RESUMO
Epidemiological studies suggest that the severity of periodontitis is higher in people with diabetes than in healthy individuals. Insulin resistance might play a crucial role in the pathogenesis of multiple diabetic complications and is reportedly induced in the gingiva of rodents with type 2 diabetes; however, the molecular mechanisms underlying the pathogenesis of diabetes-related periodontitis remain unclear. Therefore, we aimed to investigate whether endothelial insulin resistance in the gingiva may contribute to the pathogenesis of periodontitis as well as elucidate its underlying molecular mechanisms. We demonstrated that insulin treatment downregulated lipopolysaccharide (LPS)-induced or tumor necrosis factor α (TNFα)-induced VCAM1 expression in endothelial cells (ECs) via the PI3K/Akt activating pathway, resulting in reduced cellular adhesion between ECs and leukocytes. Hyperglycemia-induced selective insulin resistance in ECs diminished the effect of insulin on LPS- or TNFα-stimulated VCAM1 expression. Vascular endothelial cell-specific insulin receptor knockout (VEIRKO) mice exhibited selective inhibition of the PI3K/Akt pathway in the gingiva and advanced experimental periodontitis-induced alveolar bone loss via upregulation of Vcam1, Tnfα, Mcp-1, Rankl, and neutrophil migration into the gingiva compared with that in the wild-type (WT) mice despite being free from diabetes. We also observed that insulin-mediated activation of FoxO1, a downstream target of Akt, was suppressed in the gingiva of VEIRKO and high-fat diet (HFD)-fed mice, hyperglycemia-treated ECs, and primary ECs from VEIRKO. Further analysis using ECs transfected with intact and mutated FoxO1, with mutations at 3 insulin-mediated phosphorylation sites (T24A, S256D, S316A), suggested that insulin-mediated regulation of VCAM1 expression and cellular adhesion of ECs with leukocytes was attenuated by mutated FoxO1 overexpression. These results suggest that insulin resistance in ECs may contribute to the progression of periodontitis via dysregulated VCAM1 expression and cellular adhesion with leukocytes, resulting from reduced activation of the PI3K/Akt/FoxO1 axis.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Resistência à Insulina , Periodontite , Animais , Camundongos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais , Hiperglicemia/complicações , Insulina/metabolismo , Resistência à Insulina/fisiologia , Lipopolissacarídeos/farmacologia , Periodontite/complicações , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The properties of utricular (UT)-activated vestibular neurons that send axons to the contralateral vestibular nuclei (commissural neurons) were investigated intracellularly or extracellularly in decerebrate cats. A total of 27 vestibular neurons were orthodromically activated by stimulation of UT nerves and antidromically activated by stimulation of the contralateral vestibular nuclei. All neurons tested were classified as vestibulospinal (VS), vestibulooculospinal (VOS), vestibuloocular (VO), and unidentified vestibular neurons (V) after antidromic stimulation of the spinal cord and oculomotor/trochlear nuclei. Most UT-activated commissural neurons (20/27) received monosynaptic inputs. Twelve of 27 commissural neurons were located in the medial vestibular nucleus, 5 were in the lateral vestibular nucleus, 10 were in the descending vestibular nucleus, and no commissural neurons were recorded in the superior vestibular nucleus. Seven of 27 neurons were commissural VS neurons, 9 of 27 were commissural VOS neurons, and 11 of 27 were commissural V neurons. No commissural VO neurons were found. All VOS neurons and 3 VS neurons issued descending axons via the medial vestibulospinal tract. We also studied convergent inputs from the posterior semicircular canal (PC) nerve onto UT-activated commissural neurons. Five of 27 UT-activated commissural neurons received converging inputs from the PC nerves.
Assuntos
Vias Neurais/fisiologia , Sáculo e Utrículo/fisiologia , Núcleos Vestibulares/fisiologia , Animais , Gatos , Estado de Descerebração , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores , Canais Semicirculares/fisiologia , Nervo Vestibular/fisiologiaRESUMO
The convergence between the anterior semicircular canal (AC) and utricular (UT) inputs, as well as the convergence between the AC and saccular (SAC) inputs in single vestibular neurons of decerebrated cats were investigated. Postsynaptic potentials were recorded intracellularly after selective stimulation of each pair of vestibular nerves AC/UT or AC/SAC. Neurons were recorded from the central parts of the vestibular nuclei, where the otolith afferents mainly terminate. Of a total of 105 neurons that were activated after stimulation of the AC and UT nerves, 42 received convergent inputs. Thirty-eight of these neurons received excitatory inputs from both afferents. Convergent neurons were further classified into vestibulospinal (n=28) and vestibulooculospinal (n=6) neurons by antidromic activation from the border between the C1 and C2 spinal cord and the oculomotor or trochlear nucleus. Eight neurons that were not antidromically activated from either site were classified as vestibular neurons. Forty three percent of the convergent vestibulospinal neurons and most of the convergent vestibulooculospinal neurons projected to the spinal cord through the medial vestibulospinal tract. The remaining vestibulospinal and vestibulooculospinal neurons descended through the ipsilateral lateral vestibulospinal tract. Of a total of 118 neurons that were activated after stimulation of the AC and/or SAC nerves, 51 received convergent inputs (27 vestibulospinal, 4 vestibulooculospinal, 5 vestibuloocular and 15 vestibular neurons). Forty-two of the convergent neurons received excitatory inputs from both afferents. Thirty seven percent of the convergent vestibulospinal neurons and all of the convergent vestibulooculospinal neurons projected to the spinal cord through the medial vestibulospinal tract. The remaining vestibulospinal and vestibulooculospinal neurons descended through the ipsilateral lateral vestibulospinal tract.
Assuntos
Vias Aferentes/fisiologia , Neurônios/fisiologia , Membrana dos Otólitos/fisiologia , Canais Semicirculares/fisiologia , Nervo Vestibular/citologia , Animais , Gatos , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/classificação , Membrana dos Otólitos/inervação , Tempo de Reação , Canais Semicirculares/anatomia & histologia , Canais Semicirculares/inervação , Transmissão Sináptica , Nervo Vestibular/fisiologia , Núcleos Vestibulares/citologia , Núcleos Vestibulares/fisiologiaRESUMO
Convergent inputs from the ipsilateral semicircular canal nerves onto single vestibular nucleus neurons were investigated in decerebrate cats using intracellular recording after selective stimulation of each ampullar nerve. One hundred and seventy-four neurons were activated by stimulating the anterior semicircular (AC) and/or posterior semicircular canal (PC) nerves. These neurons were also antidromically stimulated and classified according to the pattern of their collateral projections to the oculomotor complex and the spinal cord. Four types were found: vestibulo-ocular (VO), vestibulospinal (VS), vestibulo-oculospinal (VOS), and vestibular (V) neurons, the latter of which were not activated by stimulation of either the oculomotor complex or the spinal cord. Of 174 AC- and/or PC-activated vestibular nucleus neurons, 32 (18%) received convergent inputs from both nerves. These convergent neurons included 11 VS, 6 VOS, and 15 V neurons. We found no VO neurons with convergent input. The vast majority (82%) of AC/PC-activated VS and VOS convergent neurons received excitatory inputs from both nerves, 12% received reciprocal inputs (i.e., excitatory from one and inhibitory from the other), and the remaining neurons received inhibitory inputs from both nerves. By stimulating the horizontal semicircular (HC) and/or PC nerves, 183 neurons were activated. Of these, 44 (24%) received convergent inputs from both nerves. These convergent neurons included 19 VS, 5 VOS, 2 VO, and 18 V neurons. Approximately one-half (46%) of HC/PC-activated VS and VOS convergent neurons received excitatory inputs from both nerves and 42% received reciprocal inputs, and the remaining neurons received inhibitory inputs from both nerves. In both nerve pairs, the percentage of VS neurons was higher (AC/PC, 34%; HC/PC, 43%) than that of VOS or VO neurons. Approximately half of these convergent neurons were located in the lateral nucleus. These results suggest that, during mixed angular head accelerations, the vestibulocollic reflex may be partly accomplished by VS and VOS convergent neurons.
Assuntos
Neurônios/fisiologia , Canais Semicirculares/fisiologia , Núcleos Vestibulares/fisiologia , Vias Aferentes/fisiologia , Animais , Gatos , Estado de Descerebração , Estimulação Elétrica , Inibição Neural/fisiologia , Reflexo Vestíbulo-Ocular/fisiologia , Canais Semicirculares/inervaçãoRESUMO
PURPOSE: To examine the inhibitory effects of tranilast, ketotifen fumarate, and disodium cromoglycate which are used clinically as anti-allergic agents, on the growth of bovine lens epithelial cells (LE) in culture. METHODS: LE was grown in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide(MTT) and 5-bromo-2'deoxy-uridine(BrdU). Production of collagen, transforming growth factor-beta 1(TGF-beta 1), and basic-fibroblast growth factor(b-FGF) were measured with corresponding enzyme-linked immunosorbent assay(ELISA). Apoptotic cell death was detected by TdT-mediated dUTP-biotin nick end labelling method(TUNEL technique) and the DNA ladder method. RESULTS: Both ketotifen and tranilast inhibited the growth of LE, and half-inhibitory concentrations were 200 microM and 1,000 microM, respectively. Disodium cromoglycate did not inhibit LE proliferation significantly. Ketotifen and tranilast decreased the synthesis of collagen but had no obvious effect on TGF-beta 1 and b-FGF production. Apoptotic cell death was detected in LE treated with ketotifen or tranilast. CONCLUSION: Ketotifen and tranilast may be clinically useful for the prevention of aftercataract. Apoptotic cell death may be involved in the process.
Assuntos
Antialérgicos/farmacologia , Divisão Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Cristalino/citologia , Cristalino/efeitos dos fármacos , Animais , Apoptose , Catarata/prevenção & controle , Bovinos , Células Cultivadas , Cetotifeno/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
We have previously identified the genes expressed early in the differentiation of mouse 3T3-L1 cells into adipocytes. Since these genes were isolated as small fragments, many were unknown. In this study, we have cloned two full-length cDNAs and identified them as p68 RNA helicase and mc3s5/mtCLIC. The expression of these genes was rapidly induced, and specific to the adipocyte differentiation. When the expression of p68 RNA helicase was inhibited using an inducible antisense system, the differentiation into adipocytes was partially blocked, and the expression levels of some marker genes decreased. These findings strongly indicate that the expression of the above two genes was closely related to the adipocyte differentiation, and p68 RNA helicase in particular is crucial to the differentiation.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/fisiologia , Proteínas Quinases/biossíntese , RNA Helicases/biossíntese , Células 3T3 , Adipócitos/citologia , Animais , Clonagem Molecular , RNA Helicases DEAD-box , DNA Complementar/análise , Regulação Enzimológica da Expressão Gênica , Camundongos , Proteínas Quinases/genética , RNA Helicases/genética , RNA Mensageiro/biossíntese , Fatores de TempoRESUMO
We surveyed a total of 570 cerebrospinal fluid (CSF) samples from a variety of diseases, including Alzheimer's disease (AD; n = 236), non-AD-demented and nondemented diseases (n = 239), and normal controls (n = 95) to quantitate levels of tau protein phosphorylated at serine 199 (CSF/phospho-tau199) by a recently established sandwich ELISA. The CSF/phospho-tau199 levels in the AD group were significantly elevated compared to those in all the other non-AD groups. Receiver operating characteristics curves showed that the diagnostic sensitivity and specificity for the AD group vs all the other non-AD groups using the CSF/phospho-tau199 were 85.2% and 85.0%, respectively. Furthermore, there was a significant positive correlation between CSF/phospho-tau199 and CSF/total-tau levels in the AD group. Elevated CSF/phospho-tau199 in the AD group was noted irrespective of age, gender, dementia severity, and number of apolipoprotein E4 alleles. Thus, we suggest that CSF/phospho-tau199 may be a novel and logical biomarker in supporting antemortem diagnosis of AD.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Biomarcadores/líquido cefalorraquidiano , Serina/genética , Proteínas tau/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína E4 , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Curva ROC , Reprodutibilidade dos Testes , Fatores SexuaisRESUMO
The events at the earliest stage of adipocyte differentiation are yet to be fully elucidated. Previously, we cloned the genes that are induced at the beginning of the differentiation of mouse 3T3-L1 preadipocyte cells. We found that the gene expression of regulators of G protein signaling-2 (RGS2) rapidly increased after the addition of inducers and decreased at 3-12 h. The expression pattern of RGS2 mRNAs differed among growth-arrested and proliferating 3T3-L1 cells and NIH-3T3 cells, indicating a specificity for adipogenesis. Here we report that the ectopic expression of RGS2 using a retroviral system in mouse NIH-3T3 cells promotes adipogenesis only in the presence of BRL49653, which is a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). These results strongly suggest that RGS2 play a crucial role in the program of adipocyte differentiation and may contribute to the function of PPARgamma.
Assuntos
Adipócitos/citologia , Ligantes , Proteínas RGS/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Células 3T3 , Adipócitos/metabolismo , Animais , Northern Blotting , Diferenciação Celular , Camundongos , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Rosiglitazona , Tiazóis/farmacologia , Fatores de Tempo , Vasodilatadores/farmacologiaRESUMO
We examined whether otolith-activated second- and third-order vestibular nucleus neurons received commissural inhibition from the contralateral otolithic macula oriented in the same geometric plane. For this purpose we performed intracellular recording in vestibular nucleus neurons after stimulation of the ipsi- and contralateral utricular and saccular nerves. More than half (41/72) of the utricular-activated second-order vestibular nucleus neurons received commissural inhibition from the contralateral utricular nerve. The remaining neurons (31/72) showed no visible response to contralateral utricular nerve stimulation. About half (17/36) of utricular-activated third-order neurons also received commissural inhibition from the contralateral utricular nerve. Approximately 10% (7/67) of saccular-activated second-order vestibular neurons received polysynaptic commissural inhibition, whereas 16% (11/67) received commissural facilitation. The majority (49/67) of saccular second-order vestibular neurons, and almost all (22/23) third-order neurons, showed no visible response to stimulation of the contralateral saccular nerve. The present findings suggest that many utricular-activated vestibular nucleus neurons receive commissural inhibition, which may provide a mechanism for increasing the sensitivity of vestibular neurons to horizontal linear acceleration and lateral tilt of the head. Commissural inhibition in the saccular system was less prominent than in the utricular system.
Assuntos
Neurônios/fisiologia , Membrana dos Otólitos/fisiologia , Núcleos Vestibulares/fisiologia , Animais , Gatos , Eletrofisiologia , Tempo de Reação/fisiologia , Núcleos Vestibulares/citologiaRESUMO
The rat glutathione transferase P (GST-P) gene is strongly induced during chemical hepatocarcinogenesis, whereas mRNA of this gene is rarely expressed in normal rat liver. We previously identified a silencer region in the promoter of this gene. This silencer has several DNA binding sites and at least three proteins (Silencer factor A, -B, and -C (SF-A, SF-B, and SF-C)) bind to these sites. We previously cloned and characterized the Nuclear Factor 1 (NF1) family and the CCAAT/enhancer-binding protein (C/EBP) family as SF-A and SF-B, respectively. However, SF-C which binds to GST-P silencer 2 (GPS2) remains to be cloned. By screening using yeast one-hybrid system, several zinc finger proteins were identified as a candidate of SF-C. The gel-mobility shift analyses showed that BTEB2, EZF, LKLF, TFIIIA, TIEG1, and novel zinc finger protein MZFP bound to GPS2 with different affinities. Several proteins of these are known to be transcriptional activators or repressors, suggesting that zinc finger proteins bind to GPS2 and regulate GST-P expression in the rat liver.
Assuntos
Inativação Gênica , Glutationa Transferase/genética , Dedos de Zinco , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/metabolismo , Primers do DNA , Glutationa Transferase/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Ligação Proteica , Ratos , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: It has long been suspected that duodenogastric reflux plays a role in the pathogenesis of intestinal metaplasia (IM), although recent studies have demonstrated a close association between Helicobacter pylori infection and gastroduodenal diseases, including IM. The objective of this study was to investigate the relation among IM and duodenogastric reflux, H pylori infection, and smoking. METHODS: Subjects with "marked" characteristics of IM, all with extensive prepyloric distribution at endoscopy that was confirmed histologically, were studied as an IM group (27 men, 26 women; mean age, 64 years). A control group was comprised by subjects without characteristics of IM (29 men, 28 women; mean age, 63 years). Fasting pH, total bile acid concentration, and ammonia concentration were measured in the gastric juice of all participants. Histologic examination endoscopic biopsy specimens were evaluated histologically. H pylori infection was determined by serum antibody and urease testing, and by histology. Serum gastrin and pepsinogen concentrations, and gastric emptying time were measured. Dietary, drinking, and smoking habits were recorded. Comparisons were made between groups and analyzed statistically. RESULTS: The pH and total bile acid concentrations were significantly higher in the IM group than the control group (p < 0.01). No significant difference in H pylori infection was found between the IM and control group. Smoking was associated with IM (odds ratio [OR], 15.74; 95% CI, 3.96 to 62.50). CONCLUSIONS: A high pH and total bile acid concentration and smoking were associated with "marked" IM, suggesting that these factors may play a role in the development of IM.
Assuntos
Refluxo Duodenogástrico/complicações , Gastrite/etiologia , Gastrite/patologia , Infecções por Helicobacter , Helicobacter pylori , Antro Pilórico/patologia , Fumar/efeitos adversos , Feminino , Humanos , Masculino , Metaplasia , Pessoa de Meia-IdadeRESUMO
The peroxisome-proliferator-activated receptor gamma (PPARgamma) is a member of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma forms a heterodimer with the retinoid X receptor alpha (RXRalpha) and binds to a common consensus response element consisting of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1 motif). However, other hexamer configurations for binding of PPARgamma have not been considered. By using PCR-mediated random site selection, the DNA sequence preferences for PPARgamma binding were examined. In this study, we have demonstrated that PPARgamma has dual DNA-binding specificity; binding to both the DR1 motif and a palindromic sequence with three bases as spacers (Pal3 motif). The consensus sequence selected by equimolar amounts of PPARgamma and RXRalpha was a perfect DR1 motif, whereas a relatively large population of Pal3 was observed when a 30-fold molar excess of PPARgamma over RXRalpha was used. Gel-shift analysis revealed that the PPARgamma homodimer could bind to Pal3 and that the affinity constant of the PPARgamma homodimer for Pal3 was nearly the same as that of the PPARgamma/RXRalpha heterodimer for DR1. The addition of RXRalpha decreased the binding affinity of PPARgamma for Pal3, indicating that the DNA-binding specificity of PPARgamma could be altered by heterodimer formation with RXRalpha.
Assuntos
DNA/metabolismo , Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Baculoviridae , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , Dimerização , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
The components of the vestibular ascending pathway that transmit otolith information to the thalamus were studied electrophysiologically in anesthetized cats. Thalamic-projecting vestibular neurons (confirmed antidromically) were recorded extracellularly in the various vestibular nuclei. Otolith inputs to these neurons were examined with selective stimulation of the utricular (UT) or the saccular (SAC) nerves. Vestibular nerve branches other than the tested nerve were transected. Of 40 UT-activated vestibulothalamic neurons, 40% (16/40) were activated by UT nerve stimulation with latencies ranging between 0.9-1.4 ms, suggesting they were second-order neurons from the UT nerve. UT-activated vestibulothalamic neurons were recorded in the medial vestibular nucleus (MVN; 24/40), the lateral vestibular nucleus (LVN; 9/40), the descending vestibular nucleus (DVN; 6/40), and the superior vestibular nucleus (SVN; 1/40). Most of the neurons (38/40) were antidromically activated by focal stimulation of the ventral part of the ipsilateral thalamus. Antidromic stimulation of the pontine area revealed that trajectories of the ascending axons (14 of 38 neurons) to the ipsilateral thalamus passed through the pontine reticular formation, ventral to the ascending tract of Deiters (ATD) and the medial longitudinal fasciculus (MLF). Only three SAC-activated vestibulothalamic neurons were encountered in the LVN. All these neurons were second-order neurons from the SAC nerve and were antidromically activated by stimulation of the contralateral thalamus, in marked contrast to the UT-activated vestibulothalamic neurons. Only three UT-activated and two SAC-activated neurons sent descending collaterals to the spinal cord.
Assuntos
Axônios/fisiologia , Vias Neurais/fisiologia , Membrana dos Otólitos/fisiologia , Equilíbrio Postural/fisiologia , Tálamo/fisiologia , Nervo Vestibular/fisiologia , Núcleos Vestibulares/fisiologia , Potenciais de Ação/fisiologia , Animais , Axônios/ultraestrutura , Gatos , Estimulação Elétrica , Potenciais Evocados/fisiologia , Vias Neurais/citologia , Membrana dos Otólitos/citologia , Ponte/citologia , Ponte/fisiologia , Tempo de Reação/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Transmissão Sináptica/fisiologia , Tálamo/citologia , Nervo Vestibular/citologia , Núcleos Vestibulares/citologiaRESUMO
Low density lipoprotein (LDL) receptor-related protein (LRP) gene polymorphisms located in the 5' region and in exon 3, and the apolipoprotein E (APOE) genotype were determined in 100 Japanese patients affected by late-onset Alzheimer's disease (AD). We matched 246 controls for age and found no association between the polymorphism located in the 5' region of the LRP gene. The distribution of LRP exon 3 genotypes and alleles did not differ between AD and the control groups. However, the frequency of T allele in the Alzheimer's group having APOE-epsilon4 was lower than that in the control group having APOE-epsilon4, but it was only marginally significant (p = 0.022). Age of onset was significantly younger in the patients with CC genotype than those carrying the T allele (p = 0.03), and this trend was more evident among non-APOE-epsilon4 carriers (p = 0.008). These results support the possibility that ApoE and LRP may contribute to the development of AD.
Assuntos
Doença de Alzheimer/genética , Polimorfismo Genético , Receptores Imunológicos/genética , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Alelos , Apolipoproteína E4 , Apolipoproteínas E/genética , Estudos de Casos e Controles , Éxons , Genótipo , Humanos , Japão , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Pessoa de Meia-IdadeRESUMO
Nuclear factor 1 (NF1) proteins are encoded by at least four genes (NF1-A, B, C, X). Although DNA-binding and the transcription regulation domains of these proteins are well characterized, the nuclear localization signals (NLSs) are still unknown in all NF1s. We have identified two NLSs in NF1-A, and both are required for full translocation to the nucleus, although one of them itself has a partial translocation ability. These two NLSs are conserved in all four NF1s. Interestingly, three isoforms of NF1-A (NF1-A1, A2, A4) have two NLSs and translocate completely to the nucleus. In contrast, NF1-A3 lacks the second NLS and partially stays in the cytoplasm. Since NF1s construct homodimer and heterodimer, these findings indicate the differential regulations of the NF1 translocation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Sinais de Localização Nuclear/fisiologia , Fatores de Transcrição , Sequência de Aminoácidos , Aminoácidos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Éxons , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Peptídeos/metabolismo , Transporte Proteico , Transfecção , Proteína 1 de Ligação a Y-BoxRESUMO
Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/biossíntese , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Animais , Antioxidantes/farmacologia , Sequência de Bases , Hidroxianisol Butilado/farmacologia , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Antagonistas de Estrogênios/farmacologia , Genes Reporter , Glutationa S-Transferase pi , Substâncias de Crescimento/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Hidroquinonas/farmacologia , Insulina/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Plasmídeos/metabolismo , Bifenilos Policlorados/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Transfecção , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) deficiency is caused by a mutant allele in the Mongoloids. To examine whether genetic constitutions affecting aldehyde metabolism influence the risk for late-onset Alzheimer's disease (LOAD), we performed a case-control study in the Japanese population on the deficiency in ALDH2 caused by the dominant-negative mutant allele of the ALDH2 gene (ALDH2*2). In a comparison of 447 patients with sex, age, and region matched nondemented controls, the genotype frequency carrying the ALDH2*2 allele was significantly higher in the patients than in the controls (48.1% vs 37.4%, P = 0.001). Logistic regression analysis indicates that carriage of the ALDH2*2 allele is an independent risk for LOAD of the epsilon4 allele of the apolipoprotein E gene (APOE-epsilon4) (P = 0.002). Moreover, the odds ratio for LOAD in carriers of the ALDH2*2 allele was almost twice that in noncarriers, irrespective of status with regard to the APOE-epsilon4 allele. Among patients homozygous for the APOE-epsilon4 allele, age at onset of LOAD was significantly lower in those with than without the ALDH2*2 allele. In addition, dosage of the ALDH2*2 allele significantly affected age at onset of patients homozygous for the APOE-epsilon4 allele. These results indicate that the ALDH2 deficiency is a risk for LOAD, synergistically acting with the APOE-epsilon4 allele.
Assuntos
Aldeído Desidrogenase/deficiência , Aldeído Desidrogenase/genética , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Predisposição Genética para Doença/genética , Mitocôndrias/enzimologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Alelos , Doença de Alzheimer/enzimologia , Apolipoproteína E4 , Apolipoproteínas E/genética , Estudos de Casos e Controles , Feminino , Dosagem de Genes , Frequência do Gene/genética , Genes Dominantes/genética , Genótipo , Humanos , Japão/epidemiologia , Modelos Logísticos , Masculino , Mitocôndrias/genética , Razão de ChancesRESUMO
alpha(2u)-Globulin is well known to be a rat protein encoded by a highly homologous multigene family with more than twenty members. We report here the cloning and identification of major alpha(2u)-globulin mRNA species expressed in various tissues. Initially, eight individual clones (PGCL1-8) were obtained from a male preputial gland cDNA library. Data base analysis with BLAST demonstrated six mRNAs to be novel, all clones being characterized by highly conserved sequence motifs as lipocalins. All cDNAs contained an open reading frame of 543 nucleotides and encode 181 amino acid proteins showing 92.5-98.7% and 87.3-98.3% nucleic and amino acid identity, respectively. Denaturing gradient gel electrophoresis (DGGE) with sequence analysis showed that PGCL4 is a major member in the female mammary gland, and in the submaxillary and lachrymal glands of both sexes, while the counterpart in male liver and the coagulate glands was found to be PGCL1. Numbers of cDNA species including PGCL1 and PGCL4 were found in preputial glands, no sex-related difference being observed. These results directly demonstrate complex tissue- and sex-specific expression of alpha(2u)-globulins in terms of mRNA species, providing useful information for understanding regulation of the alpha(2u)-globulin multigene family.
Assuntos
alfa-Globulinas/genética , Perfilação da Expressão Gênica , Família Multigênica/genética , Caracteres Sexuais , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Feminino , Biblioteca Gênica , Masculino , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico/genética , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de SequênciaRESUMO
Convergence between posterior canal (PC) and saccular (SAC) inputs in single vestibular nuclei neurons was investigated in decerebrated cats. Postsynaptic potentials were recorded intracellularly after selective stimulation of the SAC and PC nerves. Stimulation of either the SAC or PC nerve orthodromically activated 143 vestibular nuclei neurons. Of these, 61 (43%) were antidromically activated by stimulation of the C1-C2 junction, 14 (10%) were antidromically activated by stimulation of the oculomotor or trochlear nucleus, and 14 (10%) were antidromically activated by stimulation of both the oculomotor or trochlear nucleus and the spinal cord. Fifty-four (38%) neurons were not activated by stimulation of either or both. We named these neurons vestibulospinal (VS), vestibulo-ocular (VO), vestibulooculo-spinal (VOS) and vestibular (V) neurons, respectively. Both PC and SAC inputs converged in 47 vestibular nuclei neurons (26 VS, 2 VO, 6 VOS and 13 V neurons). Of these, 19 received monosynaptic excitatory inputs from both nerves. This input pattern was frequently seen in VS neurons. Approximately half of the convergent VS neurons descended to the spinal cord through the lateral vestibulospinal tract. The remaining half and all the convergent VOS neurons descended to the spinal cord through the medial vestibulospinal tract. Most of the convergent neurons were located in the lateral nucleus or descending nucleus.