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1.
Stud Health Technol Inform ; 264: 1720-1721, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438310

RESUMO

This study was designed to develop a wearable learning support system that enables novices to learn the skill for blood withdrawal while imitating its images displayed in front of them. For them to master "tacit knowledge," such as "proficient art" and "knacks," in intravenous injection and blood drawing techniques. This paper outlines a learning support system incorporating augmented reality (AR), which we have developed based on earlier studies.


Assuntos
Conhecimento , Aprendizagem , Enfermagem , Competência Clínica , Enfermagem/normas
2.
Stud Health Technol Inform ; 264: 1859-1860, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438379

RESUMO

Support for elderly people with dementia is required in the community. Many elementary school students are taking dementia kids supporter training courses, but it is difficult to nurture the ability to correctly understand and respond to dementia. By using multiple information communication technologies, we developed a practical program with concrete response method to convey knowledge on supporting of elderly people with dementia.


Assuntos
Demência , Estudantes , Idoso , Comunicação , Humanos , Instituições Acadêmicas
3.
Stud Health Technol Inform ; 250: 45-49, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29857368

RESUMO

In this study, we aimed to teach elementary school students how to practically deal with elderly people with dementia and developed and evaluated teaching materials using a communication robot called Pepper. The teaching material used the robot to model representative symptoms seen in elderly people with dementia in a realistic context. We applied the two principles of Merrill's first principle and the ARCS model to achieve learning objectives and sustain learning motivation. Furthermore, to verify the effectiveness of the teaching materials, we conducted a questionnaire survey on five experts in community welfare. As a result, it was speculated that by using Pepper, motivation for learning increases and learning can be effectively accomplished. In the future, we aim to introduce the function to the teaching materials based on the evaluation and introduce it to kids' dementia supporter training course.


Assuntos
Comunicação , Demência/enfermagem , Robótica , Materiais de Ensino , Idoso , Humanos , Aprendizagem , Ensino
4.
Artigo em Inglês | MEDLINE | ID: mdl-29857378

RESUMO

We created teaching materials for elementary school students to foster their ability to understand and support elderly people correctly, including those with dementia, without prejudice. Pediatric awareness education by DVDs can positively capture the elderly with dementia, but it was difficult to learn in practice. There, Slide materials designed to encourage positive understanding of elderly people were used together with a Pepper to increase participants' interest material. Results suggest that simultaneous use of the slide materials and a Pepper maintains participants' interest and promotes understanding of elderly people, including those with dementia.


Assuntos
Demência , Estudantes , Materiais de Ensino , Idoso , Criança , Humanos , Aprendizagem , Ensino
5.
J Biol Chem ; 286(13): 11632-48, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21266581

RESUMO

Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.


Assuntos
Digestão/fisiologia , Trato Gastrointestinal/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Neurônios/enzimologia , Fosfolipases A2 Secretórias/biossíntese , Fosfolipídeos/metabolismo , Reprodução/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Fosfolipases A2 Secretórias/genética , Fosfolipídeos/genética
6.
J Biol Chem ; 286(13): 11616-31, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21266583

RESUMO

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Folículo Piloso/enzimologia , Fosfolipases A2 Secretórias/biossíntese , Alopecia/enzimologia , Alopecia/genética , Animais , Ativação Enzimática/fisiologia , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Homeostase/fisiologia , Melaninas/genética , Melaninas/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Fosfolipases A2 Secretórias/genética , Prostaglandinas/genética , Prostaglandinas/metabolismo
7.
J Clin Invest ; 120(5): 1400-14, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20424323

RESUMO

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3-/- mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3-/- mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3-/- mice. Moreover, the gonads of Pla2g3-/- mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.


Assuntos
Epididimo/metabolismo , Fosfolipases A2 do Grupo III/metabolismo , Espermatozoides/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Fosfatidilcolinas/metabolismo , Espermatogênese , Distribuição Tecidual
8.
J Clin Invest ; 120(5): 1415-28, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20424324

RESUMO

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.


Assuntos
Reação Acrossômica , Fosfolipases A2 do Grupo X/genética , Fosfolipases A2 do Grupo X/metabolismo , Espermatozoides/metabolismo , Animais , Cruzamentos Genéticos , Feminino , Fertilidade , Fertilização , Fertilização In Vitro , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testículo/metabolismo
9.
Biochem J ; 421(1): 17-27, 2009 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-19371233

RESUMO

PLA2 (phospholipase A2) group III is an atypical sPLA2 (secretory PLA2) that is homologous with bee venom PLA2 rather than with other mammalian sPLA2s. In the present paper, we show that endogenous group III sPLA2 (PLA2G3) is expressed in mouse skin and that Tg (transgenic) mice overexpressing human PLA2G3 spontaneously develop skin inflammation. Pla2g3-Tg mice over 9 months of age frequently developed dermatitis with hyperkeratosis, acanthosis, parakeratosis, erosion, ulcer and sebaceous gland hyperplasia. The dermatitis was accompanied by infiltration of neutrophils and macrophages and by elevated levels of pro-inflammatory cytokines, chemokines and prostaglandin E2. In addition, Pla2g3-Tg mice had increased lymph aggregates and mucus in the airway, lymphocytic sialadenitis, hepatic extramedullary haemopoiesis, splenomegaly with increased populations of granulocytes and monocytes/macrophages, and increased serum IgG1. Collectively, these observations provide the first demonstration of spontaneous development of inflammation in mice with Tg overexpression of mammalian sPLA2.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Fosfolipases A2 do Grupo III/genética , Fosfolipases A2 do Grupo III/metabolismo , Inflamação/metabolismo , Animais , Dermatite/genética , Dermatite/metabolismo , Dermatite/patologia , Camundongos , Camundongos Transgênicos , Pele/patologia
10.
J Biol Chem ; 283(48): 33483-97, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18801741

RESUMO

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.


Assuntos
Aterosclerose/enzimologia , Células Espumosas/enzimologia , Fosfolipases A2 do Grupo III/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Venenos de Abelha/química , Dieta Aterogênica , Células Espumosas/patologia , Fosfolipases A2 do Grupo III/química , Fosfolipases A2 do Grupo III/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas LDL/genética , Lisofosfatidilcolinas/genética , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Transgênicos , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética
11.
Atherosclerosis ; 196(1): 81-91, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17353016

RESUMO

Secretory phospholipase A2s (sPLA2s) contribute to the hydrolysis of phospholipid. Among them, sPLA2-IIA, -V, and -X have been regarded as enhancers of lipid accumulation in arterial intima. However, the distribution and production of the other types of sPLA2 in human aortic wall remain unclear. Therefore, in this study, the distribution and production of seven types of sPLA2 including IIA, IID, IIE, IIF, III, V, and X in atherosclerosis development in the human aorta were comprehensively examined by immunohistochemistry and in situ hybridization (ISH). The extent of sPLA2s expression increased with atherosclerosis development, but only sPLA2-IIF was never observed in the normal aorta. Double-immunostaining demonstrated that sPLA2-V expression was limited to smooth muscle cells (SMCs), although the other sPLA2s were expressed in both macrophages and SMCs. ISH using sPLA2 cDNAs revealed that the expression pattern of each mRNA was consistent with the results of immunohistochemistry for each corresponding sPLA2. These results indicate that the seven types of sPLA2 are expressed with various patterns in all stages of atherosclerosis development and may play an atherogenic role through degradation of phospholipid.


Assuntos
Aterosclerose/enzimologia , Macrófagos/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfolipases A2 Secretórias/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal/metabolismo , Aorta Torácica/metabolismo , Aterosclerose/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade
12.
Biochem J ; 409(2): 429-38, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17868035

RESUMO

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.


Assuntos
Fosfolipases A2 do Grupo III/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Animais , Diferenciação Celular , Sobrevivência Celular/efeitos da radiação , DNA Complementar/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Neuritos/fisiologia , Células PC12 , RNA Interferente Pequeno/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray
13.
Biochim Biophys Acta ; 1771(11): 1389-96, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17980167

RESUMO

Of 10 mammalian secreted phospholipase A(2) (sPLA(2)) enzymes identified to date, group V and X sPLA(2)s, which are two potent plasma membrane-acting sPLA(2)s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA(2), which is structurally more similar to bee venom PLA(2) than to other mammalian sPLA(2)s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA(2) hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA(2) domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA(2) depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA(2)s target distinct phospholipid molecular species in cellular membranes.


Assuntos
Infecções por Adenovirus Humanos/prevenção & controle , Fosfolipases A2 do Grupo III/fisiologia , Infecções por Adenovirus Humanos/enzimologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/patogenicidade , Linhagem Celular , Membrana Celular/metabolismo , Fosfolipases A2 do Grupo III/química , Fosfolipases A2 do Grupo III/genética , Fosfolipases A2 do Grupo V/fisiologia , Fosfolipases A2 do Grupo X/fisiologia , Humanos , Mutação , Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray
14.
J Biol Chem ; 281(47): 36420-33, 2006 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-17008322

RESUMO

In an effort to elucidate the functions of secreted phospholipase A2 (sPLA2) enzymes in vivo, we generated transgenic (Tg) mice for group V sPLA2 (sPLA2-V) and group X sPLA2 (sPLA2-X), which act potently on phosphatidylcholine in vitro. We found that sPLA2-V Tg mice died in the neonatal period because of respiratory failure. The lungs of sPLA2-V Tg mice exhibited atelectasis with thickened alveolar walls and narrow air spaces, accompanied by infiltration of macrophages and only modest changes in eicosanoid levels. This severe pulmonary defect in sPLA2-V Tg mice was attributable to marked reduction of the lung surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol. Given that the expression of sPLA2-V is greatly elevated in human lungs with severe inflammation, our present results raise the intriguing possibility that this isozyme may contribute to ongoing surfactant hydrolysis often observed in the lungs of patients with respiratory distress syndrome. In contrast, sPLA2-X Tg neonates displayed minimal abnormality of the respiratory tract with normal alveolar architecture and surfactant composition. This unexpected result was likely because sPLA2-X protein existed as an inactive zymogen in most tissues. The active form of sPLA2-X was detected in tissues with inflammatory granulation in sPLA2-X Tg mice. These results suggest that sPLA2-X mostly remains inactive under physiological conditions and that its proteolytic activation occurs during inflammation or other as yet unidentified circumstances in vivo.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pulmão/embriologia , Fosfolipases A/biossíntese , Animais , Feminino , Genótipo , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo X , Inflamação , Pulmão/metabolismo , Pulmão/fisiologia , Pneumopatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Fosfolipases A/genética , Fosfolipases A2
15.
J Biol Chem ; 281(43): 32741-54, 2006 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-16931517

RESUMO

The mechanisms by which secretory phospholipases A(2) (PLA(2)s) exert cellular effects are not fully understood. Group IIF PLA(2) (gIIFPLA(2)) is a structurally unique secretory PLA(2) with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA(2) contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA(2) had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA(2) could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr(115), Phe(116), Val(118), and Tyr(119)) near the C-terminal extension are responsible for these activities. When gIIFPLA(2) was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA(2) mRNA was detected endogenously in human CD4(+) helper T cells after in vitro stimulation and exogenously added gIIFPLA(2) inhibited the proliferation of a T cell line, which was not seen with group IIA PLA(2). Collectively, these data suggest that unique membrane-binding properties of gIIFPLA(2) may confer special functionality on this secretory PLA(2) under certain physiological conditions.


Assuntos
Membrana Celular/metabolismo , Fosfolipases A/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/química , Escherichia coli/genética , Fosfolipases A2 do Grupo II , Humanos , Hibridomas/efeitos dos fármacos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Masculino , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosfolipases A/química , Fosfolipases A/genética , Fosfolipases A/farmacologia , Fosfolipases A2 , Fosfolipídeos/química , Fosfolipídeos/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Especificidade por Substrato , Linfócitos T/efeitos dos fármacos
16.
Biochem J ; 393(Pt 1): 97-106, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16146426

RESUMO

sPLA2 (secretory phospholipase A2) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA2s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA2s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA2s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA2s. Thus hydrolysis of the plasma membrane by these sPLA2s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA2s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA2 is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA2s may play a role in innate immunity against adenoviral infection in the respiratory tract.


Assuntos
Adenoviridae/fisiologia , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Fosfolipases A/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos/enzimologia , Fibroblastos/virologia , Regulação Enzimológica da Expressão Gênica , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo X , Humanos , Lisofosfatidilcolinas/metabolismo , Macrófagos/enzimologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A2 , RNA Mensageiro/metabolismo
17.
Biochim Biophys Acta ; 1736(3): 200-10, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16188494

RESUMO

The secretory phospholipase A2 (sPLA2) family in mammals contains more than 10 enzymes. In this study, we examined by immunohistochemistry the localization of six sPLA2s (IIA, IID, IIE, IIF, V and X) in human heart, kidney, liver and stomach. In normal hearts, sPLA2-IIA was detected in coronary vascular smooth muscle cells (VSMC) and sPLA2-V in cardiomyocytes beneath the endocardium. In infarcted hearts, expression of these two enzymes was markedly increased in damaged cardiomyocytes, and expression of sPLA2-IID and-IIE, which was undetectable in normal hearts, was elevated in damaged cardiomyocytes and VSMC, respectively. In infarcted kidneys, sPLA2-IIA and-V were markedly induced in the uriniferous tubular epithelium. In livers affected by viral hepatitis, sPLA2-IIA and-V were expressed in hepatocytes with fatty degeneration. In the gastric glands exhibiting intestinal metaplasia, sPLA2-IIA was localized in the glandular base, sPLA2-IID and-V in the glandular body epithelium, sPLA2-IIE and-IIF in goblet cells in the foveolar epithelium, and sPLA2-X in both glandular body epithelial cells and foveolar epithelial goblet cells. In the gastric submucosal tissues, sPLA2-IIA and-IIE were located in VSMC and sPLA2-V was in the interstitial fibroblasts. In addition, sPLA2-IIA,-IIE,-IIF and-X were highly expressed in gastric signet ring cell carcinoma. Thus, individual sPLA2s exhibit unique cellular localizations in each tissue, suggesting their distinct roles in pathophysiology.


Assuntos
Mucosa Gástrica/enzimologia , Rim/enzimologia , Fígado/enzimologia , Miocárdio/enzimologia , Fosfolipases A/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células em Anel de Sinete/enzimologia , Carcinoma de Células em Anel de Sinete/patologia , Linhagem Celular Tumoral , Mucosa Gástrica/patologia , Expressão Gênica/genética , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo X , Hepatite Viral Humana/enzimologia , Hepatite Viral Humana/patologia , Humanos , Imuno-Histoquímica , Infarto/enzimologia , Infarto/patologia , Rim/patologia , Fígado/patologia , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Fosfolipases A/genética , Fosfolipases A2 , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia
18.
J Biol Chem ; 280(26): 24987-98, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15863501

RESUMO

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Fosfolipases A/química , Fosfolipases A/fisiologia , Processamento de Proteína Pós-Traducional , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Ácido Araquidônico/química , Artrite Reumatoide/patologia , Northern Blotting , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Feminino , Fibroblastos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Fosfolipases A2 do Grupo III , Humanos , Imuno-Histoquímica , Inflamação , Isquemia/patologia , Lentivirus/metabolismo , Pulmão/patologia , Camundongos , Camundongos Nus , Microcirculação , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neovascularização Patológica , Mutação Puntual , Prostaglandinas/química , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Neoplasias Uterinas/patologia
19.
J Biol Chem ; 280(24): 23203-14, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15781456

RESUMO

Although individual mammalian secreted phospholipase A(2) (sPLA(2)) enzymes exhibit unique tissue and cellular distributions, the cell type-specific functions of each enzyme remain largely unknown. In this study, we found by immunohistochemistry that group X sPLA(2) (sPLA(2)-X) is uniquely located in the peripheral neuronal fibers, an observation that was supported by detection of its transcript and protein in the neuronal cell line PC12 and in primary dorsal root ganglia neurons. Adenoviral expression of sPLA(2)-X in PC12 cells facilitated neurite outgrowth, particularly when combined with a suboptimal concentration of nerve growth factor. In neuronally differentiated PC12 cells, sPLA(2)-X was preferentially localized in the Golgi apparatus and growth cones, and proteolytic conversion of the proenzyme to mature enzyme mainly occurred after the secretion process. The neurite-extending ability of sPLA(2)-X depended on the production of its catalytic product, lysophosphatidylcholine. Moreover, nerve growth factor-induced neurite extension of PC12 cells was modestly but significantly attenuated by an anti-sPLA(2)-X antibody or by a small interfering RNA for sPLA(2)-X. These observations suggest the potential contribution of sPLA(2)-X to neuronal differentiation, and possibly repair, under certain conditions.


Assuntos
Neurônios/enzimologia , Fosfolipases A/biossíntese , Fosfolipases A/metabolismo , Adenoviridae/metabolismo , Animais , Northern Blotting , Catálise , Diferenciação Celular , Linhagem Celular , Cromatografia em Camada Delgada , DNA Complementar/metabolismo , Gânglios Espinais/metabolismo , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fator de Crescimento Neural , Neurônios/metabolismo , Células PC12 , Fosfolipases A2 , RNA Interferente Pequeno/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo
20.
J Biol Chem ; 280(14): 14028-41, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15695510

RESUMO

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.


Assuntos
Membrana Celular/metabolismo , Isoenzimas/metabolismo , Fosfolipases A/metabolismo , Prostaglandinas/biossíntese , Adenocarcinoma/metabolismo , Animais , Ácido Araquidônico/metabolismo , Morte Celular , Linhagem Celular , Membrana Celular/química , Neoplasias Colorretais/metabolismo , Dinoprostona/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Fosfolipases A2 do Grupo VI , Humanos , Hidrólise , Isoenzimas/genética , Fosfolipases A/genética , Fosfolipases A2 , Fosfolipídeos/química , Fosfolipídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia
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