there is variation in the number of FISH signals between karyotypes. Except to the absence of FISH signals with probes CsSat1 and CsSat72 in two species, the other satellites occurred in species of different phylogenetic clades. Some satDNA sequences have been detected in the B chromosomes, indicating that they are rich in preexisting sequences in the chromosomes of the A complement. This comparative study provides an important advance in the knowledge on genome organization and heterochromatin composition in Cestrum, especially on the distribution of satellite fractions between species and their importance for the B chromosome composition.


Asunto(s)
Cestrum , Solanaceae , Animales , Caenorhabditis elegans/genética , Cestrum/genética , ADN Satélite/genética , Heterocromatina/genética , Filogenia , Retroelementos/genética , Solanaceae/genética
3.
Int J Mol Sci ; 21(15)2020 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-32727091

RESUMEN

The RNA-binding proteins (RBPs), some of them induced by transient receptor potential (TRP) ion channels, are crucial regulators of RNA function that can contribute to reproductive pathogenesis, including inflammation and immune dysfunction. This study aimed to reveal the influence of spermatozoa, seminal plasma, or natural mating on mRNA expression of RBPs and TRP ion channels in different segments of the internal genital tract of oestrous, preovulatory sows. Particularly, we focused on mRNA expression changes of the cold-inducible proteins (CIPs) and related TRP channels. Pre-ovulatory sows were naturally mated (NM) or cervically infused with semen (Semen-AI) or sperm-free seminal plasma either from the entire ejaculate (SP-TOTAL) or the sperm-rich fraction (SP-AI). Samples (cervix to infundibulum) were collected by laparotomy under general anaesthesia for transcriptomic analysis (GeneChip® Porcine Gene 1.0 ST Array) 24 h after treatments. The NM treatment induced most of the mRNA expression changes, compared to Semen-AI, SP-AI, and SP-TOTAL treatments including unique significative changes in CIRBP, RBM11, RBM15B, RBMS1, TRPC1, TRPC4, TRPC7, and TRPM8. The findings on the differential mRNA expression on RBPs and TRP ion channels, especially to CIPs and related TRP ion channels, suggest that spermatozoa and seminal plasma differentially modulated both protein families during the preovulatory phase, probably related to a still unknown early signalling mechanism in the sow reproductive tract.


Asunto(s)
Cuello del Útero/metabolismo , Proteínas y Péptidos de Choque por Frío/biosíntesis , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Semen/metabolismo , Conducta Sexual Animal , Porcinos/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Masculino
5.
New Phytol ; 207(1): 34-42, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25704334

RESUMEN

The temperature dependence of plant respiratory rate (R) changes in response to growth temperature. Here, we used a modified Arrhenius model incorporating the temperature dependence of activation energy (Eo ), and compared the temperature dependence of R between cold-sensitive and cold-tolerant species. We analyzed the temperature dependences of leaf CO2 efflux rate of plants cultivated at low (LT) or high temperature (HT). In plants grown at HT (HT plants), Eo at low measurement temperature varied among species, but Eo at growth temperature in HT plants did not vary and was comparable to that in plants grown at LT (LT plants), suggesting that the limiting process was similar at the respective growth temperatures. In LT plants, the integrated value of loge R, a measure of respiratory capacity, in cold-sensitive species was lower than that in cold-tolerant species. When plants were transferred from HT to LT, the respiratory capacity changed promptly after the transfer compared with the other parameters. These results suggest that a similar process limits R at different growth temperatures, and that the lower capacity of the respiratory system in cold-sensitive species may explain their low growth rate at LT.


Asunto(s)
Homeostasis , Modelos Biológicos , Desarrollo de la Planta , Plantas/metabolismo , Temperatura , Adaptación Fisiológica , Respiración de la Célula , Frío , Hojas de la Planta/fisiología , Especificidad de la Especie , Spinacia oleracea/fisiología
6.
Invertebr Reprod Dev ; 59(sup1): 11-16, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-26120246

RESUMEN

Cnidarian Hydra polyps escape senescence, most likely due to the robust activity of their three stem cell populations. These stem cells continuously self-renew in the body column and differentiate at the extremities following a tightly coordinated spatial pattern. Paul Brien showed in 1953 that in one particular species, Hydra oligactis, cold-dependent sexual differentiation leads to rapid aging and death. Here, we review the features of this inducible aging phenotype. These cellular alterations, detected several weeks after aging was induced, are characterized by a decreasing density of somatic interstitial cell derivatives, a disorganization of the apical nervous system, and a disorganization of myofibers of the epithelial cells. Consequently, tissue replacement required to maintain homeostasis, feeding behavior, and contractility of the animal are dramatically affected. Interestingly, this aging phenotype is not observed in all H. oligactis strains, thus providing a powerful experimental model for investigations of the genetic control of aging. Given the presence in the cnidarian genome of a large number of human orthologs that have been lost in ecdysozoans, such approaches might help uncover novel regulators of aging in vertebrates.

7.
J Neurophysiol ; 111(9): 1833-45, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24501261

RESUMEN

Cutaneous (CVC) and muscle (MVC) vasoconstrictor neurons exhibit typical reflex patterns to physiological stimulation of somatic and visceral afferent neurons. Here we tested the hypothesis that CVC neurons are inhibited by stimulation of cutaneous nociceptors but not of muscle nociceptors and that MVC neurons are inhibited by stimulation of muscle nociceptors but not of cutaneous nociceptors. Activity in the vasoconstrictor neurons was recorded from postganglionic axons isolated from the sural nerve or the lateral gastrocnemius-soleus nerve in anesthetized rats. The nociceptive afferents were excited by mechanical stimulation of the toes of the ipsilateral hindpaw (skin), by hypertonic saline injected into the ipsi- or contralateral gastrocnemius-soleus muscle, or by heat or noxious cold stimuli applied to the axons in the common peroneal nerve or tibial nerve. The results show that CVC neurons are inhibited by noxious stimulation of skin but not by noxious stimulation of skeletal muscle and that MVC neurons are inhibited by noxious stimulation of skeletal muscle but not by noxious stimulation of skin. These inhibitory reflexes are mostly lateralized and are most likely organized in the spinal cord. Stimulation of nociceptive cold-sensitive afferents does not elicit inhibitory or excitatory reflexes in CVC or MVC neurons. The reflex inhibition of activity in CVC or MVC neurons generated by stimulation of nociceptive cutaneous or muscle afferents during tissue injury leads to local increase of blood flow, resulting in an increase of transport of immunocompetent cells, proteins, and oxygen to the site of injury and enhancing the processes of healing.


Asunto(s)
Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Nociceptores/fisiología , Reflejo , Piel/inervación , Animales , Masculino , Inhibición Neural , Especificidad de Órganos , Ratas , Ratas Wistar , Médula Espinal/fisiología , Vasoconstricción
8.
Plants (Basel) ; 13(16)2024 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-39204648

RESUMEN

Cold stress affects the seed germination and early growth of winter rapeseed, leading to yield losses. We employed transmission electron microscopy, physiological analyses, metabolome profiling, and transcriptome sequencing to understand the effect of cold stress (0 °C, LW) on the cotyledons of cold-tolerant (GX74) and -sensitive (XY15) rapeseeds. The mesophyll cells in cold-treated XY15 were severely damaged compared to slightly damaged cells in GX74. The fructose, glucose, malondialdehyde, and proline contents increased after cold stress in both genotypes; however, GX74 had significantly higher content than XY15. The pyruvic acid content increased after cold stress in GX74, but decreased in XY15. Metabolome analysis detected 590 compounds, of which 32 and 74 were differentially accumulated in GX74 (CK vs. cold stress) and XY15 (CK vs. cold stressed). Arachidonic acid and magnoflorine were the most up-accumulated metabolites in GX74 subjected to cold stress compared to CK. There were 461 and 1481 differentially expressed genes (DEGs) specific to XY15 and GX74 rapeseeds, respectively. Generally, the commonly expressed genes had higher expressions in GX74 compared to XY15 in CK and cold stress conditions. The expression changes in DEGs related to photosynthesis-antenna proteins, chlorophyll biosynthesis, and sugar biosynthesis-related pathways were consistent with the fructose and glucose levels in cotyledons. Compared to XY15, GX74 showed upregulation of a higher number of genes/transcripts related to arachidonic acid, pyruvic acid, arginine and proline biosynthesis, cell wall changes, reactive oxygen species scavenging, cold-responsive pathways, and phytohormone-related pathways. Taken together, our results provide a detailed overview of the cold stress responses in rapeseed cotyledons.

9.
Neuropeptides ; 100: 102344, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37148733

RESUMEN

Arginine vasopressin (AVP) plays a hypothermic regulatory role in thermoregulation and is an important endogenous mediator in this mechanism. In the preoptic area (POA), AVP increases the spontaneous firing and thermosensitivity of warm-sensitive neurons and decreases those of cold-sensitive and temperature-insensitive neurons. Because POA neurons play a crucial role in precise thermoregulatory responses, these findings indicate that there is an association between the hypothermia and changes in the firing activity of AVP-induced POA neurons. However, the electrophysiological mechanisms by which AVP controls this firing activity remain unclear. Therefore, in the present study, using in vitro hypothalamic brain slices and whole-cell recordings, we elucidated the membrane potential responses of temperature-sensitive and -insensitive POA neurons to identify the applications of AVP or V1a vasopressin receptor antagonists. By monitoring changes in the resting potential and membrane potential thermosensitivity of the neurons before and during experimental perfusion, we observed that AVP increased the changes in the resting potential of 50% of temperature-insensitive neurons but reduced them in others. These changes are because AVP enhances the membrane potential thermosensitivity of nearly 50% of the temperature-insensitive neurons. On the other hand, AVP changes both the resting potential and membrane potential thermosensitivity of temperature-sensitive neurons, with no differences between the warm- and cold-sensitive neurons. Before and during AVP or V1a vasopressin receptor antagonist perfusion, no correlation was observed between changes in the thermosensitivity and membrane potential of all neurons. Furthermore, no correlation was observed between the thermosensitivity and membrane potential thermosensitivity of the neurons during experimental perfusion. In the present study, we found that AVP induction did not result in any changes in resting potential, which is unique to temperature-sensitive neurons. The study results suggest that AVP-induced changes in the firing activity and firing rate thermosensitivity of POA neurons are not controlled by resting potentials.


Asunto(s)
Arginina Vasopresina , Área Preóptica , Ratas , Animales , Potenciales de la Membrana , Temperatura , Arginina Vasopresina/farmacología , Ratas Sprague-Dawley , Neuronas
10.
Cell Metab ; 34(6): 888-901.e5, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35675799

RESUMEN

Homeostatic thermogenesis is an essential protective feature of endotherms. However, the specific neuronal types involved in cold-induced thermogenesis remain largely unknown. Using functional magnetic resonance imaging and in situ hybridization, we screened for cold-sensitive neurons and found preprodynorphin (PDYN)-expressing cells in the dorsal medial region of the ventromedial hypothalamus (dmVMH) to be a candidate. Subsequent in vivo calcium recording showed that cold temperature activates dmVMHPdyn neurons, whereas hot temperature suppresses them. In addition, optogenetic activation of dmVMHPdyn neurons increases the brown adipose tissue and core body temperature, heart rate, and blood pressure, whereas optogenetic inhibition shows opposite effects, supporting their role in homeostatic thermogenesis. Furthermore, we found that dmVMHPdyn neurons are linked to known thermoregulatory circuits. Importantly, dmVMHPdyn neurons also show activation during mouse social interaction, and optogenetic inhibition suppresses social interaction and associated hyperthermia. Together, our study describes dual functions of dmVMHPdyn neurons that allow coordinated regulation of body temperature and social behaviors.


Asunto(s)
Hipertermia Inducida , Interacción Social , Tejido Adiposo Pardo , Animales , Frío , Hipotálamo , Ratones , Neuronas/fisiología , Termogénesis/fisiología
11.
J Pain Res ; 15: 3881-3892, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36536695

RESUMEN

Background: Transient receptor potential channels are the major temperature and nociceptive receptors in the human body and transient receptor potential melastatin 8 (TRPM8) is the cold-sensitive and non-selective cation channel. In our study, we performed a bibliometric analysis of TRPM8 from 2002 to 2021 to summarize the current research status and potential research direction in the future. Methods: The TRPM8-related publications were selected from the Web of Science Core Collection SCI-EXPANDED database from 2002 to 2021. The publication details, such as authors, titles, and author keywords, were used for bibliometric analysis and network visualization to present the current state of TRPM8 research. Results: A total of 1035 articles met the inclusion criteria. The number of TRPM8-related articles has grown rapidly over the past two decades. The USA has the largest number of publications, citations, and international collaborations. The TRPM8-related articles are mainly published and cited in neurological journals, such as the Journal of Neuroscience (41 publications and 2171 local citations). Prevarskaya N. has the most publications (26), and Patapoutian A. has been cited the most (1414 local citations). The popular disciplines in TRPM8 research include Neurosciences and Neurology, Pharmacology and Pharmacy, Biochemistry, and Molecular Biology. Research hotspots are mainly TRP channel, calcium, prostate cancer, proliferation, pain, cold, nociception, and inflammation. Conclusion: Our bibliometric analysis demonstrates that the number of TRPM8 studies has increased from 2002 to 2021. The global research trends and hotspots include the activation mechanism of TRPM8 in neurons, the role of TRPM8 in neuronal and non-neuronal diseases, and therapeutic target research.

12.
Ocul Surf ; 26: 88-96, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35934279

RESUMEN

PURPOSE: Corneal nerves comprise the densest sensory network in the body. Dysfunction of the corneal cold sensitive neurons (CSN) is implicated in ophthalmic disorders, including Dry Eye Disease, the most common ocular surface disorder. The preservative Benzalkonium chloride (BAK) and the mydriatic agent Phenylephrine hydrochloride (PHE) are considered to be inactive at the level of the CSNs. The purpose of this study is to test the impacts of continuous exposures to BAK or PHE at their clinically used concentrations on corneal nerve structure and function. METHODS: In vivo extracellular electrophysiology of the rat trigeminal ganglion was used to monitor CSN functional response to stimuli mimicking physiological states and stressors of the cornea. Corneal nerve structure was evaluated by immunostaining. RESULTS: Among the tested stimuli, cold probe receptive field stimulation and hyperosmolar stress were the most sensitive methods of detecting activity changes. CSN activity was attenuated after 30 min exposure to either PHE or BAK. After an hour-long washout period, BAK-treated neurons failed to recover activity while PHE-treated neurons showed signs of functional recovery. Intraepithelial nerve density was reduced and nerve fragmentation was increased in BAK-treated corneas, while PHE exposure left corneal nerves structurally intact. CONCLUSIONS: Our study suggests that prolonged ocular instillations of BAK or PHE alter CSN activity through two different processes - irreversible neuronal damage in the case of BAK vs. reversible attenuation in the case of PHE.


Asunto(s)
Compuestos de Benzalconio , Síndromes de Ojo Seco , Ratas , Animales , Compuestos de Benzalconio/toxicidad , Conservadores Farmacéuticos , Córnea/inervación , Síndromes de Ojo Seco/inducido químicamente , Soluciones Oftálmicas
13.
Gene ; 764: 145090, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32861880

RESUMEN

Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world. The species often suffers from cold stress. To understand the molecular mechanism of cold tolerance, we performed transcriptomic analysis on two contrasting cultivars of L. vannamei, namely, cold-tolerant Guihai 2 (GH2) and cold-sensitive Guihai1 (GH1), under a control temperature (28 °C), cold stress (16 °C), and recovery to 28 °C. A total of 84.5 Gb of sequences were generated from 12 L. vannamei hepatopancreas libraries. The de-novo assembly generated a total of 143,029 unigenes with a mean size of 1,052 bp and an N50 of 2,604 bp, of which 34.08% were annotated in the Nr database. We analyzed the differentially expressed genes (DEGs) between nine comparison groups and detected a total of 21,026 DEGs. KEGG pathways, including lysosome, sphingolipid metabolism and nitrogen metabolism, were significantly enriched by DEGs between different temperatures in GH2. Furthermore, eight of the most significantly DEGs under cold stress from the transcriptomic analysis were selected for quantitative real-time PCR (qPCR) validation. Overall, we compared gene expression changes under cold stress in cold-tolerant and cold-sensitive L. vannamei for the first time. The results may further extend our understanding of the cold stress-response mechanism in L. vannamei.


Asunto(s)
Aclimatación/genética , Respuesta al Choque por Frío/genética , Hepatopáncreas/metabolismo , Penaeidae/fisiología , Mariscos , Animales , Acuicultura , China , Frío/efectos adversos , Regulación de la Expresión Génica/fisiología , Anotación de Secuencia Molecular , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/genética
14.
Geroscience ; 42(4): 1119-1132, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32578072

RESUMEN

Freshwater polyps of the genus Hydra do not age. However, temperature stress induces aging and a shift from reproduction by asexual budding to sexual gamete production in a cold-sensitive (CS) strain of H. oligactis. We sequenced the transcriptome of a male CS strain before and after this life history shift and compared changes in gene expression relative to those seen in a cold-resistant (CR) strain that does not undergo a life history shift in response to altered temperature. We found that the switch from non-aging asexual reproduction to aging and sexual reproduction involves upregulation of genes not only involved in gametogenesis but also genes involved in cellular senescence, apoptosis, and DNA repair accompanied by a downregulation of genes involved in stem cell maintenance. These results suggest that aging is a byproduct of sexual reproduction-associated cellular reprogramming and underscore the power of these H. oligactis strains to identify intrinsic mechanisms of aging.


Asunto(s)
Hydra , Envejecimiento/genética , Animales , Masculino , Reproducción/genética , Reproducción Asexuada , Células Madre
15.
Front Cell Neurosci ; 14: 598678, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33424555

RESUMEN

Chronic tear deficiency enhances the excitability of corneal cold-sensitive nerves that detect ocular dryness, which can lead to discomfort in patients with dry eye disease (DED). However, changes in corneal nerve excitations through the polymodal nociceptor "transient receptor potential vanilloid 1" (TRPV1) and the potential link between this receptor and symptoms of DED remain unclear. In this study, we examined the firing properties of corneal cold-sensitive nerves expressing TRPV1 and possible contributions of chronic tear deficiency to corneal nerve excitability by TRPV1 activation. The bilateral excision of lacrimal glands in guinea pigs decreased the tear volume and increased the frequency of spontaneous eyeblinks 1-4 weeks after surgery. An analysis of the firing properties of the cold-sensitive nerves was performed by single-unit recordings of corneal preparations 4 weeks after surgery in both the sham-operated and gland-excised groups. Perfusion of the TRPV1 agonist, capsaicin (1 µM), transiently increased the firing frequency in approximately 46-48% of the cold-sensitive nerves characterized by low-background activity and high threshold (LB-HT) cold thermoreceptors in both groups. Gland excision significantly decreased the latency of capsaicin-induced firing in cold-sensitive nerves; however, its magnitude was unchanged. Calcium imaging of cultured trigeminal ganglion neurons from both groups showed that intracellular calcium elevation of corneal neurons induced by a low concentration of capsaicin (0.03 µM) was significantly larger in the gland excision group, regardless of responsiveness to cold. An immunohistochemical study of the trigeminal ganglion revealed that gland excision significantly increased the proportion of corneal neurons enclosed by glial fibrillary acidic protein (GFAP)-immunopositive satellite glial cells. Topical application of the TRPV1 antagonist, A784168 (30 µM), on the ocular surface attenuated eye-blink frequency after gland excision. Furthermore, gland excision enhanced blink behavior induced by a low concentration of capsaicin (0.1 µM). These results suggest that chronic tear deficiency sensitizes the TRPV1-mediated response in the corneal LB-HT cold thermoreceptors and cold-insensitive polymodal nociceptors, which may be linked to dry eye discomfort and hyperalgesia resulting from nociceptive stimuli in aqueous-deficient dry eyes.

16.
G3 (Bethesda) ; 9(5): 1777-1783, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-30914423

RESUMEN

Essential genes cannot be deleted from the genome; therefore, temperature-sensitive (ts) mutants and cold-sensitive (cs) mutants are very useful to discover functions of essential genes in model organisms such as Schizosaccharomyces pombe and Saccharomyces cerevisiae To isolate ts/cs mutants for essential genes of interest, error-prone mutagenesis (or random mutagenesis) coupled with in vitro selection has been widely used. However, this method often introduces multiple silent mutations, in addition to the mutation responsible for ts/cs, with the result that one cannot discern which mutation is responsible for the ts/cs phenotype. In addition, the location of the responsible mutation introduced is random, whereas it is preferable to isolate ts/cs mutants with single amino acid substitutions, located in a targeted motif or domain of the protein of interest. To solve these problems, we have developed a method to isolate ts/cs mutants with single amino acid substitutions in targeted regions using site-directed mutagenesis. This method takes advantage of the empirical fact that single amino acid substitutions (L/S -> P or G/A -> E/D) often cause ts or cs. Application of the method to condensin and cohesin hinge domains was successful: ∼20% of the selected single amino acid substitutions turned out to be ts or cs. This method is versatile in fission yeast and is expected to be broadly applicable to isolate ts/cs mutants with single amino acid substitutions in targeted regions of essential genes. 11 condensin hinge ts mutants were isolated using the method and their responsible mutations are broadly distributed in hinge domain. Characterization of these mutants will be very helpful to understand the function of hinge domain.


Asunto(s)
Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Relación Estructura-Actividad , Temperatura
17.
Exp Anim ; 66(4): 337-343, 2017 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-28626113

RESUMEN

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is associated with sensitivity to cold sensation in mammals. A previous study demonstrated that TRPM8 was overexpressed in the skin of ovariectomized (OVX) rats due to the loss of estrogen. In the present study, we investigated whether estrogen replacement restricts overexpression of the TRPM8 channel in the skin of OVX rats. We divided 15 Sprague Dawley rats into three groups: a non-operated group (NON-OPE), an ovariectomy group (OVX), and a group subjected to estrogen replacement during 4 weeks beginning 7 days after ovariectomy (OVX + E2). Five weeks later, TRPM8 channel mRNA and protein in lumbar skin were quantified by real-time RT-PCR, protein ELISA, and immunohistochemistry. The OVX + E2 group exhibited a trend for decreased expression of the TRPM8 channel in the lumbar skin in comparison with the OVX group, whereas ELISA data and immunohistochemistry data and immunohistochemistry graphs relating to TRPM8 protein did not show any obvious differences between the OVX group and the OVX + E2 group. Estrogen replacement may restrict the overexpression of TRPM8 in the dermis of OVX rats.


Asunto(s)
Frío , Estradiol/administración & dosificación , Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Expresión Génica/efectos de los fármacos , Ovariectomía , Sensación/genética , Sensación/fisiología , Piel/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Animales , Femenino , Posmenopausia/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley
18.
Temperature (Austin) ; 2(2): 158-9, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-27227016

RESUMEN

The Transient Receptor Potential Ankyrin 1 ion channel is heat-sensitive in invertebrate and ancestral vertebrates, cold-sensitive in rodents, and temperature-insensitive in primates. This remarkable divergence in temperature sensitivity is in contrast to its role in sensing electrophilic compounds, which is conserved during animal evolution.

19.
Virology ; 446(1-2): 293-302, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24074593

RESUMEN

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal-terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279-316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase-portal orientation fits better to a proposed "DNA crunching" compression packaging motor and to portal determined DNA headful cutting.


Asunto(s)
Bacteriófago T4/enzimología , Bacteriófago T4/fisiología , Proteínas de la Cápside/metabolismo , Empaquetamiento del ADN , Endodesoxirribonucleasas/metabolismo , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Biología Computacional , Análisis Mutacional de ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Alineación de Secuencia
20.
Artículo en Coreano | WPRIM | ID: wpr-112979

RESUMEN

BACKGROUND: Cold-stress test is used for the objective diagnosis of Raynaud's disease and phenomenon, and the value of such test based on detection of recovery time of finger skin temperature, finger systolic blood pressure and finger color. We evaluate the finger skin temperature of recovery time after cold-stress test for Raynaud's disease and nonspecific cold sensitive patients. METHODS: Thirty-six subjects participated in the study: 9 Raynaud's disease (group 1), 13 nonspecific cold-sensitive patients (group 2) and 14 healthy adults (group 3). Subjects were adapted for 10 min in the test room, temperature 22-24degrees C, prior to measurements. Thermistor probe were attached to tips of both middle phalanges. After recording the baseline temperature, both hands were immersed to the level of the wrist in water controlled at 4degrees C. After 1 min immersion, both hands were removed from the water, recording the skin temperature at 5 min interval. RESULTS: Group 1 had the two types of recovery pattern. One; group 1 (-) was delayed recovery and the other; group 1 (+) was early recovery and hyperemic response. Group 2 showed delayed recovery. Recovery time to preimmersion temperature in group 3 were 20 min. CONCLUSIONS: Unlike results of prior report, some of Raynaud's disease (44%) with typical tricolor change had early recovery and hyperemic response after cold stress test in our study. It is suggested that Raynaud's disease has a different pathophysiology from nonspecific cold-sensitive patients. And appropriate check time is 20 min after cold immersion for the evaluation of vascular dysfunction of cold sensitive patients.


Asunto(s)
Adulto , Humanos , Presión Sanguínea , Diagnóstico , Prueba de Esfuerzo , Dedos , Mano , Inmersión , Enfermedad de Raynaud , Temperatura Cutánea , Piel , Agua , Muñeca
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