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1.
J Neuropathol Exp Neurol ; 79(2): 194-208, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774489

RESUMO

There are reports that depression induced by frontal lobe injury (FLI) has a devastating effect on human mental health. We previously reported that fasciculation and elongation protein zeta-1 (FEZ1) was essential for astrocytic protection of dopamine neurons. Studies of glutamate-glutamine cycle in mental illness have been reported, whereas not from the perspective of astrocytes. This study was designed to investigate the roles of astrocytic FEZ1 and glutamate-glutamine cycle after FLI. A model of FLI was established by inserting a blade into the right frontal lobe of rats. Behavioral tests were used to observe the behavioral changes of FLI rats. Neuropathologic examinations, including immunohistochemistry, were conducted. Behavioral tests showed that FLI decreased exploratory activity. Western blot analysis revealed that the expression of astroglial proteins overall decreased in the initial injury stage, as well as FEZ1. Immunohistochemistry showed a shift of FEZ1 localization from neurons in sham-lesioned rats to astrocytes in FLI rats, and showed the expression profile of glutamate transporter 1 and glutamine synthetase (GS) was consistent with Western blot observation. Our results indicate that astrocytic FEZ1 and glutamate-glutamine cycle dysfunction may be involved in the pathogenesis of depression after FLI.

2.
Life Sci ; : 116866, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518606

RESUMO

Neural stem cells (NSCs) are pluripotent cells that are capable of differentiating into neurons and considered as the most promising cell source for cell replacement therapy. However, the difficulty in inducing neuronal differentiation and maturation from NSCs is a major challenge for their clinical application. Clarifying the molecular mechanisms underlying the neuronal differentiation of NSCs can provide a basis for expanding their uses. Brain 4 (Brn4) is a member of the POU domain family of transcription factors and can induce the neuronal differentiation of NSCs, but its precise function in NSCs is unclear. To address this question, in this study we isolated and expanded radial glial cells (RGCs), a type of NSC, from the cerebral cortex of 14-day embryonic rats and used lentivirus carrying the human Brn4 gene to overexpress Brn4 in these cells. This induced the differentiation of RGCs into neurons and inhibited the expression of C-terminal binding protein 2 (CtBP2), a transcriptional co-repressor. CtBP2 overexpression in RGCs suppressed their differentiation into neurons, whereas CtBP2 knockdown had the opposite effect. These results indicated that Brn4 promoted the neuronal differentiation of NSCs via inhibition of CtBP2 and is a potential tool for generating neurons in cell replacement therapy of neurodegenerative diseases and brain injury.

3.
Int J Oncol ; 51(5): 1439-1448, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29048614

RESUMO

Gliomas are the most common primary brain tumors affecting adults. Four grades of gliomas have been identified, namely, grades I-IV. Brain lipid-binding protein (BLBP), which functions in the intracellular transport of fatty acids, is expressed in all grades of human gliomas. The glioma cells that are cultured in vitro are grouped into the BLBP-positive and BLBP-negative cell lines. In the present study, we found that C6 rat glioma cells was a distinct type of BLBP-negative cell line. Our results confirmed that in the C6 cells, the expression of exogenous BLBP increased the proliferation and percentage of cells in the S phase, in the culture medium containing 10 or 1% FBS. Moreover, exogenous BLBP was found to downregulate the tumor suppressors p21 and p16 in the 1% FBS culture medium, but only p21 in the 10% FBS culture medium. The results of the xenograft model assay showed that exogenous BLBP also stimulated tumor formation and downregulated p21 and p16. In conclusion, our study demonstrated that exogenous BLBP promoted proliferation of the C6 cells in vitro and facilitated tumor formation in vivo. Therefore, BLBP expression in glioma cells may promote cell growth by inhibiting the tumor suppressors.


Assuntos
Neoplasias Encefálicas/genética , Proliferação de Células/genética , Proteína 7 de Ligação a Ácidos Graxos/genética , Glioma/genética , Adulto , Animais , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Glioma/patologia , Humanos , Camundongos , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genética
4.
PLoS One ; 12(1): e0169038, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28052098

RESUMO

AIM OF STUDY: Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. MATERIALS AND METHODS: Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. RESULTS: We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. CONCLUSION: Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tirosina Quinase da Agamaglobulinemia , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células , Fluoruracila/farmacologia , Glioma/genética , Glioma/metabolismo , Glutationa/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação/genética , NADP/metabolismo , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética
5.
Surg Radiol Anat ; 37(9): 1049-54, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25944253

RESUMO

PURPOSE: Conventional surgical therapy for an intercondylar humerus fracture might result in multiple potential complications. Our study was conducted to evaluate the modified anconeus flap approach by adequately exposing the distal humeral articular surface, avoiding osteotomy of the olecranon and transection of the main part of the triceps brachial tendon from the olecranon. METHODS: Preparations of 20 upper limb specimens from adult cadavers were used in this study. We investigated the anatomical features of the distal tendon of the triceps brachii. Then, we designed a modified anconeus flap approach in cadaver specimens combined with the medial paratricipital approach, and we compared the extent of exposure of the distal humeral articular surface between the triceps-reflecting anconeus pedicle approach and this modified approach. RESULTS: The downward neurovascular bundles supplying the anconeus were located far from the intramuscular tendon of the triceps brachii. In addition, the medial head of the triceps was continuous with the anconeus near the lateral epicondyle of the humerus. These anatomical properties could assist in reducing adverse events in surgery. The percentage of the exposed humerus distal articular surface was 42.7% by applying the modified anconeus flap approach combined with the medial paratricipital approach. The modified anconeus flap approach can overcome the shortcomings of osteotomy or triceps transverse and fulfill reduction and internal fixation of most distal humerus intercondylar fractures. CONCLUSIONS: The present study has demonstrated a new approach for adequately exposing the distal humeral articular surface during surgery for an intercondylar humerus fracture. With this modified approach, osteotomy of the olecranon and the separation or transection of the main part of the triceps brachial tendon from the olecranon are not necessarily required. Therefore, we suggest that this novel approach could be applied as the primary surgical approach in intercondylar humerus fracture surgeries if the surgeons are familiar with the regional features of distal tendon of the triceps brachii and anconeus.


Assuntos
Articulação do Cotovelo/anatomia & histologia , Articulação do Cotovelo/cirurgia , Úmero/anatomia & histologia , Olécrano/anatomia & histologia , Retalhos Cirúrgicos , Adulto , Cadáver , Humanos , Úmero/cirurgia , Olécrano/cirurgia
6.
Biochem Cell Biol ; 93(4): 351-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26008822

RESUMO

The study of the genes that are related to the pathogenesis of Parkinson's disease (PD) will improve our understanding of the mechanisms that underlie the development of PD. α-Synuclein is a major protein component of Lewy bodies, which are characteristic structures of PD pathology. Mutations in α-synuclein are closely related to the early onset of autosomal dominant PD. Transgenic flies with mutant α-synuclein (A53T) display neurodegenerative changes that include movement dysfunctions and a loss of dopaminergic neurons in the brain. In the present study, we measured reactive oxygen species (ROS) levels in α-synuclein transgenic flies by monitoring the fluorescence levels of redox-sensitive indicators based on GFP (roGFP) in flies co-expressing roGFP and mutant α-synuclein. We found that the ROS levels were significantly increased in the mutant α-synuclein flies. The elevations in ROS levels were also proportionate to the behavioral disorders and the losses of dopaminergic neurons. We also found that CDDO-Me inhibited the increases in ROS levels in the A53T flies and improved the neurodegenerative changes by activating the Nrf2/antioxidant response element signaling pathway. Selective expression of the Nrf2 homologous gene cncC in the dopaminergic neurons effectively protected against the neurodegenerative phenotype of the A53T α-synuclein flies, compared to the flies that expressed cncC in all neurons. These results indicate that the reductions in oxidative stress that are mediated by the activation of the antioxidant signaling pathway can effectively attenuate the neurotoxicity caused by mutations in α-synuclein.


Assuntos
Proteínas de Drosophila/genética , Fator 2 Relacionado a NF-E2/genética , Doenças Neurodegenerativas/genética , Proteínas Repressoras/genética , alfa-Sinucleína/genética , Animais , Animais Geneticamente Modificados , Drosophila , Feminino
7.
Acta Neurobiol Exp (Wars) ; 74(1): 33-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24718042

RESUMO

Oligodendrocyte Precursor Cells (OPCs) can revert to multipotential Neural Stem-Like Cells (NSLCs) which can self-renew and give rise to neurons, astrocytes and oligodendrocytes when exposed to certain extracellular signals. This is a significant progress to understand developmental neurobiology, in particularly the possibility of converting glia to stem cells for the treatment of neurological disorders. Similarly, recent findings revealed that brain-resident microglias (MGs) can be converted to multipotential state through de-differentiation. In this study, we investigated the role of SRY (sex-determining region)-box 2 (SOX2), a high-mobility group DNA binding domain transcription factor, in the reprogramming of OPCs and MGs and molecular pathways involved in these process. Immunocytochemical analyses demonstrated that expression of SOX2 was upregulated in the reprogrammed MGs and OPCs as well as other neural stem cell markers such as CD15 and nestin. Western blot and double immunostaining analyses further confirmed that activation of bone morphogenetic proteins (BMPs) signaling partnering with SOX2 might be one of the molecular pathways involved in lineage reprogramming of OPCs which is also true in the reversion of MGs. Taken together, these results indicated that lineage reprogramming of OPCs and MGs are both controlled by the same signaling pathway and glia can be reprogrammed in culture by inducing expression of neurogenic transcription factors to transgress their lineage restriction and can stably acquire a neuronal identity. Our results suggested innovative perspectives for cell therapy with glia cells.


Assuntos
Proliferação de Células/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Microglia/metabolismo , Oligodendroglia/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Fatores de Crescimento de Fibroblastos/farmacologia , Antígenos CD15/metabolismo , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Nestina/metabolismo , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos
8.
Exp Ther Med ; 7(3): 615-620, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24520255

RESUMO

Human embryonic germ cells (hEGCs) are stem cells cultured from primordial germ cells, which reside in human embryonic genital ridges in vivo. In this study, hEGCs were induced to differentiate into cardiomyocytes by treatment with ascorbic acid in vitro and the effects of hEGC transplantation on rat models of acute myocardial infarction (AMI) were investigated. hEGCs were incubated with differentiation medium containing ascorbic acid at various concentrations. Levels of GATA-4 expression were measured to identify the optimal concentration of the inductor. Immunofluorescence microscopy was used to detect the expression of Cx43 on the induced cells. The hEGCs were injected into the myocardium of rats with AMI. The expression levels of MAB1281 and GATA-4 were used to indicate the survival, migration, distribution and differentiation of transplanted cells. The results revealed the positive expression of GATA-4, Cx43 and cardiac troponin T (cTnT) in differentiated cells, and immunocytochemistry showed that transplanted cells highly expressed GATA-4 and MAB1281. hEGCs were successfully induced to differentiate into cardiomyocytes by ascorbic acid in optimal concentrations in vitro and the transplanted hEGCs survived and differentiated into cardiomyocytes.

9.
Clin Anat ; 27(4): 631-6, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23526687

RESUMO

The aim of this study was to investigate the occurrence and patterns of the communicating branches of cords of the brachial plexus (BPs). This study was performed with 50 fixed adult cadavers (all 100 sides). The BPs were exposed, the presence of the communicating branches of BPs were determined, measured, and photographed. The communicating branches were identified in 27 sides of the BPs. According to enthesis, the communicating branches between the medial and lateral cords (25 sides) were divided into five types. The most common branches connected the lateral cord with the medial root of the median nerve (16 sides). All the communicating branches between the lateral and medial cords obliquely crossed anterior to the axillary artery and passed below the thoracoacromial artery trunk. The distance of the communicating branch with the origin of thoracoacromial artery trunk was 1.60 ± 0.64 cm. The length, transverse diameter, and anteroposterior diameter of communicating branch were 1.67 ± 0.62 cm, 1.77 ± 0.63 mm, and 1.91 ± 0.34 mm, respectively. These anatomical data about the communicating branches will be helpful for surgeons who perform surgical procedures in the cervical and axillary regions.


Assuntos
Plexo Braquial/anatomia & histologia , Feminino , Humanos , Masculino , Valores de Referência
10.
Cell Mol Neurobiol ; 32(6): 1003-10, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22410671

RESUMO

The important role of neuroinflammation in many chronic and acute pathological conditions of the central nervous system is widely recognized. Curcumin is a major component of turmeric and reportedly has anti-inflammatory and anti-oxidant effects. This study investigated the inhibitory effect of curcumin on lipopolysacharide (LPS)-induced chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1) production and whether the effect is mediated by mitogen-activated protein kinases (MAPKs) in the rat astrocytoma cell C6. We observed that LPS (1 µg/ml) induced the upregulation of CCL2 mRNA and protein in C6. Treatment with curcumin (2.5, 10, and 25 µM) decreased the expression of CCL2 mRNA and protein in a dose-dependent manner under treatment with LPS. Additionally, the c-jun N-terminal kinase (JNK) inhibitor (SP600125) dose-dependently inhibited LPS-induced CCL2 upregulation, whereas the MAPK kinase (MEK) inhibitor (PD98059) only had a mild effect and the p38 MAPK inhibitor (SB203580) had no effect. Finally, western blot showed that LPS induced rapid JNK activation and curcumin reduced LPS-induced phosphoJNK (pJNK) expression at 30 min after LPS stimulation. These data suggest that the anti-neuroinflammatory effect of curcumin relates to the downregulation of CCL2 expression through the JNK pathway in astrocytoma cells, which indicates a possible benefit from the use of curcumin in the treatment of neuroinflammation-associated disorders.


Assuntos
Astrocitoma/enzimologia , Quimiocina CCL2/metabolismo , Curcumina/farmacologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Astrocitoma/genética , Astrocitoma/patologia , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
11.
Clin Anat ; 23(7): 811-4, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20533510

RESUMO

The aim of this study was to provide a detailed characterization of the rami communicantes between the stellate (or cervicothoraic) ganglion (CTG) and brachial plexus (BP). Rami communicantes of 33 fixed adult cadavers were macroscopically observed, and connection between CTG and spinal nerves and branching was investigated. In all cases, except one, the hibateral medial rami communicantes was found to be positioned symmetrically between the CTG and C7, C8 spinal nerves. Gray rami communicantes arising from the CTG joined C8, C7, C6 nerve roots on 66, 63, and 6 sides, respectively, and branched from the rami communicantes to C7, C6, C5 nerve roots lying on 51, 41, and 2 sides, respectively. Forty-five sides of the branches from rami communicantes derived from CTG to C8 were observed to ascend through the transverse foramina of the C7 nerve. The branches from rami communicantes derived from CTG to C7 to the C6 nerve were observed ascending through the foramen transversarium of the six cervical vertebrae along with the vertebral artery and joining the C6 spinal nerve in 41 sides. Knowledge about the general distribution and individual variations of the rami communicantes between CTG and BP will be useful toward studies involving the inference of sympathetic nerve stimulation of the upper limbs and could be important for surgeons who perform surgical procedures in the cervical region or medical blockade of nerve fibers.


Assuntos
Plexo Braquial/anatomia & histologia , Gânglio Estrelado/anatomia & histologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Mol Cell Biochem ; 335(1-2): 127-36, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19760487

RESUMO

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT-PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.


Assuntos
Lesões Encefálicas/enzimologia , Lipase/metabolismo , Animais , Encéfalo/enzimologia , Imunofluorescência , Lipase/genética , Masculino , Neurônios/metabolismo , Pâncreas/enzimologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
13.
Mol Cell Biochem ; 325(1-2): 159-67, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19199094

RESUMO

Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear. A DNA microarray was used to identify gene expression in cultured T1As and T2As isolated from postnatal day 1 rat cortex. Ninety-nine of the 138 differentially expressed genes were involved in a diverse number of processes. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes. RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1 and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus. The results contribute to a clearer understanding of the two types of astrocytes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Astrócitos/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Western Blotting , Células Cultivadas , Primers do DNA , Perfilação da Expressão Gênica , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Glycoconj J ; 25(7): 685-701, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18512149

RESUMO

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of beta1,4-galactosylation is performed by a family of beta1,4-galactosyltransferases (beta1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. beta1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides and play roles in sciatic nerve regeneration after sciatic nerve injury. In the present study, the expression of beta1,4-galactosyltransferase (beta1,4-GalT) I, V mRNAs and Galbeta1-4GlcNAc group were examined in rat gastrocnemius muscles after sciatic nerve crush and transection. Real time PCR revealed that beta1,4-GalT I and V mRNAs expressed at a high level in normal gastrocnemius muscles and decreased gradually from 6 h, reached the lowest level at 2 weeks, then restored gradually to relatively normal level at 4 weeks after sciatic nerve crush. In contrast, in sciatic nerve transection model, beta1,4-GalT I and V mRNAs decreased gradually from 6 h, and remained on a low level at 4 weeks in gastrocnemius muscles after sciatic nerve transection. In situ hybridization indicated that beta1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellite cells under normal conditions and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 4 weeks after sciatic nerve transection. Furthermore, lectin blotting showed that the expression level of the Galbeta1-4GlcNAc group decreased from 6 h, reached the lowest level at 2 weeks, and restored to relatively normal level at 4 weeks after sciatic nerve crush. RCA-I lectin histochemistry demonstrated that Galbeta1-4GlcNAc group localized in numerous membranes of myocytes and muscle satellite cells in normal and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 2 and 4 weeks after sciatic nerve transection. These results indicated that the expressions of beta1,4-GalT I, V mRNAs and Galbeta1-4GlcNAc group were involved in the process of denervation and reinnervation, which suggests that beta1,4-GalT I, V mRNAs and Galbeta1-4GlcNAc group may play an important role in the muscle regeneration.


Assuntos
Amino Açúcares/metabolismo , Galactosiltransferases/genética , Músculo Esquelético/enzimologia , Músculo Esquelético/inervação , Neuropatia Ciática/enzimologia , Neuropatia Ciática/genética , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/metabolismo , Injeções Intramusculares , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Lectinas de Plantas/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/enzimologia , Células Satélites de Músculo Esquelético/patologia , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Fatores de Tempo
15.
J Mol Histol ; 39(3): 317-28, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18320333

RESUMO

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of beta-1,4-galactosylation is performed by a family of beta-1,4-galactosyltransferases (beta-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. beta-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the beta-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of beta-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for beta1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that beta1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galbeta1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, beta1,4-GalT I and V might play important roles in the regeneration of the injured sciatic nerve, and upregulation of Galbeta1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.


Assuntos
Acetilglucosamina/metabolismo , Galactosiltransferases/genética , Compressão Nervosa , Nervo Isquiático/enzimologia , Nervo Isquiático/patologia , Animais , Galactosiltransferases/metabolismo , Regulação Enzimológica da Expressão Gênica , Glicoproteínas/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lectinas/metabolismo , Modelos Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
16.
Cell Mol Neurobiol ; 28(2): 223-36, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17712626

RESUMO

beta-1,4-galactosyltransferase I (beta-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte-endothelial cell interaction. The expression of beta-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-alpha (TNF-alpha). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of beta-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected beta-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed beta-1,4-GalT I mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce beta-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected beta-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of beta-1,4-GalT I mRNA in response to inflammation.


Assuntos
Astrócitos/enzimologia , Encefalite/fisiopatologia , Galactosiltransferases/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Astrócitos/citologia , Astrócitos/imunologia , Células Cultivadas , Relação Dose-Resposta a Droga , Encefalite/imunologia , Encefalite/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Lipopolissacarídeos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia
17.
Brain Res ; 1184: 28-37, 2007 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-17980351

RESUMO

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney, etc., indicating a possible role of SSeCKS in inflammatory process. In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of SSeCKS mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected SSeCKS mRNA expression in a time- and dose-dependent manner. Now that LPS induces SSeCKS expression in type-2 astrocytes and type-1 astrocytes are well known to play a pivotal role in immunity, we compared SSeCKS mRNA expression in type-1 astrocytes with type-2 astrocytes after LPS stimulation. Real-time PCR showed that SSeCKS mRNA level was higher in normal untreated type-2 astrocytes than that in normal untreated type-1 astrocytes, increased significantly after 0.1-100 ng/ml LPS stimulation in type-2 astrocytes, but increased weakly after 10-100 ng/ml LPS stimulation in type-1 astrocytes. By using siRNA knockdown of SSeCKS expression, LPS-induced TNF-alpha synthesis and secretion in type-2 astrocytes were partly inhibited, which indicated that SSeCKS played a role in the TNF-alpha biosynthesis in type-2 astrocytes during the stimulation with LPS. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed SSeCKS mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce SSeCKS mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected SSeCKS mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of SSeCKS mRNA in response to inflammation. In addition, SSeCKS production was also drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that type-2 astrocytes which regulated SSeCKS expression after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Astrócitos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Animais , Animais Recém-Nascidos , Astrócitos/classificação , Astrócitos/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Gangliosídeos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/farmacologia , Lipopolissacarídeos/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
18.
J Mol Neurosci ; 33(2): 155-62, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17917074

RESUMO

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of beta-1,4-galactosylation is performed by a family of beta-1, 4-galactosyltransferases (beta-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. beta-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. beta-1,4-GalT I and V mRNAs are present in control astrocytes and affected by TNF-alpha and lipopolysaccharide (LPS) stimuli. In this study, we examined the regulatory mechanisms of tumor necrosis factor-alpha (TNF-alpha)-affected production of beta-1,4-GalT I and V mRNAs. We show here that cultured astrocytes express TNF-alpha receptor 1 (TNFR1) and increased slightly after exposure to LPS. TNF-alpha and TNFR2 are not detected in control astrocytes and upregulated significantly with LPS stimulation and that activation of these receptors by TNF-alpha affects expressions of beta-1,4-GalT I and V mRNAs. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by astrocytes affected beta-1,4-GalT I and V mRNAs production in astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of beta-1,4-GalT I and V mRNA in response to inflammation.


Assuntos
Astrócitos/fisiologia , Galactosiltransferases , RNA Mensageiro/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Astrócitos/citologia , Células Cultivadas , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Lipopolissacarídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética
19.
Int J Oncol ; 23(2): 353-61, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12851684

RESUMO

Astrocytomas are very common intracranial glial cell neoplasms with an inherent tendency to progress. However, the heterogeneity of the morphological features and clinical behavior of the tumors makes accurate prognosis based on the histopathological grading system very difficult. Studies demonstrated that astrocytes have two distinctive cell lineages, and tumors arisen from these two astrocytic lineages have been speculated to have different biological and clinical manifestations. The present study aimed to delineate these two astrocytic lineages in human astrocytomas by using different immunohistochemical markers and to correlate the cell lineages of the tumors with their recurrence. Three markers were used, namely the A2B5 antigen, which is present in type 2 astrocytes but absent in type 1 astrocytes, glial fibrillary acidic protein (GFAP), a marker for astrocytes, and galactocerebroside (GC), a marker for oligodendrocytes. It was found that astrocytomas sharing the A2B5+ lineage (A2B5 positive and GFAP positive) have a significantly higher recurrence rate than the tumors of the A2B5- lineage (A2B5 negative and GFAP positive). Immunohistochemical staining and PCR-single-stranded conformational polymorphism analysis showed that p53 overexpression and p53 mutations were closely associated with the recurrent astrocytomas, and p53 abnormalities were more frequently detected in astrocytomas of the A2B5+ lineage. Quantification of proliferation by counting argyrophil nucleolar organizer regions (AgNORs) indicated a higher AgNOR count in the A2B5+ lineage than the A2B5- lineage. Our findings thus suggest that astrocytomas share similar antigenicity with astrocytes, and that the A2B5+ lineage exhibited a higher recurrence rate than the A2B5- lineage. The higher recurrence rate of the A2B5+ tumors may be in part related to the higher frequency of p53 abnormalities found in the tumors and the higher proliferative activity as reflected by the higher AgNOR count of the tumors.


Assuntos
Antígenos de Neoplasias/metabolismo , Astrocitoma/classificação , Neoplasias Encefálicas/classificação , Linhagem da Célula , Recidiva Local de Neoplasia/patologia , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Divisão Celular , Galactosilceramidas/metabolismo , Gangliosídeos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Técnicas Imunoenzimáticas , Região Organizadora do Nucléolo/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Prata , Proteína Supressora de Tumor p53/metabolismo
20.
Microsc Res Tech ; 60(6): 600-13, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12645008

RESUMO

S100 calcium binding proteins have long been known to express in the adult nervous system, but their distribution in the developing brain, especially the human fetal brain, is largely unknown. We used an immunohistochemical method to determine the expression of three S100 proteins, namely S100A4, S100A5, and S100A13, in the human fetal hippocampus and temporal cortex from 12 to 33 weeks of gestation. At 12 weeks, S100A5 was strongly expressed in the cells and fibers of the polymorphic, pyramidal, and molecular layers of the hippocampus. Thereafter, its expression decreased with age. In the temporal cortex, S100A5 expression was detected from 12 weeks onwards, peaked at 20 to 24 weeks, and then decreased with age. The horizontal fibers of the marginal zone were immunoreactive at all stages examined. S100A13 immunoreactivity was also detected in both cells and fibers of the hippocampus at 12 weeks, became slightly stronger at 20 weeks, and then decreased with age. In the temporal cortex, S100A13 immunoreactivity was also strong in all cellular layers at 12 to 24 weeks before it declined with age from 28 weeks onwards. Among the three proteins examined, S100A4 showed the weakest expression, which was detected in the cells and fibers of the hippocampus and the temporal cortex at all stages examined. Our results have demonstrated for the first time, in the human fetal hippocampus and temporal cortex, specific spatio-temporal patterns of expression of these proteins, all of which are likely to have different roles to play during development despite their pronounced sequence homology.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/metabolismo , Proteínas S100/metabolismo , Lobo Temporal/metabolismo , Feto , Idade Gestacional , Hipocampo/embriologia , Humanos , Imuno-Histoquímica , Proteína A4 de Ligação a Cálcio da Família S100 , Lobo Temporal/embriologia
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