RESUMO
This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (VH & VL). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.
Assuntos
Actinina/imunologia , Anticorpos Monoclonais/imunologia , Molécula 1 de Adesão Celular/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Linhagem Celular , Reações Cruzadas , Células Hep G2 , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , ImunoprecipitaçãoRESUMO
An outbreak of enterohaemorrhagic Escherichia coli O157 occurred in multiple prefectures of Japan in November 2009. We conducted two case-control studies with trace-back and trace-forward investigations to determine the source. The case definition was met by 21 individuals; 14 (66.7%) were hospitalised, but no haemolytic uraemic syndrome, acute encephalopathy or deaths occurred. Median age was 23 (range 12-48) years and 14 cases were male (66.7%). No significant associations with food were found in a case-control study by local public health centres, but our matched case-control study using Internet surveys found that beef hanging tender (or hanger steak), derived from the diaphragm of the cattle, was significantly associated with illness (odds ratio = 15.77; 95% confidence interval, 2.00-124.11). Pulsed-field gel electrophoresis analysis of isolates from patients and the suspected food showed five different patterns: two in faecal and food samples, and another three in patient faecal samples only, although there were epidemiological links to the meat consumed at the restaurants. Trace-back investigation implicated a common food processing company from outside Japan. Examination of the logistics of the meat processing company suggested that contamination did not occur in Japan. We concluded that the source of the outbreak was imported hanging tender. This investigation revealed that Internet surveys could be useful for outbreak investigations.
Assuntos
Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Internet , Carne Vermelha/microbiologia , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Criança , Eletroforese em Gel de Campo Pulsado , Fezes/microbiologia , Feminino , Manipulação de Alimentos , Microbiologia de Alimentos , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , RestaurantesRESUMO
The spin transfer torque is essential for electrical magnetization switching. When a magnetic domain wall is driven by an electric current through an adiabatic spin torque, the theory predicts a threshold current even for a perfect wire without any extrinsic pinning. The experimental confirmation of this 'intrinsic pinning', however, has long been missing. Here, we give evidence that this intrinsic pinning determines the threshold, and thus that the adiabatic spin torque dominates the domain wall motion in a perpendicularly magnetized Co/Ni nanowire. The intrinsic nature manifests itself both in the field-independent threshold current and in the presence of its minimum on tuning the wire width. The demonstrated domain wall motion purely due to the adiabatic spin torque will serve to achieve robust operation and low energy consumption in spintronic devices.
RESUMO
The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.
Assuntos
Apoptose , Proteínas de Ligação a DNA/genética , DNA/biossíntese , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insetos/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Bromodesoxiuridina/metabolismo , Divisão Celular , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Drosophila/fisiologia , Deleção de Genes , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas de Insetos/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Modelos Genéticos , Mutação , Fenótipo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Complexo Repressor Polycomb 1 , Ligação Proteica , Fase SRESUMO
Rupture of a fibrous strand of a tricuspid aortic valve is extremely rare. We describe a patient with aortic regurgitation due to spontaneous rupture of the fibrous strands that suspend the aortic valve leaflets. The fibrous strands were recognized between the left and right coronary cusps, and between the left coronary and non-coronary cusps during the operation. The fibrous strand between the left coronary and non-coronary cusps had been ruptured, and thus the left coronary and non-coronary cusps were prolapsed into the left ventricle. Since a degenerative change was observed histologically in the resected specimen, these fibrous strands were considered to be an embryonic remnant of the aortic valve.
Assuntos
Insuficiência da Valva Aórtica/etiologia , Valva Aórtica/anormalidades , Valva Aórtica/cirurgia , Implante de Prótese de Valva Cardíaca , Idoso , Valva Aórtica/patologia , Insuficiência da Valva Aórtica/cirurgia , Fibrose , Humanos , Masculino , Ruptura Espontânea/complicaçõesRESUMO
The role of red blood cells during cold blood cardioplegia was studied using an intravital microscope in 13 isolated canine hearts perfused with diluted blood containing potassium chloride. The coronary microcirculation on the left ventricular epicardial surface was observed while the perfusate temperature was varied between 37 degrees C and 10 degrees C. Considerable sludging of red blood cells occurred during hypothermia. The percentage of capillaries perfused by red blood cells (percentage change) significantly decreased as perfusate temperature was reduced (100, 56, and 31% at 37, 20, and 10 degrees C respectively). This was caused by occlusion of microvessels due to sludging and by functional closure due to hypothermia. There was incomplete recovery of perfusion of capillaries at the end of rewarming (60%). The diameters of venules were reduced to 76% of control value at 10 degrees C because of the decrease in the numbers of feeding capillaries, but this value returned to 91% at the end of rewarming. Coronary vascular resistance (mmHg.ml-1.min-1 (kPa.s.litre-1] significantly decreased from 2.0(0.2) at 37 degrees C to 1.2(0.12) at 10 degrees C, but it increased to 2.4(0.24) at the end of rewarming. The finding in this study that sludging occurred which was slow to clear is a definite disadvantage of perfusion with red blood cells during hypothermia.
Assuntos
Circulação Coronária , Eritrócitos/fisiologia , Parada Cardíaca Induzida , Animais , Capilares/patologia , Cães , Calefação , Hipotermia Induzida , Microcirculação , Miocárdio/patologia , Resistência VascularRESUMO
We investigated whether long-term infusion of kallikrein would attenuate renal injury in salt-induced hypertension in Dahl salt-sensitive rats. A subdepressor dose of purified rat urinary kallikrein (700 ng/d IV) was infused by osmotic minipump for 4 weeks in male Dahl salt-sensitive rats fed a high salt (2% NaCl) diet. This dose did not affect the time-dependent elevation of blood pressure; however, urinary protein excretion was significantly decreased, and glomerular filtration rate was increased. These beneficial effects were reflected morphologically by an attenuation of glomerulosclerotic lesions and tubular injury seen in the hypertensive Dahl salt-sensitive rats. Kallikrein infusion increased urinary excretion of bradykinin and stimulated excretion of cyclic GMP, suggesting that the kallikrein-kinin-prostaglandin and nitric oxide axes were enhanced by rat urinary kallikrein infusion. The alterations induced by kallikrein infusion were potentiated by the concomitant administration of the angiotensin-converting enzyme inhibitor alacepril. These studies indicated that long-term replacement with rat tissue kallikrein attenuates renal injury in hypertensive Dahl salt-sensitive rats.
Assuntos
Hipertensão/induzido quimicamente , Hipertensão/patologia , Calicreínas/administração & dosagem , Rim/efeitos dos fármacos , Rim/patologia , Cloreto de Sódio , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Bradicinina/urina , Captopril/análogos & derivados , Captopril/farmacologia , Creatina/sangue , Creatina/urina , GMP Cíclico/urina , Eicosanoides/urina , Hemodinâmica/efeitos dos fármacos , Hipertensão/fisiopatologia , Calicreínas/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
OBJECTIVE: To investigate whether and how renin-angiotensin inhibition attenuates renal injury seen in salt-induced hypertension in Dahl salt-sensitive (Dahl-S) rats. METHODS: Dahl-S rats fed a high-salt (4% sodium chloride) diet for 6 weeks were treated with the angiotensin converting enzyme (ACE) inhibitor alacepril or the angiotensin receptor antagonist losartan for 4 weeks. Functional and morphological alterations in the kidney were investigated. RESULTS: Alacepril decreased systolic blood pressure (SBP). This SBP reduction was associated with the attenuation of cardiac and aortic wall hypertrophy and that of proteinuria and urinary N-acetyl-beta-D-glucosaminidase excretion. Kidney injuries, e.g. glomerular, arterial and tubular damage, were improved with alacepril treatment. Losartan decreased SBP to the same extent as alacepril, but neither renal function nor morphological structure was improved as was the case with alacepril. The response of the renal eicosanoid system to alacepril was inadequate, but cyclic GMP excretion, an indicator of nitric oxide formation, was significantly enhanced and lipid peroxidation in the kidney was decreased. CONCLUSIONS: The beneficial effects of ACE inhibition on the renal injury in Dahl-S rats outrange those induced by the receptor antagonism. This might be due to multiple factors including an increased vasodepressor eicosanoid system, enhanced nitric oxide formation and possible inhibition of oxygen radical generation in the injured renal tissues.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Rim/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Sangue/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Captopril/análogos & derivados , Captopril/farmacologia , Hemodinâmica/efeitos dos fármacos , Imidazóis/farmacologia , Rim/patologia , Losartan , Masculino , Ratos , Ratos Endogâmicos/fisiologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Tetrazóis/farmacologia , Urina/químicaRESUMO
OBJECTIVE: To investigate the effects of subpressor doses of angiotensin II (Ang II) on the progression of renal injuries in Dahl salt-sensitive (Dahl-S) rats with hypertension. METHODS: Rats were fed a high-salt (4% NaCl) diet and given an Ang II infusion (10 or 50 ng/kg per min, subcutaneously) for 4 weeks. RESULTS: The plasma Ang II concentration achieved in the high-dose Ang II infusion was lower than that in low-salt (0.3% NaCl) normotensive rats. The Ang II infusion did not affect systolic blood pressure, cardiac hypertrophy or weight of thoracic aorta. However, the high-dose Ang II infusion increased proteinuria by 58%, enhanced the urinary N-acetyl-beta-D-glucosaminase index by 77% and reduced the glomerular filtration rate by 37%. The impaired renal function was associated with a progression of glomerulosclerotic lesions. Neither tubular nor arterial lesions were exacerbated. The infusion did not influence prostacyclin production or urinary cyclic GMP excretion. CONCLUSION: A subpressor dose of Ang II infusion impairs renal function with progression of glomerulosclerosis, and these alterations may be due to increased susceptibility of the glomerulus in Dahl-S rats to Ang II-induced injuries. Such a mechanism might underlie a predisposition to hypertension-induced organ damage seen in Dahl-S rats with salt-induced hypertension.
Assuntos
Angiotensina II/toxicidade , Hemodinâmica/efeitos dos fármacos , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Angiotensina II/sangue , Animais , AMP Cíclico/urina , GMP Cíclico/urina , Eicosanoides/urina , Taxa de Filtração Glomerular/efeitos dos fármacos , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Hipertensão/metabolismo , Hipertensão/patologia , Injeções Subcutâneas , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Peróxidos Lipídicos/metabolismo , Radioimunoensaio , RatosRESUMO
The antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 micrograms/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 micrograms/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation. Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 micrograms/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 +/- 5.2 min) or sodium ozagrel (9.4 +/- 3.0 min) compared with saline (13.3 +/- 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model. These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.
Assuntos
Fibrinolíticos/farmacologia , Fragmentos Fab das Imunoglobulinas/imunologia , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombose/tratamento farmacológico , Animais , Anticorpos Monoclonais , Aspirina/farmacologia , Tempo de Sangramento , Relação Dose-Resposta Imunológica , Fibrinolíticos/imunologia , Macaca mulatta , Metacrilatos/farmacologia , Fotoquímica , Inibidores da Agregação Plaquetária/imunologia , Contagem de Plaquetas , Distribuição Aleatória , Saimiri , Trombose/etiologiaRESUMO
We characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) maintained in primary culture by measuring uptake of 45Ca2+ or Mn2+ from a normal balanced salt solution and the extracellular Ca(2+)-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) in a medium [high pH (pH 8.8)/high Mg2+ (20 mM) medium containing a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin] that inhibits removal of Ca2+ from the cytoplasm. Such measurements in the presence or absence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive voltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under resting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in these SMCs resulted in activation of neither DHP-sensitive nor -insensitive Ca2+ entry, but rather resulted in marked suppression of the former. Utilizing the DHP-sensitive extracellular Ca(2+)-induced increase in [Ca2+]i as a monitor of activity of the DHP-sensitive VGCCs, we investigated the effects of protein kinase activators and phosphatase inhibitors on the regulation of these VGCCs. We found that the DHP-sensitive VGCCs were inhibited by endothelin-1 through the activation of protein kinase C. We also found that they were inhibited by 8Br-cGMP, okadaic acid, and calyculin A.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Cálcio/farmacocinética , Canais de Cálcio/fisiologia , Radioisótopos de Cálcio , Células Cultivadas , Di-Hidropiridinas/farmacologia , Endotelinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fluorescência , Fura-2 , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Cinética , Masculino , Manganês/farmacocinética , Músculo Liso Vascular/citologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ratos , Ratos Endogâmicos WKYRESUMO
BACKGROUND: Various blood management strategies can be used to reduce the need for allogeneic blood in cardiac surgery. In anemic patients, however, avoidance of allogeneic blood transfusion is difficult to achieve. This study was performed to assess the safety and effectiveness of preoperative blood collection using recombinant human erythropoietin (rHuEPO) for reducing the exposure to allogeneic blood in anemic patients. METHODS: Thirty-two anemic patients undergoing cardiac surgery at our hospital between January 1994 and October 1997 were divided into two groups according to preoperative strategies: 3-week treatment with rHuEPO and blood donation (group 1, n = 16) or iron supplementation alone (group 2, n = 16). RESULTS: There were no statistically significant differences between the two groups in patients' characteristics and surgical data. The number of reticulocytes was increased at just before surgery in group 1, whereas group 2 showed no significant increase. The estimated hemoglobin increases in group 1 were higher at 7 days and just before surgery. The mean number of required allogeneic blood for patients during surgery was 0.59 +/- 1.12 U in group 1 and 5.01 +/- 2.63 U in group 2. In 75% of group 1 patients, allogeneic blood transfusion was successfully avoided, whereas all patients in group 2 received allogeneic blood. CONCLUSIONS: This study suggests that the combination of rHuEPO administration and autologous blood donation can reduce the need for allogeneic blood in anemic patients.
Assuntos
Anemia/sangue , Transfusão de Sangue Autóloga , Ponte de Artéria Coronária , Eritropoetina/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Anemia/etiologia , Feminino , Humanos , Ferro/administração & dosagem , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Contagem de ReticulócitosRESUMO
We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.
Assuntos
Ácido Aurintricarboxílico/administração & dosagem , Fibrinolíticos/farmacologia , Peptídeos/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Trombose/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Masculino , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Intravascular platelet aggregation and subsequent thrombus formation were produced in the microvasculature by irradiation using filtered light from a mercury lamp in combination with the intravascular administration of a fluorescent dye. The effects of several factors, such as light intensity, dye concentration, and vessel diameter were examined in venules and arterioles of the rat mesentery with respect to the time required for the initiation of thrombus formation; the time to totally occlude the lumen; and the volume of the thrombus formed. It was found that the combined conditions of light intensity and dye concentration at specific values had a critical effect on the rate of platelet aggregation. In the arterioles and venules examined, the thrombus formation time was prolonged as a function of increasing vessel diameter. The time required for the initiation of thrombus formation and complete occlusion of the vessel lumen was greater in arterioles than in venules. This result may suggest the dependence of the kinetics of intravascular thrombus formation on blood flow conditions.
Assuntos
Fluoresceínas/toxicidade , Luz/efeitos adversos , Oclusão Vascular Mesentérica/etiologia , Trombose/etiologia , Animais , Arteríolas/patologia , Velocidade do Fluxo Sanguíneo , Fluoresceína , Microcirculação , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos da radiação , Ratos , Ratos Endogâmicos , Estresse Mecânico , Vênulas/patologiaRESUMO
Diet including mold oil from a lipid accumulative fungus, containing gamma-linolenic acid, showed an inhibitory effect on thrombus formation in the microvessels of rats by the light/dye method of the authors. Male Wistar rats were fed for 3 to 4 weeks with two series of experimental diets and were examined for thrombus formation. The thrombus formation times to totally occlude, ts, were 347 sec for (mold + soybean)-oil and 236 sec for (palm + soybean)-oil in the first series of diets and 1288 sec for mold oil, 538 sec for olive oil and 575 sec for safflower oil in the second series of diets. Fatty acid composition of plasma, erythrocyte and liver lipids showed an increase in arachidonate content with the diet including the mold oil. Higher arachidonate content seem favorable in inhibiting thrombus formation with increasing PGI2 formation. In terms of the level of lipid hydroperoxides, indicated as a desaturation index of constituent fatty acids, the higher desaturation index with safflower oil gave shorter ts, which suggested some oxygen derived free radicals from polyunsaturated fatty acids were involved in the mechanism of thrombogenesis study by this method.
Assuntos
Gorduras na Dieta/farmacologia , Ácidos Linolênicos/farmacologia , Oclusão Vascular Mesentérica/prevenção & controle , Óleos de Plantas/farmacologia , Trombose/prevenção & controle , Animais , Pressão Sanguínea/efeitos dos fármacos , Gorduras na Dieta/uso terapêutico , Fluoresceína , Fluoresceínas/toxicidade , Radicais Livres , Fungos/análise , Luz/efeitos adversos , Ácidos Linolênicos/uso terapêutico , Peroxidação de Lipídeos , Masculino , Oclusão Vascular Mesentérica/etiologia , Óleos de Plantas/isolamento & purificação , Óleos de Plantas/uso terapêutico , Ratos , Ratos Endogâmicos , Trombose/etiologia , Aumento de Peso/efeitos dos fármacos , Ácido gama-LinolênicoRESUMO
The antithrombotic effect of a synthetic symmetrical triglyceride having eicosapentaenoic acid (EPA) at positions 1 and 3, and gamma-linolenic acid (GLA) at position 2 was investigated. Administration of the triglyceride significantly increased thrombus formation time and thrombotic occlusion time induced by light irradiation and a fluorescent dye in guinea pigs after 14 days administration compared to that of soybean oil. The antithrombotic effect of the triglyceride was similar to that of EPA ethyl ester. Administration of the triglyceride increased GLA, dihomo-gamma-linolenic acid (DGLA) and EPA contents in plasma and the liver, and the ratio of DGLA to arachidonic acid. These results might be responsible for this antithrombotic effect.
Assuntos
Ácido Eicosapentaenoico/química , Fibrinolíticos/uso terapêutico , Lipídeos/sangue , Oclusão Vascular Mesentérica/prevenção & controle , Mesentério/irrigação sanguínea , Triglicerídeos/uso terapêutico , Ácido gama-Linolênico/química , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/uso terapêutico , Ácidos Graxos/sangue , Fibrinolíticos/química , Fluoresceína , Fluoresceínas/efeitos da radiação , Fluoresceínas/toxicidade , Cobaias , Luz , Fígado/metabolismo , Masculino , Oclusão Vascular Mesentérica/sangue , Oclusão Vascular Mesentérica/induzido quimicamente , Estrutura Molecular , Fotoquímica , Óleo de Soja/uso terapêutico , Relação Estrutura-Atividade , Triglicerídeos/química , Vênulas/efeitos dos fármacosRESUMO
Time course changes in the cell shape and in the patterns of microfilament distribution were analyzed quantitatively using cultured porcine aortic endothelial cell monolayers before and after a shear flow exposure. Geometrical parameters of the cell and of the microfilament were measured on fluorescent photomicrographs of the cells stained with rhodamine-phalloidin. After the shear flow exposure (20 dyn cm-2, 0-24 h), the endothelial cells on glass were elongated and oriented to the direction of the flow. Under the no-flow condition, F-actin filaments were mainly localized at the periphery of the cell, although some filaments were seen in the more central portion. The angles of the filaments were randomly distributed. After 3 h, the stress fiber-like structure of an F-actin bundle was formed in the central part of the cells, and these filaments were oriented to the direction of the flow. The degree of orientation increased as the time of exposure to shear stress became longer. This change in F-actin preceded cell elongation and orientation; these changes were statistically significant only after 6 h. After 24 h, peripheral filaments were again observed, and the fluorescence intensity of rhodamine-phalloidin-stained cells was enhanced. These findings suggest that the redistribution of F-actin filaments is one of the early cellular responses to the onset of shear stress and that it is one of the most important factors controlling cell elongation and orientation to the direction of the flow.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Aorta/ultraestrutura , Endotélio Vascular/ultraestrutura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Actinas/análise , Actinas/fisiologia , Animais , Aorta/citologia , Aorta/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Microscopia de Fluorescência , Reologia , Estresse Mecânico , Suínos , Fatores de TempoRESUMO
The viscoelastic properties of cultured endothelial cells exposed to shear stress were measured by the micropipette technique and analyzed using a standard linear viscoelastic model. Cells from porcine aorta were cultured on glass coverslips. A shear stress of 2 Pa was applied using a parallel-plate flow chamber. After flow exposure, the cells were detached from the coverslips and suspended in culture medium. The micropipette experiment was performed on single cells under an inverted microscope. The desired negative pressure was applied stepwise to the tip of the micropipette by opening a solenoid valve. The deformation process of cells in the micropipette was observed through a TV camera and recorded on a videotape. To obtain the viscoelastic parameters, a half-space model of an endothelial cell was used. The cell was assumed to be a homogeneous and incompressible material, and a standard linear viscoelastic model was employed to account for the viscoelastic response. Cells exposed to shear stress for 6 h became spherical in shape after detachment from the substrate. In the case of a 24 h exposure, about half of the detached cells retained an elongated shape upon detachment, with the others taking on a spherical shape. The elastic constants, as determined based on the model, were approximately two times higher for the elongated cells than for control cells from static culture, no-flow conditions, indicating that the elongated cells became stiffer. Enhanced viscous properties also were observed for the elongated cells. These viscoelastic properties are considered to be closely related to cytoskeletal structure. Spherical cells upon detachment, even those that had been exposed to shear stress for 24 h, did not show such significant changes in viscoelastic mechanical properties.
Assuntos
Aorta/fisiologia , Endotélio Vascular/fisiologia , Animais , Aorta/citologia , Fenômenos Biomecânicos , Elasticidade , Endotélio Vascular/citologia , Modelos Cardiovasculares , Estresse Mecânico , Suínos , ViscosidadeRESUMO
The effects of somatosensory nociceptive pinch stimulation of the hindpaw on mesenteric microvascular hemodynamics and systemic circulatory parameters were investigated in anesthetized rats using an intravital microscope-television system. Blood flow velocity in the terminal (18-40 microm in diameter) or precapillary (10-20 microm in diameter) arterioles of the mesentery was monitored by the dual sensor method developed by the authors. In the proximal terminal arterioles, blood flow velocity decreased substantially along with arteriolar constriction induced by pinching of the hindpaw for 30 s. In the distal terminal arterioles and precapillary arterioles, blood flow velocity increased after pinching. In the proximal terminal arterioles, the decrease of velocity in response to reflex vasoconstriction was abolished by intravenous injection of an alpha-blocker (phentolamine, 10 mg/kg). The increase in mesenteric precapillary arteriolar blood flow velocity (43+/-9%, p < 0.01) associated with the increase in mean arterial pressure (MAP) (22+/-1%, p < 0.01) was observed within a few seconds after the onset of the stimulation, and then the response in blood flow velocity returned to the baseline ahead of MAP response recovery after the end of the stimulus. These responses were diminished by alpha-adrenergic receptor blockade. The heart rate (HR) increase (4+/-1%, p < 0.01) induced by pinching was abolished by beta-adrenergicreceptor blockade (propranolol, 3 mg/kg, i.v.). There was a strong correlation between the increase in MAP and the decrease in renal blood flow measured by laser Doppler flowmeter (r = 0.87-0.98). Pinch stimulation of the rat hindpaw evoked changes in mesenteric arteriolar blood flow velocity that were mediated via the somato-sympathetic reflex vasoconstriction and the pressor response.
Assuntos
Hemodinâmica/fisiologia , Nociceptores/fisiologia , Circulação Esplâncnica/fisiologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Arteríolas/fisiologia , Velocidade do Fluxo Sanguíneo/fisiologia , Pressão Sanguínea/fisiologia , Hemodinâmica/efeitos dos fármacos , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Estimulação Física , Ratos , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacosRESUMO
The dietary effect of 1,3-biseicosapentaenoyl-2-gamma-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-E) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, EPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-gamma-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1- and 3-series eicosanoids.