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1.
Nat Commun ; 12(1): 6300, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728609

RESUMO

Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αß-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αß-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

2.
J Pharmacol Sci ; 147(4): 340-347, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34663516

RESUMO

We investigated whether peripheral combination treatment of a sodium-glucose cotransporter 2 (SGLT2) inhibitor and leptin improves glucose metabolism in insulin-dependent diabetes mellitus (IDDM) model mice. Twelve-week-old male C57BL6 mice were intraperitoneally administered a high dose of streptozotocin to produce IDDM. IDDM mice were then divided into five groups: SGLT2 inhibitor treatment alone, leptin treatment alone, leptin and SGLT2 inhibitor co-treatment, untreated IDDM mice, and healthy mice groups. The blood glucose (BG) level at the end of the dark cycle was measured, and a glucose tolerance test (GTT) was performed and compared between the five groups. Leptin was peripherally administered at 20 µg/day using an osmotic pump, and an SGLT2 inhibitor, ipragliflozin, was orally administered at 3 mg/kg/day. Monotherapy with SGLT2 inhibitor or leptin significantly improved glucose metabolism in mice as evaluated by BG and GTT compared with the untreated group, whereas the co-treatment group with SGLT2 inhibitor and leptin further improved glucose metabolism as compared with the monotherapy group. Notably, glucose metabolism in the co-treatment group improved to the same level as that in the healthy mice group. Thus, peripheral combination treatment with leptin and SGLT2 inhibitor improved glucose metabolism in IDDM mice without the use of insulin.

3.
iScience ; 24(10): 103140, 2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34632335

RESUMO

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

4.
Sci Rep ; 11(1): 19296, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588513

RESUMO

Previous studies suggest that signaling by the gamma-aminobutyric acid (GABA) type B receptor (GABABR) is involved in the regulation of binge eating, a disorder which might contribute to the development of obesity. Here, we show that intermittent access to a high fat diet (HFD) induced binge-like eating behavior with activation of dopamine receptor d1 (drd1)-expressing neurons in the caudate putamen (CPu) and nucleus accumbens (NAc) in wild-type (WT) mice. The activation of drd1-expressing neurons during binge-like eating was substantially increased in the CPu, but not in the NAc, in corticostriatal neuron-specific GABABR-deficient knockout (KO) mice compared to WT mice. Treatment with the GABABR agonist, baclofen, suppressed binge-like eating behavior in WT mice, but not in KO mice, as reported previously. Baclofen also suppressed the activation of drd1-expressing neurons in the CPu, but not in the NAc, during binge-like eating in WT mice. Thus, our data suggest that GABABR signaling in CPu neurons expressing drd1 suppresses binge-like consumption during a HFD in mice.

5.
Biochemistry ; 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34323477

RESUMO

Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl aG) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αß-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the RC. The geranylgeraniol side chains of BChl aG coordinated by LH1 ß-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αß-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

6.
Sci Rep ; 11(1): 12873, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145364

RESUMO

The reward system, which consists of dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and caudate-putamen in the striatum, has an important role in the pathogenesis of not only drug addiction but also diet-induced obesity. In the present study, we examined whether signaling through glucocorticoid receptors (GRs) in the reward system affects the rewarding value of a high-fat diet (HFD). To do so, we generated mice that lack functional GRs specifically in dopaminergic neurons (D-KO mice) or corticostriatal neurons (CS-KO mice), subjected the mice to caloric restriction stress conditions, and evaluated the rewarding value of a HFD by conditioned place preference (CPP) test. Caloric restriction induced increases in serum corticosterone to similar levels in all genotypes. While CS-KO as well as WT mice exhibited a significant preference for HFD in the CPP test, D-KO mice exhibited no such preference. There were no differences between WT and D-KO mice in consumption of HFD after fasting or cognitive function evaluated by a novel object recognition test. These data suggest that glucocorticoid signaling in the VTA increases the rewarding value of a HFD under restricted caloric stress.


Assuntos
Dieta Hiperlipídica , Neurônios/metabolismo , Receptores de Glucocorticoides/metabolismo , Recompensa , Transdução de Sinais , Área Tegmentar Ventral/metabolismo , Animais , Condicionamento Clássico , Neurônios Dopaminérgicos/metabolismo , Metabolismo Energético , Jejum , Expressão Gênica , Camundongos , Camundongos Transgênicos , Modelos Animais , Receptores de Glucocorticoides/genética , Área Tegmentar Ventral/citologia
7.
Curr Issues Mol Biol ; 43(1): 107-115, 2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065159

RESUMO

The effects of amino acid variants encoded by the human leukocyte antigen (HLA) class II on the development of classical type 1 diabetes (T1D) and latent autoimmune diabetes in adults (LADA) have not been fully elucidated. We retrospectively investigated the HLA-DRB1 and -DQB1 genes of 72 patients with classical T1D and 102 patients with LADA in the Japanese population and compared the frequencies of HLA-DRB1 and -DQB1 alleles between these patients and the Japanese populations previously reported by another institution. We also performed a blind association analysis with all amino acid positions in classical T1D and LADA, and compared the associations of HLA-DRB1 and -DQB1 amino acid positions in classical T1D and LADA. The frequency of DRß-Phe-13 was significantly higher and those of DRß-Arg-13 and DQß-Gly-70 were significantly lower in patients with classical T1D and LADA than in controls. The frequencies of DRß-His-13 and DQß-Glu-70 were significantly higher in classical T1D patients than in controls. The frequency of DRß-Ser-13 was significantly lower and that of DQß-Arg-70 was significantly higher in LADA patients than in controls. HLA-DRß1 position 13 and HLA-DQß1 position 70 could be critical amino acid positions in the development of classical T1D and LADA.

8.
J Struct Biol ; 213(3): 107748, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34033899

RESUMO

In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S.cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 Å, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S.cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

9.
Sci Rep ; 11(1): 8052, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850226

RESUMO

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother's embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.


Assuntos
Bombyx , Morus , Fotoperíodo , Temperatura , Animais , Ritmo Circadiano , Diapausa , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo
10.
PLoS One ; 16(3): e0248065, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33730054

RESUMO

Excessive sodium salt (NaCl) or fat intake is associated with a variety of increased health risks. However, whether excessive NaCl intake accompanied by a high-fat diet (HFD) affects glucose metabolism has not been elucidated. In this study, C57BL/6J male mice were fed a normal chow diet (NCD), a NCD plus high-NaCl diet (NCD plus NaCl), a HFD, or a HFD plus high-NaCl diet (HFD plus NaCl) for 30 weeks. No significant differences in body weight gain, insulin sensitivity, and glucose tolerance were observed between NCD-fed and NCD plus NaCl-fed mice. In contrast, body and liver weights were decreased, but the weight of epididymal white adipose tissue was increased in HFD plus NaCl-fed compared to HFD-fed mice. HFD plus NaCl-fed mice had lower plasma glucose levels in an insulin tolerance test, and showed higher plasma glucose and lower plasma insulin levels in an intraperitoneal glucose tolerance test compared to HFD-fed mice. The ß-cell area and number of islets were decreased in HFD plus NaCl-fed compared to HFD-fed mice. Increased Ki67-positive ß-cells, and increased expression levels of Ki67, CyclinB1, and CyclinD1 mRNA in islets were observed in HFD-fed but not HFD plus NaCl-fed mice when compared to NCD-fed mice. Our data suggest that excessive NaCl intake accompanied by a HFD exacerbates glucose intolerance, with impairment in insulin secretion caused by the attenuation of expansion of ß-cell mass in the pancreas.


Assuntos
Dieta Hiperlipídica , Intolerância à Glucose/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Cloreto de Sódio na Dieta , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/metabolismo , Metabolismo Energético/fisiologia , Teste de Tolerância a Glucose , Insulina/sangue , Masculino , Camundongos
11.
Neuroscience ; 461: 72-79, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33609637

RESUMO

The reward system plays an important role in the pathogenesis of not only drug addiction, but also diet-induced obesity. Recent studies have shown that insulin and leptin receptor signaling in the ventral tegmental area (VTA) regulate energy homeostasis and that their dysregulation is responsible for obesity and altered food preferences. Although a high-fat diet (HFD) induces inflammation that leads to insulin and leptin resistance in the brain, it remains unclear whether HFD induces inflammation in the VTA. In the present study, we placed male mice on a chow diet or HFD for 3, 7, and 28 days and evaluated the mRNA expression of inflammatory cytokines and microglial activation markers in the VTA. The HFD group showed significantly elevated mRNA expressions of IL1ß at 3 days; tumor necrosis factor-alpha (TNFα), IL1ß, IL6, Iba1, and CD11b at 7 days; and TNFα, IL1ß, Iba1, and CD11b at 28 days. The changes in TNFα were also confirmed in immunohistochemical analysis. Next, after administration of chow or HFD for 7 days, we selected mice with equal weights in both groups. In experiments using these mice, Akt phosphorylation in the VTA was significantly decreased after intracerebroventricular injection of insulin, whereas no change in STAT3 phosphorylation was found with leptin. Taken together, these results suggest that HFD induces inflammation at least partly associated with microglial activation in the VTA leading to insulin resistance, independently of the energy balance. Our data provide new insight into the pathophysiology of obesity caused by a dysfunctional reward system under HFD conditions.


Assuntos
Resistência à Insulina , Animais , Dieta Hiperlipídica/efeitos adversos , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade , Área Tegmentar Ventral
12.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443213

RESUMO

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.


Assuntos
Bombyx/metabolismo , Diapausa/genética , Desenvolvimento Embrionário/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Animais , Bombyx/embriologia , Bombyx/genética , Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Antagonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Proteínas de Insetos/genética , Neuropeptídeos/genética , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Filogenia , Receptores de GABA-A/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
13.
Hum Immunol ; 82(4): 226-231, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33386169

RESUMO

The effects of amino acid variants encoded by human leukocyte antigen (HLA) class II on the development of Graves' disease (GD) and Hashimoto's thyroiditis (HT) have not been fully elucidated. We investigated the HLA-DRB1 genes of 243 GD patients and 82 HT patients in the Japanese population and compared the frequencies of HLA-DRB1 alleles and HLA-DRB1 amino acid variants between these patients and the Japanese populations previously reported by another institution. The frequencies of HLA-DRB1*04:05 and -DRB1*14:03 alleles were significantly higher and those of HLA-DRB1*01:01 and -DRB1*15:02 alleles were lower in GD patients than in controls. The frequencies of HLA-DRB1*08:03 and -DRB1*09:01 alleles were significantly higher and that of the HLA-DRB1*13:02 allele was lower in HT patients than in controls. A blind association analysis with all amino acid positions identified DRß9 and DRß31 for GD and DRß9, DRß13, and DRß21 for HT. The frequency of Glu-9 was significantly higher and that of Cys-9 was lower in GD patients than in controls. The frequencies of Lys-9 and Phe-13 were significantly higher in HT patients than in controls. DRß9 and DRß13 could be critical amino acid positions in the development of GD and HT.


Assuntos
Aminoácidos/genética , Genótipo , Doença de Graves/imunologia , Cadeias HLA-DRB1/genética , Doença de Hashimoto/imunologia , Adulto , Idoso , Alelos , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Doença de Graves/genética , Doença de Hashimoto/genética , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético
14.
Kidney Int ; 98(5): 1179-1192, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33069430

RESUMO

Kidney fibrosis is the common consequence of chronic kidney diseases that inexorably progresses to end-stage kidney disease with organ failure treatable only with replacement therapy. Since transforming growth factor-ß1 is the main player in the pathogenesis of kidney fibrosis, we posed the hypothesis that recombinant thrombomodulin can ameliorate transforming growth factor-ß1-mediated progressive kidney fibrosis and failure. To interrogate our hypothesis, we generated a novel glomerulus-specific human transforming growth factor-ß1 transgenic mouse to evaluate the therapeutic effect of recombinant thrombomodulin. This transgenic mouse developed progressive glomerular sclerosis and tubulointerstitial fibrosis with kidney failure. Therapy with recombinant thrombomodulin for four weeks significantly inhibited kidney fibrosis and improved organ function compared to untreated transgenic mice. Treatment with recombinant thrombomodulin significantly inhibited apoptosis and mesenchymal differentiation of podocytes by interacting with the G-protein coupled receptor 15 to activate the Akt signaling pathway and to upregulate the expression of anti-apoptotic proteins including survivin. Thus, our study strongly suggests the potential therapeutic efficacy of recombinant thrombomodulin for the treatment of chronic kidney disease and subsequent organ failure.


Assuntos
Insuficiência Renal Crônica , Fator de Crescimento Transformador beta1 , Fibrose , Proteínas de Ligação ao GTP , Humanos , Rim/patologia , Proteínas Proto-Oncogênicas c-akt , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Transdução de Sinais , Trombomodulina/genética , Fator de Crescimento Transformador beta1/metabolismo
15.
Nat Commun ; 11(1): 4955, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009385

RESUMO

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αß-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αß-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and ß-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.


Assuntos
Cálcio/metabolismo , Microscopia Crioeletrônica , Complexos de Proteínas Captadores de Luz/ultraestrutura , Peptídeos/metabolismo , Fotossíntese , Sequência de Aminoácidos , Bacterioclorofila A/metabolismo , Sítios de Ligação , Chromatiaceae/metabolismo , Detergentes/química , Dimerização , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos/química , Peptídeos/química , Quinonas/química
16.
Biochem Biophys Res Commun ; 527(3): 716-722, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32423824

RESUMO

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.


Assuntos
Cílios/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Cílios/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p27/genética , Técnicas de Inativação de Genes , Humanos , Epitélio Pigmentado da Retina/metabolismo
17.
J Diabetes Investig ; 11(6): 1434-1447, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32279428

RESUMO

AIMS/INTRODUCTION: Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of non-large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin-induced insulin secretion (IIIS) in these islets. MATERIALS AND METHODS: Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks-of-age were isolated. The islets of fa/fa rats at 12 weeks-of-age were separated into non-large islets (≤200 µm in diameter) and enlarged islets (>300 µm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets. RESULTS: The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for ß-cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets. CONCLUSIONS: The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell-like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age-dependent increase in such islets could contribute to the pathophysiology of obese diabetes.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Incretinas/toxicidade , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Metaboloma/efeitos dos fármacos , Obesidade/fisiopatologia , Neoplasias Pancreáticas/patologia , Animais , Perfilação da Expressão Gênica , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ratos , Ratos Zucker
18.
Neurochem Int ; 136: 104733, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32222288

RESUMO

Glial cells can mediate hypothalamic inflammatory processes induced in response to a high-fat diet (HFD). We used magnetic-activated cell sorting (MACS) to isolate microglia and astrocytes from hypothalamus of mice fed HFD and examined changes in expression of inflammation-related cytokines and markers related to glial cell activation status. Hypothalamus from male C57BL6 mice fed a chow diet (chow) or HFD for 1, 3, or 28 days were collected and microglia and astrocytes were isolated by MACS. After confirming cell viability by fluorescence activated cell sorting, mRNA expression levels of inflammation-related cytokines and markers of glial cell activation status were examined by qRT-PCR, which revealed that both glial cell types isolated by MACS retained specificity. On day 3 of HFD, both CD86 and TNFα mRNA expression was significantly increased in microglia relative to the chow group. In astrocytes, TNFα mRNA expression levels were similar between the chow and HFD groups on day 3, but anti-inflammatory cytokine IL-10 levels were significantly increased. On day 7 of HFD, TNFα expression in microglia decreased to levels comparable to the chow group while that in astrocytes remained unchanged. On day 28 of HFD, TNFα levels were significantly increased in both microglia and astrocytes, which had increased mRNA expression of CD86 and MAO-B, respectively. For both glial cell types, results for TNFα expression assessed by RT-PCR and immunohistochemical analysis were similar. These results indicate that the role of microglia and astrocytes in hypothalamic inflammation under HFD conditions changed with time and these changes were accompanied by changes in the activation status of glial cells. Our data suggest that early after initiating HFD, hypothalamic astrocytes suppress diet-induced inflammation at least in part by secreting IL-10, whereas continued HFD feeding impairs this suppressive function such that both microglia and astrocytes promote hypothalamic inflammation.


Assuntos
Astrócitos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Inflamação/metabolismo , Microglia/metabolismo , Neuroglia/metabolismo , Animais , Gliose/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismo
19.
Nat Commun ; 11(1): 1539, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210242

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Bactérias/imunologia , Fibrose Pulmonar Idiopática/imunologia , Peptídeos/imunologia , Staphylococcus/imunologia , Idoso , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Voluntários Saudáveis , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/microbiologia , Fibrose Pulmonar Idiopática/patologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Peptídeos/análise , Peptídeos/metabolismo , Staphylococcus/metabolismo , Staphylococcus/patogenicidade , Exacerbação dos Sintomas , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia
20.
Biophys Rev ; 12(2): 349-354, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32162215

RESUMO

Recent advances in cryo-electron microscopy (cryo-EM) have enabled protein structure determination at atomic resolutions. Cryo-EM specimens are prepared by rapidly freezing a protein solution on a metal grid coated with a holey carbon film; this results in the formation of an ice film on each hole. The thickness of the ice film is a critical factor for high-resolution structure determination; ice that is too thick degrades the contrast of the protein image while ice that is too thin excludes the protein from the hole or denatures the protein. Therefore, trained researchers need to manually select "good" regions with appropriate ice thicknesses for imaging. To reduce the time spent on such tasks, we developed a deep learning program consisting of a "detector" and a "classifier" to identify good regions from low-magnification EM images. In our method, the holes in a low-magnification EM image are detected via a detector, and the ice image on each hole is classified as either good or bad via a classifier. The detector detected more than 95% of the holes regardless of the type of samples. The classifier was trained for different types of samples because the appropriate ice thickness varies between sample types. The accuracies of the classifiers were 93.8% for a soluble protein sample (ß-galactosidase) and 95.3% for a membrane protein sample (bovine heart cytochrome c oxidase). In addition, we found that a training data set containing ~ 2100 hole images from 300 low-magnification EM images was sufficient to obtain good accuracy, such as higher than 90%. We expect that the throughput of the cryo-EM data collection step will be greatly improved by using our method.

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