RESUMO
Formation of the acrosome during spermiogenesis is an essential process for creating fertilization-competent sperm. Of the numerous aspects required for acrosome biogenesis, adherence of the acrosomal outer membrane to the nuclear surface is mediated by the subacrosomal perinuclear theca. However, the cellular dynamics and congruent functions pertaining to these acrosomal anchoring factors are not well understood despite many of them being implicated as potential causes for human male infertility. Actin-like 7A (ACTL7A) is one such factor for which deleterious polymorphisms have recently been shown to cause human male infertility. It is thought that acrosomal attachment is coordinated by cytoskeletal associations between the acrosome and nucleus via the acroplaxome. To further illuminate the mechanistic underpinnings of ACTL7A for essential acrosome associations, in this study, we investigated its dynamic localization in the developing germline, molecular associations with other cytoskeletal components, and the cellular consequences of ablation. Our intracellular localization data show ACTL7A to be dynamically present within the nucleus and subacrosomal space and later associated with postacrosomal regions of developing spermatids. Through the generation of an Actl7a knock-out mouse model, we consistently observed disruption of acrosomal biogenesis with abnormal migration of the acrosomal granule and peeling acrosomes during spermatid elongation. Significantly, we found a complete loss of subacrosomal filamentous actin (F-actin) structures in knock-out spermatids suggesting a regulatory role for subacrosomal F-actin. Considering our reported data together with existing literature, we propose a mechanistic model explaining the essential role of ACTL7A for acroplaxome-associated F-actin, acrosomal attachment integrity, and male fertility.
Assuntos
Infertilidade Masculina , Testículo , Camundongos , Animais , Masculino , Humanos , Testículo/metabolismo , Actinas/metabolismo , Sêmen/metabolismo , Infertilidade Masculina/metabolismo , FertilidadeRESUMO
BACKGROUND: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation. OBJECTIVES: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis. MATERIALS AND METHODS: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6 months to check fertility. RESULTS: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males. DISCUSSION AND CONCLUSION: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.
Assuntos
Epididimo/metabolismo , Fertilidade/genética , Proteínas de Membrana/genética , Espermatozoides/metabolismo , Animais , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Knockout , Maturação do Esperma/fisiologia , Motilidade dos Espermatozoides/genéticaRESUMO
BACKGROUND: The purpose of this study was to produce molecules that can precisely regulate the complement and coagulation system and to assess the expression of such molecules in transgenic animals. METHODS: The CTDM gene, which is composed of the delta-1-99 amino acid (aa) C1-INH, EGF domain 4-6 of thrombomoduline (TM), short consensus repeat (SCR) 2-4 of DAF(CD55), and SCR 2-4 of MCP(CD46) was established. The codon usage for expression in mammals was adopted. The cDNA of CTDM was subcloned into the pCPI site (the human insulin promoter and a cytomegalovirus enhancer). pCPI-CTDM was transfected into pig endothelial cells (PEC). The expression of the molecule was clearly assessed by means of flow cytometry. RESULTS: BD3F1 female mice were induced to superovulate and were then crossed with BD3F1 males. Micro-injection and embryo transfer were performed by standard methods, thus generating transgenic mice that express CTDM. The mice carried the CTDM plasmid, as verified by PCR. Tissue expression levels in transgenic mouse lines generated with the constructs were follows: pancreas, 1.0; brain, 5.4; thymus, 0.3; heart, 0.2; lung, 1.2; liver, 0.1; kidney, 0.1; intestine, 0.4; and spleen, 1.6. A naive control mouse was also analyzed in the exact manner as for the transgenic mice. CONCLUSIONS: A synthetic CTDM gene with codon usage optimized to the mammalian system represents a critical factor in the development of transgenic animals.
Assuntos
Coagulação Sanguínea/genética , Proteínas do Sistema Complemento/genética , Genes Sintéticos/genética , Animais , Antígenos CD55/genética , Clonagem Molecular , DNA Complementar/genética , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Suínos , Trombomodulina/genética , Transfecção/métodos , Transplante HeterólogoRESUMO
BACKGROUND: We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). METHODS: Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. RESULTS: Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. CONCLUSIONS: Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Oxigenases de Função Mista/deficiência , Animais , Antígenos Heterófilos/metabolismo , Sequência de Bases , Células Endoteliais/imunologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Oxigenases de Função Mista/genética , Ácido N-Acetilneuramínico/metabolismo , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Sus scrofa , SuínosRESUMO
BACKGROUND: In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice. METHODS: A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method. RESULTS: The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung. CONCLUSION: A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation.
Assuntos
Animais Geneticamente Modificados/genética , Códon/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Actinas/genética , Animais , Animais Geneticamente Modificados/imunologia , Linhagem Celular , Citomegalovirus , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos/genética , Genes Sintéticos , Humanos , Células Matadoras Naturais/fisiologia , Macrófagos/fisiologia , Camundongos , Regiões Promotoras Genéticas/genética , Suínos , Transfecção , Transplante Heterólogo , Antígenos HLA-ERESUMO
STUDY HYPOTHESIS: We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization. STUDY FINDING: Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation. WHAT IS KNOWN ALREADY: Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry). MAIN RESULTS AND THE ROLE OF CHANCE: Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001). LIMITATIONS, REASONS FOR CAUTION: The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies. WIDER IMPLICATIONS OF THE FINDINGS: Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest.
Assuntos
Sequência de Bases , Glicoproteínas/genética , Infertilidade Masculina/genética , Deleção de Sequência , Interações Espermatozoide-Óvulo/genética , Espermatozoides/metabolismo , Adulto , Animais , Cálcio/metabolismo , Moléculas de Adesão Celular , Estro/genética , Éxons , Feminino , Expressão Gênica , Glicoproteínas/deficiência , Humanos , Infertilidade Masculina/fisiopatologia , Infertilidade Masculina/cirurgia , Tamanho da Ninhada de Vivíparos , Masculino , Proteínas de Membrana , Camundongos , Camundongos Knockout , Capacitação Espermática/genética , Espermatozoides/patologia , Vasectomia , Zona Pelúcida/metabolismoRESUMO
Oxidative stress, one of the most probable molecular mechanisms for neuronal impairment, is reported to occur in the affected brain regions of various neurodegenerative diseases. Recently, many studies showed evidence of a link between oxidative stress or mitochondrial damage and neuronal degeneration. Basic in vitro experiments and postmortem studies demonstrated that biomarkers for oxidative damage can be observed in the pathogenic regions of the brain and the affected neurons. Model animal studies also showed oxidative damage associated with neuronal degeneration. The molecular imaging method with positron emission tomography (PET) is expected to delineate oxidatively stressed microenvironments to elucidate pathophysiological changes of the in vivo brain; however, only a few studies have successfully demonstrated enhanced stress in patients. Radioisotope copper labeled diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) may be the most promising candidate for this oxidative stress imaging. The tracer is usually known as a hypoxic tissue imaging PET probe, but the accumulation mechanism is based on the electron rich environment induced by mitochondrial impairment and/or microsomal over-reduction, and thus it is considered to represent the oxidative stress state correlated with the degree of disease severity. In this review, Cu-ATSM PET is introduced in detail from the basics to practical methods in clinical studies, as well as recent clinical studies on cerebrovascular diseases and neurodegenerative diseases. Several other PET probes are also introduced from the point of view of neuronal oxidative stress imaging. These molecular imaging methods should be promising tools to reveal oxidative injuries in various brain diseases.
Assuntos
Encéfalo/patologia , Mitocôndrias/patologia , Doenças Neurodegenerativas/patologia , Estresse Oxidativo , Tomografia por Emissão de Pósitrons/métodos , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Antioxidantes/química , Transtornos Cerebrovasculares/diagnóstico por imagem , Radioisótopos de Cobre , Humanos , Imagem Molecular/métodos , Oxigênio/química , Doença de Parkinson/diagnóstico por imagemRESUMO
Nonodontogenic toothache is a painful condition that occurs in the absence of a clinically evident cause in the teeth or periodontal tissues. The purpose of this review is to improve the accuracy of diagnosis and the quality of dental treatment regarding nonodontogenic toothache. Electronic databases were searched to gather scientific evidence regarding related primary disorders and the management of nonodontogenic toothache. We evaluated the level of available evidence in scientific literature. There are a number of possible causes of nonodontogenic toothache and they should be treated. Nonodontogenic toothache can be categorised into eight groups according to primary disorders as follows: 1) myofascial pain referred to tooth/teeth, 2) neuropathic toothache, 3) idiopathic toothache, 4) neurovascular toothache, 5) sinus pain referred to tooth/teeth, 6) cardiac pain referred to tooth/teeth, 7) psychogenic toothache or toothache of psychosocial origin and 8) toothache caused by various other disorders. We concluded that unnecessary dental treatment should be avoided.
Assuntos
Odontalgia , Diagnóstico Diferencial , Dor Facial/complicações , Humanos , Isquemia Miocárdica/complicações , Síndromes da Dor Miofascial/complicações , Neuralgia/complicações , Sinusite/complicações , Odontalgia/classificação , Odontalgia/diagnóstico , Odontalgia/etiologia , Odontalgia/terapiaRESUMO
In order to elucidate the mechanisms of post-exercise acute renal failure, one of the complications of hereditary renal hypouricemia, we have targeted the mouse Slc22a12 gene by the exchange of exons 1-4 with pMC1neo-polyA. The knockout mice revealed no gross anomalies. The concentration ratio of urinary urate/creatinine of the knockout mice was significantly higher than that of wildtype mice, indicating an attenuated renal reabsorption of urate. The plasma levels of urate were around 11 muM and were similar among the genotypes. Although the fractional excretion of urate of knockout mice was tend to higher than that of wildtype mice, the urate reabsorption ability remained in the kidney of knockout mice, indicating a urate reabsorptive transporter other than Urat1.
Assuntos
Camundongos Knockout , Transportadores de Ânions Orgânicos/genética , Alantoína/urina , Animais , Northern Blotting , Western Blotting , Cromatografia Líquida de Alta Pressão , Creatinina/urina , Camundongos , Transportadores de Ânions Orgânicos/metabolismo , Ácido Úrico/sangue , Ácido Úrico/metabolismo , Ácido Úrico/urinaRESUMO
A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.
Assuntos
Retículo Endoplasmático/metabolismo , Ácido Glutâmico/toxicidade , Hipocampo/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Proteínas/metabolismo , Animais , Proteínas de Choque Térmico HSP70 , Heterozigoto , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , Neurônios/metabolismo , Proteínas/genéticaRESUMO
A necrotic immature mandibular second premolar with periapical involvement in a 13-year-old patient was treated. Instead of the standard root canal treatment protocol and apexification, antimicrobial agents were used in the canal, after which the canal was left empty. Radiographic examination showed the start of apical closure 5 months after the completion of the antimicrobial protocol. Thickening of the canal wall and complete apical closure was confirmed 30 months after the treatment, indicating the revascularization potential of a young permanent tooth pulp into a bacteria-free root canal space.
Assuntos
Fístula Dentária/etiologia , Necrose da Polpa Dentária/complicações , Polpa Dentária/irrigação sanguínea , Periodontite Periapical/tratamento farmacológico , Fraturas dos Dentes/complicações , Adolescente , Anti-Infecciosos Locais/uso terapêutico , Dente Pré-Molar/lesões , Ciprofloxacina/uso terapêutico , Fístula Dentária/terapia , Polpa Dentária/lesões , Necrose da Polpa Dentária/etiologia , Necrose da Polpa Dentária/cirurgia , Dentina Secundária/metabolismo , Feminino , Humanos , Mandíbula , Metronidazol/uso terapêutico , Neovascularização Fisiológica , Periodontite Periapical/complicações , Periodontite Periapical/etiologia , Pulpectomia , Fraturas dos Dentes/fisiopatologiaRESUMO
We previously targeted EGFP (a mutant of green fluorescent protein) to the lumen of the mouse sperm acrosome and reported the time course of EGFP release during the acrosome reaction. In the study reported here, we estimated the pH within the mouse sperm acrosome utilizing the pH-dependent nature of EGFP fluorescence. The average intra-acrosomal pH was estimated to be 5.3 +/- 0.1 immediately after sperm preparation, gradually increasing to 6.2 +/- 0.3 during 120 min of incubation in TYH media suitable for capacitation. Spontaneous acrosome reactions were noted to increase concomitantly with acrosomal alkalinization during incubation. We also demonstrated that acrosomal antigens detected by monoclonal antibodies MN7 and MC41 did not dissolve following the acrosome reaction in pH 5.3 media, but dissolved at pH 6.2. These data suggest that acrosomal alkalinization during incubation conducive for sperm capacitation may function to alter acrosomal contents and prepare them for release during the acrosome reaction.
Assuntos
Acrossomo/metabolismo , Proteínas Luminescentes/metabolismo , Capacitação Espermática , Reação Acrossômica , Animais , Antígenos/metabolismo , Cálcio/farmacologia , Feminino , Proteínas de Fluorescência Verde , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos TransgênicosRESUMO
The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.
Assuntos
Elementos de DNA Transponíveis , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Genes Reporter , Proteínas de Fluorescência Verde , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MutagêneseRESUMO
The aim of this study was to evaluate the effects of combination psychotropic drug treatment on heart rate variability (HRV), which was mainly controlled by the parasympathetic nervous system. Mean R-R intervals (mRR) and coefficient of variation (CV), an index of HRV, were studied in 22 psychiatric patients and 21 age- and sex-matched healthy controls. Next, in the patient group focusing on both anticholinergic and antidopaminergic properties, combination psychotropic drug daily doses were converted into biperiden milligram equivalents (BPDeq) and chlorpromazine milligram equivalents (CPZeq), respectively. The relationship between mRR and CV and these equivalent dosages was examined. A significant reduction in both mRR (P < 0.05) and CV (P < 0.05) was found in the patient group. In addition, significant negative correlations were observed between the dose of BPDeq and mRR (P < 0.05), and between the dose of BPDeq and CV (P < 0.005). In contrast, no significant correlations were observed between the dose of CPZeq and either parameter. These findings suggest that the effects of combination psychotropic drug treatment on HRV are mainly due to their anticholinergic properties. Therefore, CV is a useful indicator to assess the parasympathetic activity of psychiatric patients under combination psychotropic drug treatment.
Assuntos
Sistema Nervoso Autônomo/efeitos dos fármacos , Antagonistas Colinérgicos/efeitos adversos , Frequência Cardíaca/efeitos dos fármacos , Transtornos Mentais/tratamento farmacológico , Psicotrópicos/farmacologia , Adulto , Idoso , Estudos de Casos e Controles , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Psicotrópicos/efeitos adversosRESUMO
The purpose was to examine the effect of brief intrusive forces on human pulpal blood flow (PBF). Laser Doppler flowmetric measurements were made from 17 vital upper left central incisors of 17 participants who had clinically healthy tooth crowns and periodontal tissues. Brief intrusive forces (0.5,1,5 N; duration 20 s) were applied to the incisal edges of the examined teeth, and apical displacement of the teeth and the PBF were measured simultaneously. Recordings were made with and without an opaque rubber dam applied to the examined teeth. Intrusive force significantly reduced PBF flux both with and without the dam (P<0.05, Friedman analysis). The results indicate that transient apical displacement can reduce PBF.
Assuntos
Polpa Dentária/irrigação sanguínea , Análise do Estresse Dentário/instrumentação , Técnicas de Movimentação Dentária , Adulto , Análise do Estresse Dentário/métodos , Humanos , Incisivo , Fluxometria por Laser-Doppler , Diques de Borracha , Estatísticas não ParamétricasRESUMO
Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.
Assuntos
Ciclinas/metabolismo , Dano ao DNA , Fase G2 , Mitose , Tolerância a Radiação , Animais , Ciclina G , Ciclina G1 , Ciclinas/genética , Relação Dose-Resposta à Radiação , Raios gama , Camundongos , Camundongos Mutantes , Mutagênese Insercional , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Irradiação Corporal TotalRESUMO
The human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenic mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Delta1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Delta1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenic pigs with high levels of expression.
Assuntos
Antígenos CD55/biossíntese , Antígenos CD55/genética , Células CHO/metabolismo , Genes Sintéticos/genética , Camundongos Transgênicos/genética , Suínos/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Códon/genética , Cricetinae , Endotélio/citologia , Endotélio/metabolismo , Expressão Gênica/genética , Mamíferos/genética , Camundongos , Camundongos Transgênicos/metabolismo , Pâncreas/metabolismo , Transfecção/métodos , Transplante Heterólogo/métodosRESUMO
Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607-611). In the present study, we demonstrate that calmegin -/- sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin -/- sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin beta -/- sperm, we investigated the interaction of calmegin with fertilin beta. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin alpha and beta, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin alpha and beta. In the calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm. The data not only explain why the calmegin and fertilin beta knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.