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2.
F S Sci ; 3(3): 210-216, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35661817

RESUMO

OBJECTIVE: To evaluate the developmental competency of mouse metaphase II oocytes and the pattern of mitochondrial positioning through cytoplasmic streaming in mouse metaphase II oocytes. DESIGN: We observed cytoplasmic streaming as movement indicated by fluorescently stained mitochondria using a newly developed method in which the spindle is translocated to the opposite site of the oocyte. This method is termed as intracytoplasmic spindle translocation (ICST). SETTING: University research laboratory. ANIMALS: Female B6D2F1 mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fresh oocytes, postovulatory-aged oocytes, and oocytes treated with cytochalasin B were classified based on the presence of cytoplasmic streaming induced by ICST. The pattern of redistributed mitochondria and developmental competence caused by parthenogenetic activation were evaluated in oocytes with or without cytoplasmic streaming. RESULT(S): Induced cytoplasmic streaming occurred in 84% of the fresh oocytes but not in the postovulatory-aged oocytes and the oocytes treated with cytochalasin B. Abnormal mitochondrial aggregation was observed in oocytes in which cytoplasmic streaming was not induced. Furthermore, the developmental competence was significantly lower in oocytes without cytoplasmic streaming. CONCLUSION(S): Cytoplasmic streaming induced by ICST contributes to developmental competence through the redistribution of mitochondria and may be a valuable criterion for predicting early developmental competence in mouse oocytes.


Assuntos
Mitocôndrias , Oócitos , Animais , Citocalasina B/farmacologia , Corrente Citoplasmática , Feminino , Humanos , Camundongos , Partenogênese
3.
Cell Transplant ; 26(7): 1301-1313, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28933223

RESUMO

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease's evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI.


Assuntos
Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/patologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inflamação/genética , Inflamação/patologia , Percussão , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/enzimologia , Forma Celular , Córtex Cerebral/enzimologia , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/complicações , Inflamação/enzimologia , Masculino , Neuroglia/enzimologia , Neuroglia/patologia , Neurônios/enzimologia , Neurônios/patologia , Ratos Wistar
4.
J Histochem Cytochem ; 55(6): 585-95, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17312012

RESUMO

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.


Assuntos
Núcleo Celular/metabolismo , Nucleoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Espermátides/metabolismo , Espermatozoides/metabolismo , Ubiquitina/metabolismo , Animais , Western Blotting , Núcleo Celular/ultraestrutura , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Imunoeletrônica , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/fisiologia , Ratos , Ratos Wistar , Cabeça do Espermatozoide/metabolismo , Espermátides/crescimento & desenvolvimento , Espermátides/ultraestrutura , Espermatozoides/ultraestrutura
5.
J Forensic Sci ; 52(2): 355-63, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17316232

RESUMO

This study presents a reliable method that uses high-fidelity long-range PCR and optimized primers to assess polymorphism and to genotype human mitochondrial DNA (mtDNA). This method was used to analyze polymorphic sites in the human mtDNA control region, including hypervariable regions I, II, and III (HVI, HVII, and HVIII), from 124 unrelated Japanese individuals. In HVI, HVII, and HVIII, 80, 37, and 14 polymorphic sites were identified, respectively, excluding those in the homopolymeric cytosine stretch (C-stretch) regions. The region between HVI and HVII also contained 15 polymorphic sites. On the other hand, C-stretch length heteroplasmy in HVI or HVII was observed in 66 of 124 Japanese individuals (53%), which is much higher than in Caucasian populations. The variants in the C-stretch regions were characterized by counting the number of heteroplasmic peaks split from the single peak in homoplasmic sequences (i.e., 16244G and 16255G in HVI and 285G in HVII). Including the C-stretch length heteroplasmy, the 124 Japanese mtDNA samples were classified into 116 distinct haplotypes. The random match probability and the genetic diversity were estimated to be 0.95% and 0.998581, respectively, indicating that the method presented here has higher discrimination than the conventional method for mtDNA typing using HVI and HVII. [Correction added after publication 30 January 2007: in the preceding sentence random match probability and genetic diversity estimates were corrected from 0.95 and 0.998581%, respectively, to 0.95% and 0.998581, respectively.] The haplogroups and their frequencies observed in this study (i.e., D4; 13.7%, M7a1; 11.3%, D4a; 9.7% and M7b2; 8.9%) were similar to those observed in other studies of Japanese mtDNA polymorphism. The method described here is suitable for forensic applications, as shown by successful analysis of tissues from highly putrefied remains of an infant, which allowed maternal relationship to be determined via mtDNA haplotyping.


Assuntos
Impressões Digitais de DNA/métodos , DNA Mitocondrial/genética , Povo Asiático/genética , Regiões Determinantes de Complementaridade/genética , Genética Forense , Haplótipos , Humanos , Lactente , Japão , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNA
6.
Electrophoresis ; 28(3): 309-16, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17177245

RESUMO

Human complement factor H (factor H) is polymorphic, with five previously reported FH alleles and three previously reported HF alleles (HF*A, HF*B, and HF*Q0). The relationship between the FH and HF alleles is not clear, and the genetic basis of factor H phenotypes has not yet been identified. In this study, nucleotide sequence analysis of complementary DNA (cDNA) from individuals with each HF phenotype identified seven mutated sites in the factor H gene. However, in four cases, the same cDNA sequence was observed in individuals with two different HF phenotypes. Western blotting and 2-DE also showed that a 160 kDa protein corresponding to factor H was expressed in individuals with HF phenotypes. In addition, factor H cross-reacting 45 and 42 kDa polypeptides were detected in individuals with HF A, HF B, or HF AB phenotypes, but not in individuals with the HF Q0 (a null allele) phenotype. Thus, HF phenotype did not correlate well with factor H gene or protein structural variation. Evidence is provided to support the hypothesis that the HF phenotypes do not correspond to polymorphism in factor H, but instead correspond to polymorphism in factor H-related protein 1. A novel PCR-RFLP method was developed and used to detect four polymorphisms (G257A, G1492A, A2089G, and G2881T) in the factor H gene in 54 unrelated Japanese individuals. This method could be useful for studies on genetic disease associated with these mutations.


Assuntos
Alelos , Genoma Humano , Fenótipo , Polimorfismo Genético , Fator H do Complemento/genética , DNA Complementar/genética , Humanos , Polimorfismo de Fragmento de Restrição
7.
Tohoku J Exp Med ; 210(2): 137-44, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17023767

RESUMO

Selection of good quality oocytes is important for improvement of assisted reproductive technology. Here, we studied the relationship of the mitochondrial distribution in metaphase II stage (MII) oocytes with fertility, since mitochondria in ooplasm are essential for energy production required for fertilization and embryo development. To observe mitochondria non-invasively, we used oocytes from a transgenic mouse, in which enhanced green fluorescent protein is targeted to the mitochondrial matrix and thus fluorescence is observed exclusively in the mitochondria. Control oocytes with mitochondria distributed around the nucleus showed normal embryo developmental competence, whereas oocytes with abnormal diffuse and fragmented mitochondria showed a significantly lower rate of embryo development after activation by intracytoplasmic sperm injection or strontium, which is a very effective agent for activation of mouse oocytes. Also, we showed that the reduced developmental competence of oocytes with diffuse and fragmented mitochondria caused by vitrification and thawing is similar to that of oocytes with abnormal mitochondrial foci obtained naturally. These findings suggest that abnormal mitochondrial distribution in oocytes at MII is a cause of developmental retardation and therefore normal mitochondrial distribution could be used as a criterion for selection of good oocytes.


Assuntos
Desenvolvimento Embrionário , Mitocôndrias/patologia , Oócitos/citologia , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Metáfase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/genética , Oócitos/patologia
8.
Neuropsychopharmacology ; 31(12): 2619-26, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16823390

RESUMO

The therapeutic use of interferon-alpha (IFN-alpha), a proinflammatory cytokine, is known to cause various neuropsychiatric adverse effects. In particular, depression occurs in 30-45% of patients, frequently interrupting treatment. IFN-alpha-treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by IFN-alpha remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Therefore, we investigated the effect of subchronic IFN-alpha treatment on neurogenesis in the adult rat dentate gyrus (DG). Immediately after the administration of IFN-alpha for 1 week, a decrease in the number of 5-bromo-deoxyuridine-labeled proliferating cells was observed in the DG; however, no effect was detected on the expression of mature neuronal phenotype in the newly formed cells 3 weeks later. Also, an increase in the level of interleukin-1beta (IL-1beta), a major proinflammatory cytokine, was observed in the hippocampus following the administration of IFN-alpha. Furthermore, coadministration of an IL-1 receptor antagonist completely blocked the IFN-alpha-induced suppression of the cell-proliferative activity in the DG. Our results indicate that IFN-alpha suppresses neurogenesis in the DG, and that IL-1beta plays an essential role in the suppression. The decreased cell proliferation caused by IFN-alpha-induced IL-1beta may be responsible, at least in part, for IFN-alpha-induced depression.


Assuntos
Proliferação de Células/efeitos dos fármacos , Giro Denteado/efeitos dos fármacos , Interferon-alfa/efeitos adversos , Interleucina-1beta/agonistas , Neurônios/efeitos dos fármacos , Animais , Bromodesoxiuridina , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Giro Denteado/imunologia , Giro Denteado/fisiopatologia , Transtorno Depressivo/induzido quimicamente , Transtorno Depressivo/imunologia , Transtorno Depressivo/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Fatores Imunológicos/efeitos adversos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Masculino , Neurônios/imunologia , Ratos , Ratos Wistar , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologia
9.
J Neurosurg ; 104(2): 264-71, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16509500

RESUMO

OBJECT: It remains unclear whether malignant glioma cells can deliver tumor antigens efficiently to major histocompatibility complex (MHC) Class I molecules. To elucidate the mechanism of antigen presentation in malignant gliomas, the authors examined the expression of the transporter associated with antigen processing 1 (TAP1), which transports antigens to MHC Class I molecules, and low-molecular-mass polypeptide 2 (LMP2), which is a subunit of a proteasome. They also analyzed the effects of interferon (IFN)-gamma and IFN-beta on the expression of these molecules. METHODS: Five glioma cells expressed undetectable or very low levels of TAP1 protein and did not express TAP1 messenger (m)RNA. Normal brain tissue and glioma tissue specimens also showed undetectable levels of TAP1 protein and the same levels of LMP2 protein as lymphoblastoid B cells. Treatments of the tumor cells with IFN-gamma, or -beta enhanced the expression of both TAP1 protein and mRNA as well as the expression of MHC Class I molecules. The expression of LMP2 protein was not altered by the IFN treatments. The authors analyzed structural alterations in the TAP1 promoter region in eight malignant glioma cell lines. Single nucleotide polymorphism was found in 446 bp up-stream of the translation start site of the TAP1 gene, and a point mutation was found in 34 bp upstream of the TAP1 gene. CONCLUSIONS: Malignant glioma cells may be less immunogenic due to low levels of TAP1 expression. Upregulated expression of TAP1 and MHC Class I molecules by IFN-gamma and -beta may enhance antigen presentation in malignant glioma cells.


Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Apresentação de Antígeno , Neoplasias Encefálicas/imunologia , Glioma/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Humanos , Interferon beta/fisiologia , Interferon gama/fisiologia , Complexo Principal de Histocompatibilidade/imunologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas , Regulação para Cima
10.
J Neurooncol ; 77(1): 25-32, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16132527

RESUMO

Invasion of tumor cells into the surrounding normal brain tissues is a prominent feature of malignant gliomas. Malignant glioma cells secrete thrombospondin-1 which participates in the motility of glioma cells and binds cell surface heparan sulfate proteoglycan. To clarify the invasion mechanism of tumor cells, expression of the syndecans (syndecan-1, -2, -3, and -4), a major cell surface heparan sulfate proteoglycan family, was analyzed in malignant gliomas. Involvement of nuclear factor-kappaB (NF-kappaB) on syndecan-1 expression was also investigated. Using reverse transcription-PCR, the authors analyzed the expression of syndecan-1, -2, -3, and -4 in 10 malignant glioma cell lines, 2 glioblastoma specimens, and 2 normal brain specimens. All malignant glioma cell lines and glioblastoma specimens expressed all types of syndecan mRNA, except in one glioma cell line that lacked syndecan-3 expression. On the other hand, normal brain specimens expressed syndecan-2, -3, and -4 mRNA, but did not syndecan-1 mRNA. Syndecan-1 protein was localized in the cell surface of all malignant glioma cell lines by flow cytometry. Various levels of active nuclear factor-kappa B (NF-kappaB) was detected in all malignant glioma cell lines using immunoblotting. The expression of active NF-kappaB and syndecan-1 increased in U251 glioma cells after tumor necrosis factor-alpha or interleukin-1beta treatment, which can activate NF-kappaB. The amplification of active NF-kappaB and syndecan-1 by tumor necrosis factor-alpha or interleukin-1beta was suppressed by an inhibitor of NF-kappaB activation (emodin). Emodin also downregulated the expression of syndecan-1 mRNA in U251 cells. These results indicate that malignant glioma cells express all types of syndecans and suggest that NF-kappaB participates in the upregulation of the syndecan-1 expression at the transcriptional level, and increased expression of syndecan-1 could associate with extracellular matrices including thrombospondin-1.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Proteoglicanas de Heparan Sulfato/classificação , Proteoglicanas de Heparan Sulfato/genética , Humanos , Linfotoxina-alfa/fisiologia , Glicoproteínas de Membrana/classificação , Glicoproteínas de Membrana/genética , Proteoglicanas/classificação , Proteoglicanas/genética , Proteoglicanas/metabolismo , RNA Mensageiro/análise , Sindecana-1 , Sindecana-2 , Sindecana-3 , Sindecana-4 , Sindecanas , Trombospondina 1/metabolismo
11.
J Histochem Cytochem ; 53(4): 455-65, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15805420

RESUMO

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.


Assuntos
Corpos de Inclusão/ultraestrutura , Organelas/ultraestrutura , Testículo/ultraestrutura , Animais , Biomarcadores/metabolismo , Imuno-Histoquímica , Corpos de Inclusão/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Organelas/metabolismo , Ratos , Ratos Wistar , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Espermatogênese , Testículo/metabolismo
12.
Curr Genet ; 47(5): 265-72, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15776236

RESUMO

We previously reported that there were three copies of ATP1 coding for F1-alpha and two copies of ATP3 coding for F1-gamma on the left and right arm of chromosome II, respectively. In this study, we present evidence that there are three closely linked copies of ATP2 encoding the beta subunit of the F1F0-ATPase complex on the right arm of chromosome X in several laboratory strains, including Saccharomyces cerevisiae strain S288C, although it was reported by the yeast genome project that ATP2 is a single-copy gene. Chromosome X fragmentation, long-PCR, chromosome-walking and ATP2-disruption analysis using haploid wild-type strains and prime clone 70645 showed that the three copies of ATP2 are present on the right arm of chromosome X, like those of ATP1 on chromosome II. Each was estimated to be approximately 4 kb apart. We designated the ATP2 proximal to the centromere as ATP2a, the middle one as ATP2b and the distal one as ATP2c. The region containing the three ATP2s is composed of two repeated units of approximately 7 kb; that is, both ends (ATP2a, ATP2c) accompanying the ATP2-neighboring ORFs are the same. A part of YJR119c, YJR120w, YJR122w (CAF17) and YJR123w (RP55), which were reported by the yeast genome project, are contained in the ATP2 repeated units; and the middle ATP2 of the three ATP2s, ATP2b, is located between the two repeated units. Expression of all three copies of ATP2 (ATP2a, ATP2b, ATP2c) was confirmed because a single or double ATP2-disruptant could grow on glycerol, but a triple ATP2-disruptant could not. In addition, of the three copies of ATP1 and ATP2, even if only one copy of the ATP1 and ATP2 genes remained, the cells grew on glycerol.


Assuntos
Cromossomos Fúngicos , Dosagem de Genes , ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromossomo X , Southern Blotting , Regulação Fúngica da Expressão Gênica , Ordem dos Genes , Glicerol/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Sequências de Repetição em Tandem/genética
13.
J Histochem Cytochem ; 52(11): 1393-403, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15505334

RESUMO

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.


Assuntos
Acrossomo/metabolismo , Espermatogênese , Testículo/metabolismo , Ubiquitina/metabolismo , Animais , Complexo de Golgi/metabolismo , Masculino , Microscopia Imunoeletrônica , Coelhos , Ratos , Ratos Wistar , Testículo/ultraestrutura , Vesículas Transportadoras/metabolismo
14.
Neurol Med Chir (Tokyo) ; 44(12): 637-43; discussion 644-5, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15684595

RESUMO

The highly invasive and angiogenic characteristics of malignant gliomas depend on the production of growth factors and angiogenic factors. Heparin-binding growth-associated molecule (HB-GAM) is a secreted growth factor that is mitogenic for endothelial cells. To examine the expression profile of HB-GAM in malignant glioma cells, messenger ribonucleic acid (mRNA) expression was analyzed in 10 malignant glioma cell lines, two glioblastoma tissue specimens, and two normal brain tissue specimens by the reverse transcription-polymerase chain reaction. HB-GAM mRNA was expressed in all specimens including normal brain tissue specimens. Western blot analysis revealed that HB-GAM protein contents in glioma cell lines and glioblastoma tissues were 1.8 to 6.3 times higher than those in normal brain tissues. The effect of neutralizing anti-platelet-derived growth factor (PDGF) antibody was also examined on the production of HB-GAM in malignant glioma cells, since malignant glioma cells secrete PDGF that upregulates HB-GAM expression. Treatment of U251 and T98G glioblastoma cells with the anti-PDGF antibody did not affect the HB-GAM production. These results suggest that HB-GAM is overexpressed in malignant glioma cells and is involved in tumor growth.


Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Glioma/metabolismo , Anticorpos/farmacologia , Encéfalo/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/genética , Glioma/patologia , Humanos , Fator de Crescimento Derivado de Plaquetas/imunologia , RNA Mensageiro/metabolismo
15.
Hum Cell ; 17(4): 195-201, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16035504

RESUMO

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.


Assuntos
Desenvolvimento Embrionário , Mitocôndrias/transplante , Oócitos/ultraestrutura , Animais , Células Cultivadas , Citoplasma , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Feminino , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microinjeções , Microscopia Confocal , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Reprodução Assistida
16.
Yeast ; 20(11): 943-54, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12898710

RESUMO

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.


Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos/genética , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Passeio de Cromossomo , DNA Fúngico/química , DNA Fúngico/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Eletroforese em Gel de Campo Pulsado , Dosagem de Genes , Dados de Sequência Molecular , Mutagênese Insercional , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
17.
Biol Reprod ; 69(3): 885-95, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12724282

RESUMO

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.


Assuntos
Glutationa Peroxidase/metabolismo , Espermatócitos/enzimologia , Espermatogênese/fisiologia , Espermatozoides/enzimologia , Testículo/enzimologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Perfilação da Expressão Gênica , Glutationa Peroxidase/imunologia , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ratos , Espermatócitos/citologia , Espermatócitos/ultraestrutura , Espermatozoides/citologia , Espermatozoides/ultraestrutura , Frações Subcelulares/enzimologia , Testículo/citologia , Fatores de Tempo
18.
J Histochem Cytochem ; 51(2): 215-26, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12533530

RESUMO

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.


Assuntos
Isocitrato Desidrogenase/metabolismo , Rim/enzimologia , Mitocôndrias/enzimologia , Miocárdio/enzimologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação com Gradiente de Concentração , Imuno-Histoquímica , Rim/ultraestrutura , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Mitocôndrias Cardíacas/enzimologia , Miocárdio/ultraestrutura , Especificidade de Órgãos , Ratos
19.
Reprod Med Biol ; 1(2): 41-47, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29699072

RESUMO

In eukaryotic cells, mitochondria are the major site of ATP production, which is achieved through the electron-transport chain and oxidative phosphorylation, according to the energy demand. Mitochondria contain their own genome (mitochondrial DNA, mtDNA) on which a limited number of genes are encoded. In the human sperm, mitochondria helically wrap the midpiece of the tail and supply the energy for the driving force of motility. While various mutations in mtDNA in somatic cells are found to be associated with a wide spectrum of diseases, it is also reported that the abnormal mtDNA causes astenozoospermia and male infertility. At fertilization, the paternal mitochondria and mtDNA are rapidly degraded early in embryogenesis, thus, only maternal mtDNA is transmitted to the descendant. We briefly review here the basic characteristics of mtDNA and its maternal transmission during fertilization, as well as male infertility. (Reprod Med Biol 2002; 1: 41-47).

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